Biological reactors

Curr Top Dev Biol, 2004, 60, 91 - 126
Mechanical signals as regulators of stem cell fate; Estes BT et al.; In recent years, stem cells have shown significant promise for their potential to provide a source of undifferentiated progenitor cells for therapeutic applications in tissue or organ repair . Significant questions still remain, however, as to the genetic and epigenetic signals that regulate the fate of stem cells . It is now well accepted that the micro-environment of the stem cell can have a significant influence on its differentiation and phenotypic expression . Although emphasis has been placed in previous work on the role of soluble mediators such as growth factors and cytokines on stem cell differentiation, there is now significant evidence, both direct and indirect, that mechanical signals may also regulate stem cell fate . We review a number of in vivo and in vitro studies that have provided evidence that mechanical factors have the ability to influence the differentiation of a number of cells that have been classified as either precursor, progenitor, or stem cells . Taken together, these studies show that specific mechanical signals may promote cell differentiation into a particular phenotype, potentially having an effect on embryonic development . The use of such mechanical signals in vitro in specially designed "bioreactors" may provide important adjuncts to standard biochemical signaling pathways for promoting engineered tissue growth . A further understanding of the biomechanical and biochemical pathways involved in mechanical signal transduction by stem cells will hopefully provide new insight for the improvement of stem-cell based therapies.

Environ Pollut, 1997, 97(3), 247 - 51
Implementation of a continuous-flow stirred bioreactor system in the bioremediation of heavy metals from industrial waste water; Stoll A et al.; The industrial applicability of immobilized non-viable yeast biomass (Saccharomyces cerivisiae) in continuous-flow stirred bioreactors was tested with respect to metal removal from electroplating effluent solutions containing Cu, Cd, Cr, Ni and Zn in excess of the stipulated water quality criteria . Two systems, a dual bioreactor and triple bioreactor system in series, were tested and compared regarding their efficiency of metal removal . The system containing two bioreactors linked in series provided a more efficient solution to the problem . The introduction of a third bioreactor into the system had a marginal effect on the amount of metal removed . Subsequent to treatment within the bioreactors the levels of Cu, Cr and Ni in the effluent complied with the stipulated drinking water quality criteria and, although in excess of these criteria only 18% of the initial Zn and 17% of the Cd remained in solution.

Environ Pollut, 1998, 99(1), 87 - 92
Development of effective hydrogen sulphide removing equipment using Thiobacillus sp.IW; Oh KJ et al.; Hydrogen sulphide is one of the commonest odours emitted by chemical plants . To remove the hydrogen sulphide biologically, a three phase fluidised bed bioreactor was used in which Thiobacillus sp.IW was immobilised on activated carbon . The optimum operating conditions of the bioreactor were 30 degrees C, pH7, aspect ratio (L/D) = 1 and at these conditions, the system removed over 94% of the hydrogen sulphide in the concentration range of 100-200 ppm and flow rate of 1.0-2.0 litre min(-1) . From the upset and recovery test, the system proved stable within the moderate inlet concentration changes investigated.

Environ Pollut, 2000 Jan, 107(1), 145 - 51
Effect of immobilized microalgal bead concentrations on wastewater nutrient removal; Tam NF et al.; A unicellular green microalga, Chlorella vulgaris, entrapped in calcium alginate as algal beads were employed to remove nutrients (N and P) from simulated settled domestic wastewater . A significantly higher nutrient reduction was found in bioreactors containing algal beads (at concentrations ranging from 4 to 20 beads ml(-1) wastewater) than the blank alginate beads (without algae) . A complete removal of NH(4)(+)-N and around 95% reduction of PO(4)(3-)-P was achieved within 24 h of treatment in bioreactors having the optimal algal bead concentration (12 beads ml(-1), equivalent to 1:3 algal beads:wastewater, v/v) . The NH(4)(+)-N removal was significantly lower at low (around 4 beads ml(-1)) and high (>15 beads ml(-1)) algal bead concentrations . On the other hand, the effect of bead concentration on phosphate removal was less obvious, and bead concentrations ranging from 8 to 20 beads ml(-1) showed comparable percentages of phosphate reduction . Algal uptake and adsorption on alginate gels were found to be the major processes involved in the removal of N and phosphate in the present study . In addition, NH(4)(+)-N could be lost via ammonia volatilization while PO(4)(3-)-P was removed by chemical precipitation, as alkaline pH was recorded in the immobilized microalgal treatment system.

Environ Pollut, 2000 Apr, 108(1), 91 - 101
Tracing the transformation of labelled {1-13C}phenanthrene in a soil bioreactor; Richnow HH et al.; {1-(13)C}-labelled phenanthrene was incubated in a closed bioreactor to study the flux and biotransformation of polycyclic aromatic hydrocarbon (PAH) in contaminated soils on a bulk and molecular level . The degradation of extractable phenanthrene was observed by GC-MS measurements and the mineralisation was monitored by (13)CO(2) production . The transformation of the (13)C-label into non-extractable soil-bound residues was determined by carbon isotopic measurements . With these data we were able to calculate a carbon budget of the (13)C-label . Moreover, the chemical structure of non-extractable bound residues was characterised by applying selective chemical degradation reactions to cleave xenobiotic subunits from the macromolecular organic soil matrix . The obtained low molecular weight products yielded (13)C-labelled compounds which were identified using IRM (isotope ratio monitoring)-GC-MS and structurally characterised with GC-MS . Most of the (13)C-labelled products obtained by chemical degradation of non-extractable bound residues are well-known metabolites of phenanthrene . Thus, metabolites of {1-(13)C}phenanthrene formed during biodegradation appear to be reactive components which are subsequently involved in the bound residue formation . Hydrolysable amino acids of the soil residues were significantly labelled with (13)C as confirmed by IRM-GC-MS measurements . Therefore, phenanthrene-derived carbon was transformed by anabolic microbial processes into typical biologically derived compounds . These substances are likely to be incorporated into humic-like material after cell death.

Environ Pollut, 1990, 66(3), 237 - 52
Bacterial leaching of heavy metals from sewage sludge-bioreactors comparison; Couillard D et al.; More than 50% of municipal sewage sludges cannot be used on agricultural land because of their heavy metals content . Therefore, microbial leaching of heavy metal from municipal sludge was studied in a continuously stirred tank reactor without recycling (CSTR) or with sludge recycling (CSTRWR) at residence times of 1, 2, 3 and 4 days . The reactor CSTRWR is supposed to be more efficient for bacterial process due to the recycling of active bacteria from the settling tank to the reactor . The CSTRWR and the CSTR with 1 g litre(-1) FeSO(4).7H(2)O addition were equally efficient because of copper reprecipitation or recomplexation in the settling tank of the CSTRWR . In the CSTR, about 62% of copper and about 77% of zinc were dissolved in 3 days residence time compared to 50% of copper and 64% of zinc in the CSTRWR, if 3 g litre(-1) FeSO(4).7H(2)O was added . Thus with larger amount of substrate, the CSTR was more efficient than the CSTRWR . Residence time and pH were the main factors for zinc solubilization while for copper, the redox potential was also a major factor . The effect of FeSO(4).7H(2)O concentration on bacterial activity to solubilize heavy metals was also studied, increased concentration of FeSO(4).7H(2)O yielded better copper solubilization while it had no effect or a negative effect on zinc . This supports the hypothesis of a direct mechanism for zinc solubilization and of an indirect mechanism for copper solubilization.

Environ Pollut, 1993, 81(3), 229 - 49
Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): A review; Wilson SC et al.; Polynuclear aromatic hydrocarbons (PAHs) constitute a group of priority pollutants which are present at high concentrations in the soils of many industrially contaminated sites . Criteria established for the removal or treatment or both of soils contaminated with PAHs vary widely within and between nations . The bioremediation of contaminated soils with in-situ, on-site, and bioreactor techniques is reviewed, together with the factors affecting PAH degradation . Current in-situ remediation techniques are considered ineffective for the removal of most PAHs from contaminated soil . On-site 'landforming' methods have been used successfully (and within a reasonable period of time) to degrade only those PAHs with three or fewer aromatic rings . Bioreactors have proved most effective for soil remediation, since conditions for enhanced degradation can be achieved most readily . However, bioreactors are still at the development stage, and further research is required to optimise their efficiency and economy for routine use . Degradation of the more recalcitrant high-molecular-weight PAHs is contaminated soil has not been particularly successful to date . Further research needs are identified to help develop bioremediation into a most cost-effective technology . The importance of full site assessments and treatability studies for successful application in the field is emphasised.

Adv Biochem Eng Biotechnol, 2004, 86, 215 - 53
Hematopoietic stem cells: clinical requirements and developments in ex-vivo culture; Mukhopadhyay A et al.; Ex vivo expansion of hematopoietic stem cells is a promising technology for many potential applications from marrow reconstitution to gene therapy . Considerable progress has been made during the past ten years in understanding the biology of hematopoietic stem cells and its ex vivo expansion; despite this, the cultured cell is still between pre-clinical and phase I clinical trials . This review summarizes recent progress in the ex vivo expansion of hematopoietic stem cells and its requirements for clinical applications . The second section covers hematopoiesis and the bone marrow microenvironment . The third and fourth sections deal with therapeutic applications of stem cells and transplantation requirements, respectively . Biological alteration of expanded stem cells, molecular control of hematopoiesis, characterization of cells, and bioreactors for culture of stem cells and its operational parameters are the subjects of the fifth section . The next section covers pre-clinical and clinical studies on expanded stem cells . Ex vivo expansion of stem cells in three-dimensional culture system is the subject matter for the last section.

Adv Biochem Eng Biotechnol, 2004, 86, 1 - 45
Investigations of the production of cephalosporin C by Acremonium chrysogenum; Tollnick C et al.; A review is given on the morphology of Acremonium chrysogenum and the biosynthesis of cephalosporin C based on the published references . Investigations are presented on the comparison of cultivation media carried out by means of shake flask cultures . The process performance of a standard cultivation in well controlled bioreactor is presented and compared with other cultivations, which were executed with the same strain and bioreactor, but with various carbon-, nitrogen- and sulphur-sources keeping the concentrations of the key components at definite levels . Also the influence of dilution and enrichment of the medium on the process performance is explored . Mathematical models for the growth of Acremonium chrysogenum and production of cephalosporin C are reviewed and their application for control of industrial processes with complex cultivation media are discussed.

Water Res, 2004 Apr, 38(8), 2166 - 72
Mechanism of textile metal dye biotransformation by Trametes versicolor; Blanquez P et al.; The biodegradation of Grey Lanaset G, which consists of a mixture of metal complexed dye, was studied . Experiments were carried out in a bioreactor with retained pellets of the fungus Trametes versicolor that was operated under conditions of laccase production . Although decolorization was highly efficient (90%), no direct relationship to extracellular enzyme was apparent . Moreover, the extracellular enzyme was found to be unable to degrade the dye in vitro . The process involves several steps . Thus, the initial adsorption of the dye and its transfer into cells is followed by breaking of the metal complex bond in the cells release of the components . The metal (Cr and Co) contents of the biomass and treated solutions, and their closer relationship to intracellular enzyme and degradation of the dye, confirm the initial hypothesis.

Wound Repair Regen, 2004 Mar-Apr, 12(2), 148 - 56
Autologous cultured keratinocytes on porcine gelatin microbeads effectively heal chronic venous leg ulcers; Liu JY et al.; We have established a specific bioreactor microcarrier cell culture system using porcine gelatin microbeads as carriers to produce autologous keratinocytes on a large scale . Moreover, we have shown that autologous keratinocytes can be cultured on porcine collagen pads, thereby forming a single cell layer . The objective of this study was to compare efficacy and safety of autologous cultured keratinocytes on microbeads and collagen pads in the treatment of chronic wounds . Fifteen patients with recalcitrant venous leg ulcers were assigned to three groups in a single-center, prospective, uncontrolled study: five underwent a single treatment with keratinocyte monolayers on collagen pads (group 1); another five received a single grafting with keratinocyte-microbeads (group 2); and the last five received multiple, consecutive applications of keratinocyte-microbeads 3 days apart (group 3) . All patients were followed for up to 12 weeks . By 12 weeks, there was a mean reduction in the initial wound area of 50, 83, and 97 percent in the three groups, respectively . The changes in wound size were statistically significant between the first and third groups (p= 0.0003) . Keratinocyte-microbeads proved to be more effective than keratinocyte monolayers on collagen pads when the former were applied every 3 days . Rapid availability within 10-13 days after skin biopsy and easy handling represent particular advantages.

Bioprocess Biosyst Eng, 2004 Jul, 26(4), 249 - 58 Epub 2004 Apr 15.
Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor; Zelic B et al.; A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes . The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium . Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1) . Acetate was fed according to the previously developed feeding strategy . While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC) . The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively . Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term).

Mar Biotechnol (NY) . 2004 Apr 19; {Epub ahead of print}
Improved Production of Bioactive Glucosylmannosyl-Glycerolipid by Sponge-Associated Microbacterium Species; Lang S et al.; The marine Microbacterium species HP2 (DSM 12583), isolated from the sponge Halichondria panicea, is able to produce a glucosylmannosyl-glycerolipid when grown on a complex medium with glucose . Optimizing the carbon sources in shake flask experiments has shown that glycerol affords the highest specific glycoglycerolipid production . The product yield approached 300 mg/L or 25 mg/g biomass upon scaling up in a 40-L bioreactor volume . The native diglycosyl-glycerolipid GGL.2 strongly inhibited growth of the tumor cell lines HM02 and Hep G2 (50% inhibition at 0.4 to 3 micro g/mL), while the related deacylated compound (GG.2) showed a potent anti-tumor-promoting activity.

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6786 - 91 Epub 2004 Apr 14.
Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures; Zhang L et al.; Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent . Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 beta-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures . Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h) . The best line (T(3)) produced 411 mg/liter scopolamine, which was over nine times more than that in the wild type (43 mg/liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H(11) (184 mg/liter) . To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant . We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes . It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H . niger whereas the functioning of the upstream enzyme PMT is increased proportionally . This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Biotechnol Bioeng, 2004 May 5, 86(3), 308 - 16
A novel packed-bed bioreactor operating under isothermal and non-isothermal conditions; De Maio A et al.; A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed . The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate . Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively . Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions . Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value . Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration . The results have been explained within the frame of reference of the process of thermodialysis .

Biotechnol Bioeng, 2004 May 5, 86(3), 301 - 7
Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production; van de Vondervoort PJ et al.; Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants . Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures . In most cultivations mutants with fluffy (aconidial) morphology became dominant . From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle . The mutant contained a single morphological mutation, causing an increased colony radial growth rate . The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation .

J Appl Microbiol, 2004, 96(5), 1074 - 81
Beta-glucan production by Botryosphaeria rhodina in different bench-top bioreactors; Selbmann L et al.; AIMS: Evaluation of the technical feasibility of transferring beta-glucan production by Botryosphaeria rhodina DABAC-P82 from shaken flasks to bench-top bioreactors . METHODS AND RESULTS: Three different bioreactors were used: 3 l stirred tank reactor (STR-1) equipped with two different six-blade turbines; STR as above but equipped with a three-blade marine propeller plus draft-tube (STR-2); 2 l air-lift column reactor (ALR) equipped with an external loop . STR-1, tested at three different stirrer speeds (300, 500 and 700 rev min(-1)) appeared to be less suitable for beta-glucan production by the fungus, being maximum production (19.4 g l(-1)), productivity (0.42 g l(-1) h(-1)) and yield (0.48 g g(-1) of glucose consumed) markedly lower than those obtained in shaken culture (29.7 g l(-1), 1.23 g l(-1) h(-1) and 0.61 g g(-1), respectively) . Better performances were obtained with both STR-2 and ALR . With the latter, in particular, the increase of production was accompanied by reduced fermentation time (25.7 g l(-1) after only 22 h); productivity and yield were highest (1.17 g l(-1) h(-1) and 0.62 g g(-1) of glucose consumed, respectively) . CONCLUSION: Using an air-lift reactor with external loop, the scaling up from shaken flasks to bench-top bioreactor of the beta-glucan production by B . rhodina DABAC-P82 is technically feasible . SIGNIFICANCE AND IMPACT OF THE STUDY: Although culture conditions are still to be optimized, the results obtained using the ARL are highly promising.

Water Sci Technol, 2004, 49(4), 305 - 10
Ozonation of persistent DOC in municipal WWTP effluent for groundwater recharge; Schumacher J et al.; Groundwater recharge is becoming common in areas where the withdrawal of groundwater exceeds its natural recharge . Wastewater treatment plant (WWTP) effluent can be used for this purpose, but persistent organic compounds can only be partly removed during soil passage . This point was confirmed in degradation tests using soil columns in which the DOC of a membrane bioreactor (MBR) permeate could only be reduced by 15% and adsorbable organic iodine (AOI) by 2% . However, ozonation was found to improve biodegradation and at the maximum formation of biodegradable DOC at 2.5 mg O3/mg DOC0, the DOC values of MBR permeate (11-13 mg/L) could be reduced in aerobic degradation batch tests to the DOC of Berlin drinking water (3-5 mg/L) . A combination of ozonation at 1.9 g O3/g DOC0 with soil passage could adjust DOC, UVA254, colour (436 nm) and the molecular DOC size distribution to drinking water ranges, but AOI was only reduced from 143 microg/L to 92 microg/L and remained high compared to tap water (2.2 microg/L) . The extremely persistent X-ray contrast compound iopromide, which represents part of AOI, was spiked into MBR permeate at a low concentration . Iopromide was reduced by 88% during ozonation, but AOI only decreased by 23% indicating that a transformation, but not a mineralization, of iodinated organic compounds occurs.

Water Sci Technol, 2004, 49(4), 299 - 303
Effect of ozone, chlorine and hydrogen peroxide on the elimination of colour in treated textile wastewater by MBR; Brik M et al.; In treating textile wastewater, the application of membrane bioreactor (MBR) technology showed high efficiency in COD and BOD5 removal . However, insufficient colour removal was achieved for possible reuse . The aim of the work presented in this paper was to test the performance of chemical advanced oxidation on the elimination of the colour downstream of an MBR . To improve the quality of the membrane bioreactor effluent three different oxidation treatments were tested at lab-scale: ozonation, chlorination and hydrogen peroxide oxidation . Colour, COD and BOD5 were controlled in order to assess the effectiveness of each process . For chlorination, even with 250 mg/L (active chlorine) only 80% colour removal (SACin = 14; SACout = 2.8) was achieved which is considered unsatisfactory . For hydrogen peroxide, the colour removal was even poorer; it was just 10% at a concentration of 250 mg/L . In contrast, good results were obtained by ozonation . By using only 38 mg/L within 20 minutes, it was possible to achieve the reuse recommendation with a satisfactory colour removal of 93% (SACin = 14; SACout = 0.98) . The results showed that ozonation was the most promising method.

J Vasc Surg, 2004 Apr, 39(4), 859 - 67
Dog peritoneal and pleural cavities as bioreactors to grow autologous vascular grafts; Chue WL et al.; OBJECTIVE: The purpose of this study was to grow "artificial blood vessels" for autologous transplantation as arterial interposition grafts in a large animal model (dog) . METHOD AND RESULTS: Tubing up to 250 mm long, either bare or wrapped in biodegradable polyglycolic acid (Dexon) or nonbiodegradable polypropylene (Prolene) mesh, was inserted in the peritoneal or pleural cavity of dogs, using minimally invasive techniques, and tethered at one end to the wall with a loose suture . After 3 weeks the tubes and their tissue capsules were harvested, and the inert tubing was discarded . The wall of living tissue was uniformly 1-1.5 mm thick throughout its length, and consisted of multiple layers of myofibroblasts and matrix overlaid with a single layer of mesothelium . The myofibroblasts stained for alpha-smooth muscle actin, vimentin, and desmin . The bursting strength of tissue tubes with no biodegradable mesh scaffolds was in excess of 2500 mm Hg, and the suture holding strength was 11.5 N, both similar to that in dog carotid and femoral arteries . Eleven tissue tubes were transplanted as interposition grafts into the femoral artery of the same dog in which they were grown, and were harvested after 3 to 6.5 months . Eight remained patent during this time . At harvest, their lumens were lined with endothelium-like cells, and wall cells stained for alpha-actin, smooth muscle myosin, desmin and smoothelin; there was also a thick "adventitia" containing vasa vasorum . CONCLUSION: Peritoneal and pleural cavities of large animals can function as bioreactors to grow myofibroblast tubes for use as autologous vascular grafts.

J Biotechnol, 2004 Apr 29, 109(3), 287 - 94
Production of highly 13C-labeled polyphenols in Vitis vinifera cell bioreactor cultures; Aumont V et al.; The use of Vitis vinifera cells grown in a 2 l-stirred tank bioreactor for producing isotopically 13C-labeled phenolic substances is presented . Several culture parameters were optimized to achieve characteristics of growth and polyphenol metabolism similar to that recorded in shake flasks . Administration of {1-13C}L-phenylalanine (3 mM) to grape cell suspension cultures led to the production of 13C-labeled stilbenes (trans- and cis-piceids), catechins (catechin and epicatechin) and anthocyanins (delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-O-beta-glucosides) . Incorporation of {1-13C}L-phenylalanine into polyphenols was measured by means of 13C satellites in the proton NMR spectrum and EA-IRMS . The enrichment of labeling obtained for all the compounds (between 40 and 65%) is sufficient to investigate their absorption and metabolism in humans.

J Biotechnol, 2004 Apr 29, 109(3), 239 - 54
Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry; Kacmar J et al.; Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences . However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured . In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system . To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared . The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp . The other two plasmids are 2 microm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter . The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics . The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible "fingerprint" of the culture dynamics . The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability . Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability.

J Chromatogr A, 2004 Apr 2, 1032(1-2), 305 - 12
On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography; Kulp M et al.; Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses . The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit . The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions . No additional sample preparation was required . The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min . The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm . The analytes were detected at a microg/ml level with a reproducibility of about 7% . To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.

J Biotechnol, 2004 Apr 8, 109(1-2), 201 - 11
Potential of real-time measurement of GFP-fusion proteins; Jones JJ et al.; Building on the basic design concepts of Randers-Eichhorn {Biotechnol . Bioeng . 55 (1997) 921}, an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed . A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion . Cultivations were carried out at the 20l scale with the signal from the sensor being relayed directly to the control system of the bioreactors . The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression . A positive expression correlation between on-line and off-line data was obtained . Protein accretion measured off-line was quantified using both LC-MS and plate reader assays . The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein . Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct . Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed.

J Biotechnol, 2004 Apr 8, 109(1-2), 147 - 58
Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain; Calado CR et al.; By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock . However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step . Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process . From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose . This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose . Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose . The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth . Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.

Bioresour Technol, 2004 Jul, 93(3), 261 - 8
Thermal denaturation: is solid-state fermentation really a good technology for the production of enzymes?
Muller dos Santos M, Souza da Rosa A, Dal'Boit S, Mitchell DA, Krieger N.
The potential for thermal denaturation to cause enzyme losses during solid-state fermentation processes for the production of enzymes was examined, using the protease of Penicillium fellutanum as a model system . The frequency factor and activation energies for the first-order denaturation of this enzyme were determined as 3.447 x 10(59) h(-1) and 364,070 Jmol(-1), respectively . These values were incorporated into a mathematical model of enzyme deactivation, which was used to investigate the consequences of subjecting this protease to temporal temperature profiles reported in the literature for mid-height in a 34 cm high packed-bed bioreactor of 150 mm diameter . In this literature source, temperature profiles were measured for 5, 15 and 25 liters per minute of air and enzyme activities were measured as a function of time . The enzyme activity profiles predicted by the model were distributed similarly, one relative to the other, as had been found in the experimental study, with substantial amounts of denaturation being predicted when the substrate temperature exceeded 40 degrees C, which occurred at the lower two airflow rates . A mathematical model of a well-mixed bioreactor was used to explore the difficulties that would be faced at large scale . It suggests that even with airflows as high as one volume per volume per minute, up to 85% of the enzyme produced by the microorganism can be denatured by the end of the fermentation . This work highlights the extra care that must be taken in scaling up solid-state fermentation processes for the production of thermolabile products .

Biotechnol Prog, 2004 Mar-Apr, 20(2), 457 - 66
Hollow-fiber enzyme reactor operating under nonisothermal conditions; Diano N et al.; A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized . Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively . Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient . The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane . Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions . Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated . The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.

J Biomed Mater Res A, 2004 May 1, 69(2), 205 - 15
Quantitative analysis of three-dimensional fluid flow in rotating bioreactors for tissue engineering; Botchwey EA et al.; Tissue engineering has emerged as a viable alternative to the problem of organ and tissue shortage . Our laboratory has developed matrices for bone tissue engineering based on sintered spherical particles and, using bioreactor technology, has demonstrated the ability to produce highly mineralized matrices in vitro . In this study, porous microcapsule scaffolds were developed for bone tissue engineering in the high aspect ratio vessel rotating bioreactor . The motion of individual microcapsules as well as scaffolds in the bioreactor were studied by numerical simulation and in situ imaging analysis . Results show that spherical microcapsules with density less than the surrounding fluid exhibited two motions: (1) a periodic circular orbit with tangential speed equal to the free fall speed of the particle, and (2) an inward radial migration of the circular orbit toward the center of the bioreactor vessel . Lighter-than-water scaffolds were fabricated by sintering poly(lactic-co-glycolic acid) hollow microcarriers with diameter from 500 to 860 microm into a fixed three-dimensional geometry with approximately 30% pore volume and 180 to 190 microm median pore size . Scaffolds were fabricated with aggregate densities ranging from 0.65 g/mL and 0.99 g/mL by appropriate combinations of hollow and solid microcarriers within the scaffold . Scaffold velocity in the bioreactor for the above range of densities was accurately predicted by numerical simulation and ranged from 100 mm/s to 3 mm/s . Maximum shear stress estimation due to media flow over the exterior of the scaffold ranged from 0.3 N/m(2) to 0.006 N/m(2) . Internal perfusion velocity through scaffolds also was calculated and ranged from 13 mm/s to 0.2 mm/s . Estimates of maximum interior shear stress ranged from 0.03 to 0.0007 N/m(2) . These analytical methods provide an excellent vehicle for the study of bone tissue synthesis in three-dimensional culture with fluid flow .

Biotechnol Lett, 2004 Feb, 26(4), 307 - 10
Reduction of nitrobenzene with H2 using a microbial consortium; Cao HB et al.; Proof of concept was obtained that nitrobenzene can be reduced to aniline by a mixed reductive microbial culture using H2 as the sole electron donor source . In a continuous-flow anaerobic bioreactor, both pH and temperature affected nitrobenzene reduction with optima of pH 6.5-6.8 and 30 degrees C . The efficiency of nitrobenzene degradation increased with H2 up to 10% (v/v) . An increase in sulfate concentration decreased the removal rate of nitrobenzene.

J Environ Monit, 2004 Apr, 6(4), 278 - 85 Epub 2004 Mar 17.
Heavy metal speciation in solid-phase materials from a bacterial sulfate reducing bioreactor using sequential extraction procedure combined with acid volatile sulfide analysis; Jong T et al.; Heavy metal mobility, bioavailability and toxicity depends largely on the chemical form of metals and ultimately determines potential for environmental pollution . For this reason, determining the chemical form of heavy metals and metalloids, immobilized in sludges by biological mediated sulfate reduction, is important to evaluate their mobility and bioavailability . A modified Tessier sequential extraction procedure (SEP), complemented with acid volatile sulfide (AVS) and simultaneous extracted metals (SEM) measurements, were applied to determine the partitioning of five heavy metals (defined as Fe, Ni, Zn and Cu, and the metalloid As) in anoxic solid-phase material (ASM) from an anaerobic, sulfate reducing bioreactor into six operationally defined fractions . These fractions were water soluble, exchangeable, bound to carbonates (acid soluble), bound to Fe-Mn oxides (reducible), bound to organic matter and sulfides (oxidizable) and residual . It was found that the distribution of Fe, Ni, Zn, Cu and As in ASM was strongly influenced by its association with the above solid fractions . The fraction corresponding to organic matter and sulfides appeared to be the most important scavenging phases of As, Fe, Ni, Zn and Cu in ASM (59.8-86.7%) . This result was supported by AVS and SEM (Sigma Zn, Ni and Cu) measurements, which indicated that the heavy metals existed overwhelmingly as sulfides in the organic matter and sulfide fraction . A substantial amount of Fe and Ni at 16.4 and 20.1%, respectively, were also present in the carbonate fraction, while an appreciable portion of As (18.3%) and Zn (19.4%) was bound to Fe-Mn oxides . A significant amount of heavy metals was also associated with the residual fraction, ranging from 2.1% for Zn to 18.8% for As . Based on the average total extractable heavy metal (TEHM) values, the concentration of heavy metals in the ASM was in the order of Cu > Ni > Zn > Fe > As . If the mobility and bioavailability of heavy metals are assumed to be related to their solubility and chemical forms, and that they decrease with each successive extraction step, then the apparent mobility and bioavailability of these five heavy metals in ASM increase in the order of Cu < As < Ni < Fe < Zn . The SEM/AVS ratio was less than one in eight replicate ASM samples, indicating that the ASM was non-toxic with regards to having a low probability of bioavailable metals in the pore water.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 261 - 72
Immobilized enzyme studies in a microscale bioreactor; Jones F et al.; Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques . The effectiveness of these reactors was examined along with their behavior over time . Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon . The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height . The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme . Urea conversions of >90% were observed.

Lab Chip, 2004 Apr, 4(2), 98 - 103 Epub 2004 Mar 10.
Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities; Andersson H et al.; An introductory overview of the use of microfluidic devices for tissue engineering is presented . After a brief description of the background of tissue engineering, different application areas of microfluidic devices are examined . Among these are methods for patterning cells, topographical control over cells and tissues, and bioreactors . Examples where microfluidic devices have been employed are presented such as basal lamina, vascular tissue, liver, bone, cartilage and neurons . It is concluded that until today, microfluidic devices have not been used extensively in tissue engineering . Major contributions are expected in two areas . The first is growth of complex tissue, where microfluidic structures ensure a steady blood supply, thereby circumventing the well-known problem of providing larger tissue structures with a continuous flow of oxygen and nutrition, and withdrawal of waste products . The second, and probably more important function of microfluidics, combined with micro/nanotechnology, lies in the development of in vitro physiological systems for studying fundamental biological phenomena.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 9 - 17 Epub 2004 Mar 27.
A survey of computational and physical methods applied to solid-state fermentation; Lenz J et al.; During the last decade, significant effort has been made to apply computational and physical methods to solid-state fermentation (SSF) . This had positive impact both on our understanding of the basic principles underlying this old technology, and on the latest progress made in industrial bioengineering . Guidelines on bioreactor design and operation including scale-up, new methods for biomonitoring and advanced control strategies are among the most important outcomes of practical use . Nevertheless, there still is a lack of experimental data, which hampers parameter identification and thus broader use of mathematical modeling . More attention should therefore be paid to combining and concentrating modern physical techniques and computational approaches in order to allow better model validation and thus further progress in rational bioengineering of SSF.

Biosens Bioelectron, 2004 May 15, 19(10), 1269 - 75
Direct electrochemistry and electrocatalysis of hemoglobin immobilized on carbon paste electrode by silica sol-gel film; Wang Q et al.; Direct electrochemical and electrocatalytic behaviors of hemoglobin (Hb) immobilized on carbon paste electrode (CPE) by a silica sol-gel film derived from tetraethylorthosilicate (TEOS) were investigated for the first time . Hb/sol-gel film modified electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for Hb Fe(III)/Fe(II) redox couple at about -0.312 V (versus Ag/AgCl) in a pH 7.0 phosphate buffer . The formal potential of Hb heme Fe(III)/Fe(II) couple varied linearly with the increase of pH in the range of 5.0-10.0 with a slope of 49.44 mV pH(-1), which suggests that a proton transfer is accompanied with each electron transfer (ET) in the electrochemical reaction . The immobilized Hb displayed the features of peroxidase and gave excellent electrocatalytic performance to the reduction of O2, NO2(-) and H2O2 . The calculated apparent Michaelis-Menten constant was 8.98 x 10(-4)M, which indicated that there was a large catalytic activity of Hb immobilized on CPE by sol-gel film toward H2O2 . In comparison with other electrodes, the chemically modified electrodes, used in this direct electrochemical study of Hb, are easy to be fabricated and rather inexpensive . Consequently, the Hb/sol-gel film modified electrode provides a convenient approach to perform electrochemical research on this kind of proteins . It also has potential use in the fabrication of the third generation biosensors and bioreactors.

Bioprocess Biosyst Eng, 2004 Jul, 26(4), 239 - 48 Epub 2004 Mar 26.
Fully integrated L-phenylalanine separation and concentration using reactive-extraction with liquid-liquid centrifuges in a fed-batch process with E . coli; Ruffer N et al.; A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E . coli F-4/pF81 is presented . The strain was rationally constructed for the production of the aromatic amino acid . Glucose and tyrosine control is used . A reactive extraction system consisting of kerosene, the cation-selective carrier D(2)EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate . Permeate is drained off from the bioreactor bypass . Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28% relative glucose/L-phe yield increase is observed . Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.

Protein Sci, 2004 Apr, 13(4), 913 - 24
Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations; Pioselli B et al.; The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors . A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products . The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities . This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes . Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase . This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

Bioprocess Biosyst Eng, 2004 Jul, 26(4), 231 - 8 Epub 2004 Mar 20.
Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors . 1 . Simulated fermentation broths; Galaction AI et al.; Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors . It can be described and analyzed by means of the mass transfer coefficient, k(L) a . The k(L) a values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties . The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration . The experimental results obtained for simulated broths indicated a considerable increase of k(L) a in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen . The magnitude of the positive effect of n-dodecane depends both on the broths' characteristics and operational conditions of the bioreactor.

Trends Biotechnol, 2004 Apr, 22(4), 189 - 94
Milking of microalgae; Hejazi MA et al.; The low productivity of algal cultures in the production of high-value compounds is the most significant bottleneck for commercialization of this technology . Cultures in which cell mass is reused for continuous production are proposed as a solution to overcome this problem . Recently, a method was developed in which beta-carotene was harvested from the microalga Dunaliella salina grown in a two-phase bioreactor . This raises the question of whether this technique could also be used in the mass production of secondary metabolites . Understanding the mechanism of the milking process and its relationship to the product formation pathway should reveal whether other products can be milked from various species of microalgae.

Trends Biotechnol, 2004 Apr, 22(4), 174 - 80
Life and death in mammalian cell culture: strategies for apoptosis inhibition; Arden N et al.; Mammalian cell culture is widely used to produce valuable biotherapeutics including monoclonal antibodies, vaccines and growth factors . Industrial cell lines such as Chinese hamster ovary (CHO), mouse myeloma (NS0), baby hamster kidney (BHK) and human embryonic kidney (HEK)-293 retain many molecular components of the apoptosis cascade . Consequently, these cells often undergo programmed cell death upon exposure to stresses encountered in bioreactors . The implementation of strategies to control apoptosis and enhance culture productivities represents a major goal of biotechnologists . Fortunately, previous research has uncovered many intracellular proteins involved in activating and inhibiting apoptosis . Here, we summarize three apoptotic pathways and discuss different environmental and genetic methodologies implemented to limit cell death for biotechnology applications.

Biotechnol Appl Biochem, 2004 Apr, 39(Pt 2), 151 - 7
Shear-stress preconditioning and tissue-engineering-based paradigms for generating arterial substitutes; Baguneid M et al.; In situ tissue engineering using shear-stress preconditioning and adhesive biomolecules is a new approach to autologous tissue engineering . In the present study, novel tissue-engineering grafts (TEGs) were preconditioned within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), to establish whether low-shear-stress conditions promoted endothelial cell (EC) retention and differentiation . TEGs ( n =24) were generated by the contraction and compaction of collagen(I) by porcine aortic smooth-muscle cells (SMCs) on to a compliant polyester graft scaffold . ECs were radiolabelled with {(111)In}indium tropolonate and seeded on to the luminal surface of the TEGs . Following organ culture in a bioreactor (7 days), TEGs were split into four groups ( n =six TEGs per group): Group A acted as controls with TEGs unmodified and seeded with radiolabelled ECs; Group B underwent luminal pre-coating with FN (75 microg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 10-20 microN (1-2 dyn)/cm(2) for 7 days prior to EC seeding, and Group D TEGs were preconditioned for 7 days at 1-2 dyn/cm(2), followed by luminal pre-coating with FN prior to EC seeding . The resistance to physiological shear stress of the seeded ECs was assessed using a gamma-radiation counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 93.2 microN (9.32 dyn)/cm(2) . Environmental scanning electron microscopy (ESEM) was used to determine the distribution and degree of differentiation of the attached Ecs, and tissue-type-plasminogen-activator (tPA) assays provided a measure of function and viability . EC resistance to shear stress at 93.2 microN/cm(2) was significantly enhanced by a period of preconditioning (Group C) at 10-20 microN/cm(2), surface modification with FN (Group B), or both (Group D) when compared with control grafts (Group A) . However, TEGs coated with FN whether preconditioned (Group D) or not (Group B) demonstrated the best results for EC retention . ESEM demonstrated near-confluent differentiated flattened ECs in both these cases . EC function was demonstrated by a steady increase in tPA production . Low-shear-stress preconditioning of TEGs enhances EC retention in vitro with an additional advantage demonstrated by pre-treatment with FN prior to endothelialization . These findings may be exploited in the development of tissue-engineered constructs to maintain a confluent endothelial lining.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(2), 493 - 505
Computer simulation of leachate quality by recirculation in a sanitary landfill bioreactor; Chanthikul S et al.; Sanitary Landfills are the most widely used method of solid waste disposal around the world . Modern sanitary landfills are designed with impervious liners, and leachate collection, removal, and treatment systems to minimize the potential for groundwater contamination . Leachate recycle through the landfill is an effective method of leachate treatment, and to enhance solid waste stabilization . A mathematical model is developed to simulate the release of contaminants from solid wastes, and their movement into the percolating liquid . Two differential equations are used that express the mass balance of the contaminants in the percolating water and those in the solid wastes . These simultaneous linear differential equations are solved numerically using a fourth-order Runge-Kutta algorithm with many physical and process parameters . The model results are used to estimate the active life of landfill with and without leachate recirculation . Such information is valuable in the operation, maintenance, and closure plan of a sanitary landfill.

Environ Technol, 2004 Jan, 25(1), 101 - 9
Chromate reduction at low sulphate concentration in hydrogen-fed bioreactors; Battaglia-Brunet F et al.; Aimed at developing a bioremediation process to treat Cr(VI)-bearing water at low sulphate concentration in order to reduce excess sulphide production, the highly toxic, mutagenic, and soluble Cr(VI) was reduced to the less toxic and insoluble Cr(III) in 2-litre fixed-bed reactors inoculated with the sulphate-reducing bacterium (SRB) Desulfomicrobium norvegicum, capable of performing direct enzymatic Cr(VI) reduction . H2 was used as the electron source . The fixed-films were developed on three different supports: a PVC cross-flow material, a pozzolana, and a ceramic granulate . The phased experiments began with a progressive increase of the Cr(VI) concentration in the feed to the column reactors, followed by a progressive decrease of the sulphate concentration . Inhibition by Cr(VI) was less pronounced with pozzolana than with the other supports; when the pozzolana column was fed with a medium containing 100 mg l(-1) Cr(VI) and only 250 mg l(-1) sulphate, the lowest residence time that could be applied for complete Cr(VI) reduction was 16 h . The molar ratio between the sulphate and Cr(VI) reduction rates was decreased down to 1.5, suggesting that indirect reaction with HS was not the sole mechanism of Cr(VI) reduction.

Indian J Gastroenterol, 2003 Dec, 22 Suppl 2, S69 - 74
Bioartificial liver support for fulminant hepatic failure; Anand AC; Mortality from fulminant hepatic failure (FHF) is high (50%-80%), although survivors have absolutely normal liver function . The only treatment option that is curative is liver transplantation . However, because of shortage of cadaveric organ donors and/or delay in their availability, only 10% of FHF patients ultimately receive a transplant . This has led to development of artificial liver support systems with an idea to bridge the time to transplantation and/or recovery from FHF . Initial support systems were based on the principles of hemodialysis, hemofiltration, plasma-exchange, and hemoperfusion through adsorbent media (e.g., charcoal) . However, lack of clinical efficacy, problems of bioincompatibility and fear of loss of circulating hepatocyte-regeneration factors led to the search for alternate strategies . With the successful long-term propagation and culturing of human and pig hepatocytes, and the development of adequate biocompatible microcarrier modules, it is now possible to achieve sufficient density of hepatocytes per unit volume to develop bioartificial liver systems . These can be implanted transperitoneally but are subject to early destruction because of inadequate vascularization and immune attack from the host . Thus the major thrust is now to develop bioreactors, e.g., Extracorporeal Liver Assist Device (ELAD), Bioartificial Liver (BAL), etc . These contain human or pig hepatocytes implanted on hollow-fiber ultrafiltration cartridges . The patient's blood or plasma circulates through these bioreactors and after clearance of toxic compounds (via ultrafiltration and metabolism in hepatocytes) and addition of synthesized products, is returned to the patient . This article reviews the genesis, the pros and cons, and the clinical experience of BAL support for FHF.

Osteoarthritis Cartilage, 2004 Apr, 12(4), 306 - 13
Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes; Malda J et al.; OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets . DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation . Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase . Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau) . In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically . RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold . Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I . After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO . After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO . Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture . After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions . The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions . CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Feb, 21(1), 146 - 50
{Bioartificial liver support system: state of the art}; Xiong W et al.; The temporary function replacement and intensive support play a pivotal role in the treatment of patients with acute or chronic end-stage organ failure . The hemodialysis and peritoneal dialysis have become routine techniques in the management of acute or chronic renal failure . But for the complexity of hepatic function, e.g . metabolism, biosynthesis and biotransformation for endogenous or exogenous substrates, the simulation or partial replacement of hepatic function is a great dream of bionic technologists . With the development of cell biology and biomedical material, the combination of biomaterial and the hepatocyte cultured ex vivo could provide a range of human liver-specific functions . The combination of biomaterial and viable cell was called hybrid or bioartificial liver support system (BALSS) . It is a chimera of biomaterials and hepatocytes . The bionic technologists study the cell and its culture in vitro, which is the main component of BASLL . Many types of BALSS were translated into the early clinical stage . In this overview, we review the hepatocyte culture and the design of different bioreactors . It includes the immune obstacles in xeno-hemoperfusion and how to assess pre-clinical and clinical effectiveness.

Biomaterials, 2004 Aug, 25(17), 3681 - 8
Tissue engineering of biphasic cartilage constructs using various biodegradable scaffolds: an in vitro study; Wang X et al.; Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue . Biphasic implants consisting of pre-fabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements . Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro . Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-L-lactide {P(L)LA}, poly-d,l-lactide {P(D,L)LA} or Collagen-hydroxyapatite {Col-HA} and were cultured for 15 weeks . Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation . P(D,L)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape . Little or no breakdown or chondrocyte colonisation was evident in P(L)LA . Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial . These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.

Water Res, 2004 Mar, 38(6), 1405 - 18
Column experiments for microbiological treatment of acid mine drainage: low-temperature, low-pH and matrix investigations; Tsukamoto TK et al.; The lifetime of traditional sulfate-reducing bacteria (SRB) bioreactors that utilize a source of reducing equivalents contained within the matrix (e.g . manure) is limited by the amount of readily available reducing equivalents within that matrix . In order to extend bioreactor lifetime indefinitely, the addition of known concentrations of alternative reducing equivalents (methanol and ethanol) to a depleted matrix was tested at low pH and low temperatures . Following acclimation, up to 100% efficiencies of reducing equivalents were directed toward sulfate reduction . Alcohol was added in stoichiometric concentrations to remove 50% of the added sulfate (900 mg/L), producing sufficient sulfide to precipitate all of the iron from solution . An average of 42% of the sulfate was removed following acclimation, reflecting 84% efficiency . An average of 93% of the iron was removed (93 mg/L) . Bacteria acclimated to ethanol more rapidly than methanol, although both alcohols were effective as carbon sources . Efficient treatment was observed at the lowest temperatures (6 degrees C) and lowest pHs (pH=2.5) tested . The use of ethanol-fed, highly permeable bioreactor matrices of wood chip, pulverized plastic and rock was also examined to determine which of these porous matrices could be implemented in a field bioreactor . Results indicated that >95% of the 100mg/L iron added was removed by all matrices . Sufficient reducing equivalents were added to remove 450 mg/L of sulfate, wood and rock matrices removed approximately 350 mg/L plastic removed approximately 225 mg/L . A study comparing rock size indicated that small rocks removed iron and sulfate more efficiently than medium- and large-size rocks . The results suggest that wood and rock in conjunction with ethanol are viable alternatives to traditional bioreactor matrices . These findings have direct application to semi-passive sustained operation of SRB bioreactors for treatment of acidic drainage at remote sites.

Water Res, 2004 Mar, 38(6), 1368 - 75
Interspecies electron transfer in methanogenic propionate degrading consortia; de Bok FA et al.; Propionate is a key intermediate in the conversion of complex organic matter under methanogenic conditions . Oxidation of this compound requires obligate syntrophic consortia of acetogenic proton- and bicarbonate reducing bacteria and methanogenic archaea . Although H(2) acts as an electron-carrier in these consortia, evidence accumulates that formate plays an even more important role . To make energy yield from propionate oxidation energetically feasible for the bacteria and archaea involved, the concentrations of H(2) and formate have to be extremely low . On the other hand, the diffusion distance of these carriers has to be small to allow high propionate conversion rates . Accordingly, the high conversion rates observed in methanogenic bioreactors are due to the fact that the propionate-oxidizing bacteria and their methanogenic partners form micro-colonies within the densely packed granules.

J Appl Microbiol, 2004, 96(4), 742 - 9
Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica; Fickers P et al.; AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors . METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations . The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase . SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y . lipolytica mutant . Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium . Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration . We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media . CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y . lipolytica.

Zhonghua Wai Ke Za Zhi, 2004 Jan 22, 42(2), 110 - 3
{Preliminary study on in vitro tendon engineering using tenocytes and polyglycolic acids}; Cao DJ et al.; OBJECTIVE: To find out the feasibility of tendon engineering in vitro using expanded tenocytes and polyglycolic acids (PGA) . METHODS: Tenocytes were isolated using tissue explant method and expanded in vitro . Tenocytes (20 x 10(6)) at the second passage were collected and then seeded onto PGA unwoven fibers to form a cell-scaffold construct in a shape of tendon . The constructs were cultured in DMEM with 20% FBS for 1 week . The cell-scaffold constructs were then cultured under constant tension generated by a U-shaped spring (n = 5), which served as experimental group, or cultured without tension (n = 4), which served as control group 1 . PGA fibers alone were cultured (n = 3), which served as control group 2 . Small fragments at the end of the constructs were harvested at 2, 4 and 6 weeks respectively for histological and immunohistochemistry (IHC) analysis . Six-week samples were also evaluated by transmission electron microscope (TEM) and mechanical test . RESULTS: No obvious difference was observed among the three groups at 2 weeks grossly and histologically as the constructs remained to be mainly undegraded PGA fibers . By 4 weeks, a neo-tendon was formed in the experimental group and control group 1 grossly, and histology and IHC revealed the formation of collagen fibers . In contrast, PGA fibers alone in control group 2 were mostly degraded . At 6 weeks, tendons of control group 1 were much thicker {(2.55 +/- 0.18) mm in diameter} than those of experimental group {(1.44 +/- 0.13) mm in diameter} . Periodical striae were observed in collagen fibers of experimental group and control group 1 by TEM . However, histology of tendons in experimental group revealed longitudinally aliened collagen fibers, which resembled the structure of normal tendon more closely than that of control group 1 tendons . Furthermore, the maximum tensile stress (N/mm(2)) of experimental group (1.107 +/- 0.327) was greater than that of control group 1 (0.294 +/- 0.138) (P < 0.05) . CONCLUSION: It is possible to use an engineering to construct tendon tissue in vitro . Periodical strain generated by bioreactor may be the optimal mechanical stimulation, which is currently under investigation.

Tissue Eng, 2004 Jan-Feb, 10(1-2), 215 - 29
Three-dimensional in vitro model of adipogenesis: comparison of culture conditions; Fischbach C et al.; In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat . Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis . Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated . Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates . At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions . Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs . Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.

Tissue Eng, 2004 Jan-Feb, 10(1-2), 165 - 74
Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes; Fiegel HC et al.; Maintenance of liver-specific function of hepatocytes in culture is still difficult . Improved culture conditions may enhance the cell growth and function of cultured cells . We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures . Hepatocytes were harvested from Lewis rats . Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system . Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix . Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period . Culture under flow condition significantly enhanced cell numbers . An additional improvement of this effect was observed, when matrix coating was used . Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture . Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function . Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect . Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

Tissue Eng, 2004 Jan-Feb, 10(1-2), 93 - 100
Osteoclastogenesis on tissue-engineered bone; Nakagawa K et al.; Bone remodeling plays an important role in bone function . To date, bone tissue-engineering research has focused primarily on bone formation from osteoblasts . This study demonstrates that osteoclastogenesis can occur on a mineralized polymer scaffold . Porcine bone marrow-derived mesenchymal stem cells (pMSCs) and hematopoietic cells were isolated from the bone marrow of Yucatan minipigs (n = 3) and cultured separately . pMSCs were differentiated into osteoblasts, seeded on porous poly(D,L-lactic-co-glycolic acid) foams, and cultured in a rotating oxygen-permeable bioreactor system . Once the cell-polymer constructs had started to mineralize, the hematopoietic cells were added and cocultured to include osteoclastogenesis . The cultured constructs were evaluated by histochemical and microscopic examination . Our results show that osteoblasts and osteoclasts were successfully differentiated from bone marrow on the scaffolds . This is the first demonstration of osteoclast formation on mineralized polymer surfaces.

Tissue Eng, 2004 Jan-Feb, 10(1-2), 33 - 41
In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nanofibrous scaffold; Shin M et al.; The objective of this study was to assess bone formation from mesenchymal stem cells (MSCs) on a novel nanofibrous scaffold in a rat model . A highly porous, degradable poly(epsilon-caprolactone) (PCL) scaffold with an extracellular matrix-like topography was produced by electrostatic fiber spinning . MSCs derived from the bone marrow of neonatal rats were cultured, expanded, and seeded on the scaffolds . The cell-polymer constructs were cultured with osteogenic supplements in a rotating bioreactor for 4 weeks, and subsequently implanted in the omenta of rats for 4 weeks . The constructs were explanted and characterized by histology, immunohistochemistry, and scanning electron microscopy . The constructs maintained the size and shape of the original scaffolds . Morphologically, the constructs were rigid and had a bone-like appearance . Cells and extracellular matrix (ECM) formation were observed throughout the constructs . In addition, mineralization and type I collagen were also detected . This study establishes the ability to develop bone grafts on electrospun nanofibrous scaffolds in a well-vascularized site using MSCs.

Ann Biomed Eng, 2004 Feb, 32(2), 212 - 22
Tissue engineering of ovine aortic blood vessel substitutes using applied shear stress and enzymatically derived vascular smooth muscle cells; Opitz F et al.; Compared to native blood vessels, all clinically available blood vessel substitutes perform suboptimally . Numerous approaches to tissue engineer (TE) blood vessels have been pursued using different scaffold materials, cell types, and culture conditions . Several limitations however remain to be overcome prior to the potential application in the arterial system . This study aimed at tissue engineering viable ovine blood vessels suitable for implantation into the systemic circulation of sheep . In recent studies vascular smooth muscle cells (vSMC) were derived by an explant technique . However, in this study we show that homogenous populations of differentiated vSMC were only obtained by enzymatic dispersion as characterized by immunostaining for specific vSMC marker proteins . In contrast the explant method yielded predominantly less differentiated myofibroblast-like cells . Enzymatically derived vSMC were seeded onto P-4-HB scaffolds and incubated either in a pulsatile flow bioreactor or under static conditions . Dynamically cultured TE blood vessel substitutes showed confluent layered tissue formation and were completely water resistant . They displayed significantly increased ECM synthesis, DNA, and protein content as well as vSMC marker expression . Mechanical properties of bioreactor cultured TE blood vessels approached those of native aorta . In conclusion ovine, aortic blood vessel substitutes were successfully created using enzymatically derived vSMC, bioabsorbable scaffolds, and applied shear stress.

J Biotechnol, 2004 Mar 18, 108(3), 253 - 63
Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production; Gorret N et al.; We report the successful culture of oil palm (Elaeis guineensis Jacq.) suspension cells in a bioreactor . In vitro propagation of this perennial monocotyledonous tree is an important part of the oil palm industry's approach to clonal propagation of high-yielding accessions . During culture of oil palm cells in a batch bioreactor, nutrients and extracellular metabolites were monitored, and kinetic parameters and nutrient-to-biomass conversion yields were calculated . The biomass increased approximately 3.5-fold per month, consistent with values reported for shake flask cultures . Although the carbon source was completely depleted by the end of the run, nitrogen sources remained in large excess and the sugar-to-biomass conversion yield remained low . Linear growth indicated that the cells were limited . The results obtained from the bioreactor runs indicated that we should be able to improve biomass production by carrying out optimization studies . Therefore, we initiated multi-factorial analyses using response surface experimental designs to investigate the effects of different nitrogen sources, as well as inoculum size and conditioned medium, on biomass production in flask cultures . Whereas glutamine does not have a significant effect on biomass production, ammonia has a positive effect up to an optimum concentration . Both inoculum density and conditioned medium have positive, synergistic effects on biomass production.

J Biotechnol, 2004 Mar 18, 108(3), 243 - 51
Production of the soluble human Fas ligand by Dictyostelium discoideum cultivated on a synthetic medium; Lu Y et al.; Human Fas ligand (hFasL) is of considerable interest since it is a type II transmembrane glycoprotein that induces programmed cell death, or apoptosis . In this study Dictyostelium discoideum was used to produce a soluble form of the human Fas ligand . The recombinant cells were adapted to a modified synthetic FM medium, called SIH medium . Cells adapted to the SIH medium reached about 2 times higher cell densities and hFasL concentrations on this medium compared with cells growing on the standard complex medium HL-5C . Even higher values were achieved by a dissolved oxygen-controlled fed-batch cultivation in a conventional stirred bioreactor on SIH medium . Cell densities of up to 5.5 x 10(7) ml(-1) and a maximum hFasL concentration of 148 microgl(-1) were obtained . These results were further improved by means of continuous cultivation of D . discoideum in a bioreactor equipped with cell retention by microfiltration . At low space velocity very high cell densities of up to 2.4 x 10(8) ml(-1) and hFasL concentrations of up to 205 microgl(-1) were achieved.

Clin Pharmacokinet, 2004, 43(4), 211 - 25
Pharmacokinetic considerations in development of a bioartificial liver; Iwata H et al.; We consider the pharmacokinetics of bioartificial livers (BALs) prepared using a hollow fibre module with respect to two key functions, detoxification and plasma protein supply, and present the results in a simple form . We then discuss the advantages and disadvantages of BAL therapy in comparison with the non-biological therapies of haemodialysis and plasma exchange . Nitrogenous and other potentially toxic compounds, such as ammonia, mercaptans, short-chain fatty acids and gamma-aminobutyric acid, are produced in the bowels and accumulate in the systemic blood because of impaired elimination by the ailing liver . Adrenal and gonadal steroids, including corticosteroids, estrogens, progestins and androgens, are biosynthesised, and high concentrations of these hormones become harmful . All these endogenously produced toxins require effective metabolism . In haemodialysis, toxins that permeate through the hollow fibre membrane are rapidly removed by the dialysate flow, and their concentrations decrease to almost zero . In a BAL bioreactor, the toxins are slowly metabolised by hepatocytes in the hollow fibres, and decreased to concentrations that are inversely proportional to the number of hepatocytes in the BAL, even after a long-term assist . It is difficult to rationalise the clinical usage of BAL systems containing small amounts of hepatocytes (70-100g) to remove the toxins . Concentrations of plasma proteins in a patient after long-term BAL treatment are proportional to the number of hepatocytes in the device . BAL reactors prepared using porcine hepatocytes supply porcine proteins, not human proteins, to the recipient . Plasma exchange increases protein concentrations much more effectively than BAL as long as a sufficient amount of plasma is available . The blood inflow rate to the liver is about 1500 mL/min in a normal adult . On the other hand, blood draw rates to a BAL system are restricted to the range of 100-300 mL/min . Toxins that are rapidly cleared by the liver (for example, the ammonia clearance of the normal human liver is several hundred mL/min) cannot be effectively eliminated from the systemic blood by BAL systems currently under clinical evaluation . Hepatocytes are the only elements in a BAL reactor that can metabolise toxins and synthesise proteins, and thus BAL performance increases with the increasing number of hepatocytes in the bioreactor . The human liver weighs about 1500 g and contains about 80% hepatocytes, i.e . about 1200 g of hepatocytes . The blood flow through the liver is about 1500 mL/min in a normal adult . To effectively replace liver functions would require a BAL reactor containing the functional equivalent of several hundreds of grams of human hepatocytes with an extracorporeal perfusion rate of more than 1000 mL/min . At this point, only orthotopic liver transplantation can meet these criteria.

Biotechnol Lett, 2004 Jan, 26(1), 55 - 9
Activation of proteases in an anaerobic sulphidogenic bioreactor; Whiteley C et al.; Activities of proteases were stimulated by specific sulphur metabolites during the enhanced hydrolysis of complex polymeric organic carbon in an anaerobic sulphidogenic environment . While sulphate at 1000 mg l(-1) inhibited proteases by 50%, there was a 2.5-fold increase in activity of proteases by added sulphite and a 3.6-fold increase from added sulphide . Two hypothetical models are proposed . First the sulphur species, sulphite (HSO3-) and sulphide (HS-), liberated at different times during the sulphate reduction process, directly activate the proteases, which are associated with the organic particulate matter, leading to a subsequent enhancement of hydrolysis of polymeric material . Second, they indirectly activate the proteases by neutralising the cations on the floc surface disrupting the integrity of the organic particulate floc therebye releasing further entrapped enzymes from the organic particulate matter.

Biotechnol Lett, 2004 Jan, 26(1), 21 - 6
Construction of an electro-enzymatic bioreactor for the production of (R)-mandelate from benzoylformate; Kim MH et al.; Coupling both the electrocatalytic recycling of NADH and the enzymatic reduction of the substrate was used to produce (R)-mandelate from benzoylformate using benzoylformate reductase (BFR) . The reduction of benzoylformate by BFR in combination with FAD-mediated electrolysis (at -0.5 V vs . Ag/AgCl) was complete in about 18 h and gave 47.5 mM (R)-mandelate from 50 mM substrate, while the process involving MV2+-mediated procedure (at -0.7 V vs . Ag/AgCl) produced 40 mM (R)-mandelate after 30 h . The overpotential for the NAD+ reduction could be decreased by about 0.2 V by substituting a toxic viologen derivative, MV2+, with a natural electron carrier, FAD . MV2+, however, decreased the productivity as BFR lost about 50% of its initial activity after 6 d in its presence.

Biotechnol Lett, 2004 Jan, 26(1), 1 - 10
Minibioreactors; Kumar S et al.; The performance of currently available minibioreactors with volumes below about 100 ml is reviewed . Bioreactors are characterized by their area of application, by mass transfer and mixing characteristics and by their suitability for on-line monitoring and control . The review comprises shaken bioreactors such as shake-flasks, microtiter plates and test-tubes, stirred bioreactors including spinner-flasks for the cultivation of mammalian cells and various special reactors particularly involving on-line monitoring as e.g . membrane inlet mass spectrometry and NMR.

Curr Opin Plant Biol, 2004 Apr, 7(2), 166 - 70
The moss bioreactor; Decker EL et al.; The production of recombinant proteins in moss bioreactors provides all of the benefits of molecular farming in plants but avoids many plant-specific disadvantages, such as the genetic instability of de-differentiated cells in suspension culture or the lack of containment during field production . Protein yields are in the same range as those of other cell-culture-based production systems . On top of this, the moss Physcomitrella patens is the only known plant that can be genetically modified by homologous recombination, allowing efficient targeted gene disruption . Thus, the major drawback of producing human proteins in plants, allergic reactions caused by plant-specific glycosylation, can be diminished by targeted knockout of the responsible genes in moss . Unlike all other plants, moss allows straightforward 'humanisation' of plant-derived pharmaceuticals.

Curr Opin Plant Biol, 2004 Apr, 7(2), 152 - 8
Plant-based production of biopharmaceuticals; Fischer R et al.; Plants are now gaining widespread acceptance as a general platform for the large-scale production of recombinant proteins . The first plant-derived recombinant pharmaceutical proteins are reaching the final stages of clinical evaluation, and many more are in the development pipeline . Over the past two years, there have been some notable technological advances in this flourishing area of applied biotechnology, as shown by the continuing commercial development of novel plant-based expression platforms . There has also been significant success in tackling some of the limitations of plant bioreactors, such as low yields and inconsistent product quality, that have limited the approval of plant-derived pharmaceuticals.

Biotechnol Lett, 2004 Jan, 26(2), 165 - 9
Alginate coated loofa sponge discs for the removal of cadmium from aqueous solutions; Iqbal M et al.; A biosorbent was prepared by coating the fibrous network of loofa sponge (Luffa cylindrica) with a thin film of calcium alginate . Alginate-coated loofa sponge removed Cd(II) rapidly, reaching equilibrium loading of 124 mg g(-1) in 30 min . Seventy % of equilibrium uptake was achieved in 10 min . In contrast, it took 240 min for alginate beads to reach a loading equilibrium of 88 mg g(-1) under identical conditions . The biosorption behaviour followed the Langmuir adsorption isotherm and the ACLS biosorbent was shown to be highly effective in removing Cd(II) from a 10 mg l(-1) solution in a continuous flow fixed-bed column bioreactor.

Adv Space Res, 2003, 32(8), 1569 - 76
Parathyroid hormone-related protein is a gravisensor in lung and bone cell biology; Torday JS; Parathyroid Hormone-related Protein (PTHrP) has been shown to be essential for the development and homeostatic regulation of lung and bone . Since both lung and bone structure and function are affected by microgravity, we hypothesized that 0 x g down-regulates PTHrP signaling . To test this hypothesis, we suspended lung and bone cells in the simulated microgravity environment of a Rotating Wall Vessel Bioreactor, which simulates microgravity, for up to 72 hours . During the first 8 hours of exposure to simulated 0 x g, PTHrP expression fell precipitously, decreasing by 80-90%; during the subsequent 64 hours, PTHrP expression remained at this newly established level of expression . PTHrP production decreased from 12 pg/ml/hour to 1 pg/ml/hour in culture medium from microgravity-exposed cells . The cells were then recultured at unit gravity for 24 hours, and PTHrP expression and production returned to normal levels . Based on these findings, we have obtained bones from rats flown in space for 2 weeks (Mission STS-58, SL-2) . Analysis of PTHrP expression by femurs and tibias from these animals (n=5) revealed that PTHrP expression was 60% lower than in bones from control ground-based rats . Interestingly, there were no differences in PTHrP expression by parietal bone from space-exposed versus ground-based animals, indicating that the effect of weightlessness on PTHrP expression is due to the unweighting of weight-bearing bones . This finding is consistent with other studies of microgravity-induced osteoporosis . The loss of the PTHrP signaling mechanism may be corrected using chemical agents that up-regulate this pathway . In conclusion, PTHrP represents a stretch-sensitive paracrine signaling mechanism that may sense gravity . c2003 COSPAR . Published by Elsevier Ltd . All rights reserved.

Adv Space Res, 2003, 32(8), 1467 - 72
Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor; Montufar-Solis D et al.; Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (1 g) . During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb . This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development . In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes . This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs . flattened tissues compressed by their own weight . A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development . The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system . Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology . E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape . Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor . Younger limb buds fused together during culture, so an additional set of E11.5 limb buds was cultured with and without encapsulation in alginate prior to culturing in the bioreactor . Encapsulated limbs grown in the bioreactor did not fuse together, but developed only the more proximal elements while limbs grown in culture dishes formed proximal and distal elements . Alginate encapsulation may have reduced oxygenation to the progress zone of the developing limb bud resulting in lack of development of the more distal elements . These results show that the bioreactor supports growth and differentiation of skeletal elements in entire E13 limb buds, and that a method to culture younger limb buds without fusing together needs to be developed if any morphometric analysis is to be performed . c2003 COSPAR . Published by Elsevier Ltd . All rights reserved.

Ying Yong Sheng Tai Xue Bao, 2003 Nov, 14(11), 2077 - 8
{An experimental study with bioreactor-landfill system}; Wang J et al.; In this study, a methane bioreactor-landfill system was utilized to treat municipal solid waste (MSW) . Through analyzing and detecting the pollutant(CODCr) in the bioreactor-landfill system, a simulated mathematic formula of waste degradation was established . After treated with this system, the CODCr and VFA concentrations in MSW could be decreased from more than 20000 and 7000 mg.L-1 to less than 1500 and 200 mg.L-1, respectively.

J Agric Food Chem, 2004 Mar 10, 52(5), 1357 - 63
Red wine making by immobilized cells and influence on volatile composition; Tsakiris A et al.; Red wine making using yeast cells immobilized in two types of raisin berries, at various temperatures (6-30 degrees C), was studied . A modification of the batch bioreactor was used to separate the grape skins used for color extraction from the biocatalyst and the fermenting grape must . The evaluation of the immobilized biocatalysts was made on terms of productivity and organoleptic quality, including color intensity and formation of volatiles . The immobilized cells were found capable of low-temperature wine making, producing red wines containing more than 11% v/v alcohol in 8 days at 6 degrees C . The quality of wines was examined by gas chromatography (GC) and GC-MS analysis and sensory evaluation . Higher alcohol concentrations were decreased, and ethyl acetate concentrations increased by the drop of temperature . Many esters, alcohols, carbonyls, and miscellaneous compounds were identified in wines produced by immobilized cells, revealing no significant qualitative differences as compared to wines produced by free cells . The sensory evaluation showed that the best red wine was produced at 6 degrees C.

J Agric Food Chem, 2004 Mar 10, 52(5), 1170 - 6
Cross-linking and rheological changes of whey proteins treated with microbial transglutaminase; Truong VD et al.; Modification of the functionality of whey proteins using microbial transglutaminase (TGase) has been the subject of recent studies . However, changes in rheological properties of whey proteins as affected by extensive cross-linking with TGase are not well studied . The factors affecting cross-linking of whey protein isolate (WPI) using both soluble and immobilized TGase were examined, and the rheological properties of the modified proteins were characterized . The enzyme was immobilized on aminopropyl glass beads (CPG-3000) by selective adsorption of the biotinylated enzyme on avidin that had been previously immobilized . WPI (4 and 8% w/w) in deionized water, pH 7.5, containing 10 mM dithiothreitol was cross-linked using enzyme/substrate ratios of 0.12-10 units of activity/g WPI . The reaction was carried out in a jacketed bioreactor for 8 h at 40 degrees C with continuous circulation . The gel point temperature of WPI solutions treated with 0.12 unit of immobilized TGase/g was slightly decreased, but the gel strength was unaffected . However, increasing the enzyme/substrate ratio resulted in extensive cross-linking of WPI that was manifested by increases in apparent viscosity and changes in the gelation properties . For example, using 10 units of soluble TGase/g resulted in extensive cross-linking of alpha-lactalbumin and beta-lactoglobulin in WPI, as evidenced by SDS-PAGE and Western blotting results . Interestingly, the gelling point of WPI solutions increased from 68 to 94 degrees C after a 4-h reaction, and the gel strength was drastically decreased (lower storage modulus, G') . Thus, extensive intra- and interchain cross-linking probably caused formation of polymers that were too large for effective network development . These results suggest that a process could be developed to produce heat-stable whey proteins for various food applications.

Microb Ecol, 2004 Feb, 47(2), 137 - 49 Epub 2004 Mar 04.
Microbial astronauts: assembling microbial communities for advanced life support systems; Roberts MS et al.; Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth . The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space . Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments "everything is everywhere" given enough time) . This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch . Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time . Research at the John F . Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support . Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed . In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity . Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment .

Water Sci Technol, 2004, 49(2), 237 - 44
Methods for understanding organic fouling in MBRs; Jefferson B et al.; The identification and quantification of foulants in membrane bioreactors present a major challenge due to their complexity resulting from biomass heterogeneity . Fouling is normally characterised with respect to the critical flux, this being conventionally viewed as being the flux below which a reduction in membrane permeability does not take place . However, recent studies have revealed that such fouling arises even at very low fluxes . Moreover, fouling rates can differ substantially between different experiments, trials and installations even when operated under apparently similar conditions of biomass and soluble organic carbon concentrations . The methods available for quantifying and analysing fouling are reviewed with specific reference to recent data on sub-critical flux behaviour . It is concluded that HPSEC analysis of extracted biomass fractions may provide the most valuable data towards the determination of differences in fouling propensity between different biomasses, as inferred in conventional flux step analysis.

Water Sci Technol, 2004, 49(1), 23 - 9
Fast online determination of surfactant inhibition in acidic phase bioreactors; Feitkenhauer H; Surfactants have been shown to inhibit the anaerobic digestion process severely, with the methanogenic microorganisms being the most affected . The diverse nature of surfactants used even in one (e.g . textile finishing) plant makes an online determination of surfactants sometimes very difficult and expensive . Therefore a fast online determination of inhibitory effects on the acidogenic microorganisms (first step of the degradation cascade) can help to give an early warning signal or to calculate a "pseudo"-surfactant concentration . In a two-phase system this information can be used to protect the methanogenic reactor against surfactant overloading and its long term negative effects . In this paper it is shown that the inhibition is a consequence of microbial inhibition and is not caused by an inactivation of extracellular hydrolytic enzymes (released by the cells for biopolymer cleavage) . A titration technique was successfully employed to measure the surfactant inhibition in a laboratory-scale acidification reactor . Additional experiments demonstrate (using sodium dodecyl sulfate as the model substance) how inhibitory effects (and strategies to overcome inhibitory effects) can be investigated efficiently.

J Gene Med, 2004 Feb, 6 Suppl 1, S105 - 24
State-of-the-art of the production of retroviral vectors; Merten OW; Retroviral vectors are still the vectors that are used in the majority of gene therapy trials for treatment of acquired or inherited diseases . In this review, the present state-of-the-art of the production of retroviral vectors and the most important parameters, such as the choice of the producer cell line, stability issues, medium additives, serum, type of bioreactor, that influence production issues is presented and discussed in light of an optimal vector production . The available literature data clearly indicate that, on one hand, the choice of the producer cell line is of utmost importance for obtaining a high level producer cell line, and that, on the other hand, the optimization of the medium, e.g . the replacement of glucose by fructose, has a potential for improving vector production rates and titers . Finally, the use of high-density perfusion culture systems for adherent as well as for suspension cells presents the best choice for a production system, because high cell densities imply high reactor specific production rates, which must be associated with a rapid harvest of the produced vector, thus avoiding vector inactivation due to an extended residence time . The overall optimization of the cultivation and production parameters will have a significant impact on the use of retroviral vectors for gene therapy purposes .

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 3053 - 8 Epub 2004 Feb 20.
Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics; Voutetakis A et al.; The use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns . To circumvent such issues, we have considered salivary glands (SGs) as an alternative gene therapeutics target tissue . Given the high secretory abilities of SGs, we hypothesized that administration of low doses of recombinant adeno-associated virus (AAV) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream . We administered 10(9) particles of an AAV vector encoding human erythropoietin (hEPO) directly to individual mouse submandibular SGs . Serum hEPO reached maximum levels 8-12 weeks after gene delivery and remained relatively stable for 54 weeks (longest time studied) . Hematocrit levels were similarly increased . Moreover, these effects proved to be vector dose-dependent, and even a dosage as low as 10(8) particles per animal led to significant increases in hEPO and hematocrit levels . Vector DNA was detected only within the targeted SGs, and levels of AAV copies within SGs were highly correlated with serum hEPO levels (r = 0.98) . These results show that SGs appear to be promising targets with potential clinical applicability for systemic gene therapeutics.

Nat Prod Res, 2004 Feb, 18(1), 51 - 7
Cytotoxicity of in vitro produced podophyllotoxin from Podophyllum hexandrum on human cancer cell line; Chattopadhyay S et al.; Podophyllotoxin was produced by cell culture of Podophyllum hexandrum under in vitro culture conditions . A maximum of 4.26 mg/L of podophyllotoxin was produced when P . hexandrum was cultivated in 3 L stirred tank bioreactor . The compound extracted from the cell culture was applied to the human breast cancer cell line (MCF-7) and 1 nM podophyllotoxin was able to inhibit the growth of the cancer cells by 50% . The most profound inhibitory effect of podophyllotoxin was observed when it was applied in the beginning of cell growth.

Rev Esp Enferm Dig, 2003 Dec, 95(12), 876 - 89
{Indications and therapeutic possibilities of albumin dialysis (MARS)}; Banares Canizares R et al.; Despite remarkable medical advances during the last few years, liver failure--both acute and chronic--still results in high mortality . Since liver transplant programs were developed to improve survival in numerous hepatic end-stage disorders, fewer than 15% of patients with liver failure do actually receive a transplantation, be it because of the presence of procedural contraindications (toxic habits, age, concurrent disease), or of clinical conditions that may render surgery more difficult or worsen transplant prognosis . All these circumstances encouraged the development of alternative procedures to increase liver graft availability, as is the case of liver partition techniques and living-donor transplantation . On the other hand, organ scarcity for transplantation during the 1960s encouraged the parallel development of liver support systems in an attempt to reduce mortality and to improve patient survival while waiting for a transplant . Such systems attempt to replace a number of synthesis and detoxification functions for the damaged liver parenchyma . During the past few years both bioartificial systems--also referred to as "bioartificial livers"--based on bioreactors containing functionally active living hepatocytes, and extracorporeal liver detoxification systems have been developed . The latter type includes the so-called MARS (molecular adsorbent recirculating system) system, which combines albumin-bound molecule clearance and novel dialysis membrane biocompatibility.

Biotechnol Bioeng, 2004 Mar 20, 85(6), 656 - 65
Hollow fiber bioreactor: new development for the study of contrast agent transport into hepatocytes by magnetic resonance imaging; Planchamp C et al.; The aim of our study was to develop a magnetic resonance (MR)-compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI) . We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB) . The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined . Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 x 10(7) cells/mL) . The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd-DTPA), and gadobenate dimeglumine (Gd-BOPTA), which also enters into hepatocytes . The HFB was imaged in the MR room using a dynamic T1-weighed sequence . No adsorption of CAs was detected in the perfusion system without hepatocytes . The use of a membrane with a high porosity (0.5 microm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs . The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period . The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd-BOPTA that enters into hepatocytes and Gd-DTPA that remains extracellular . Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time .

Biotechnol Bioeng, 2004 Mar 20, 85(6), 610 - 9
High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing; Su WW et al.; Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells . This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures . Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production . In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP . Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture . The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed . Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway . Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay . Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations .

Biotechnol Bioeng, 2004 Mar 20, 85(6), 589 - 600
Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions; Figueroa B Jr et al.; A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture . In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL . The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination . Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis . CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults . After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone . However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL . In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone . Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus . However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene . This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture .

Ann Biomed Eng, 2004 Jan, 32(1), 112 - 22
Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow; Meinel L et al.; We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge) . MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks . MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation . In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films . Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films . The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds . In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume . These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.

Wei Sheng Yan Jiu, 2003 Nov, 32(6), 516 - 8
{Effect evaluation of three cell culture models}; Wang A et al.; Primary rat hepatocytes were cultured using three kinds of models in vitro and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed . The results showed that the level of LDH in the medium decreased over time in the period of culture . However, on 5 days, LDH showed a significant increase in monolayer culture (MC) while after 8 days LDH was not detected in sandwich culture (SC) . The levels of AST and ALT in the medium did not change significantly over the investigated time . The basic CYP 1A activity gradually decreased with time in MC and SC . The decline of CYP 1A in rat hepatocytes was faster in MC than that in SC . This effect was partially reversed by using cytochrome P450 (CYP450) inducers such as omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than that in SC . Basic CYP 1A activity in bioreactor was keeped over 2 weeks and the highest albumin production was observed in bioreactor, and next were SC and MC . In conclusion, our results clearly indicated that there have some advantages and disadvantages in each of models in which can address different questions in metabolism of toxicants and durgs.

Calcif Tissue Int, 2004 May, 74(5), 458 - 68 Epub 2004 Feb 17.
Mechanical stimulation promotes osteogenic differentiation of human bone marrow stromal cells on 3-D partially demineralized bone scaffolds in vitro; Mauney JR et al.; Bone is a dynamic tissue that is able to sense and adapt to mechanical stimuli by modulating its mass, geometry, and structure . Bone marrow stromal cells (BMSCs) are known to play an integral part in bone formation by providing an osteoprogenitor cell source capable of differentiating into mature osteoblasts in response to mechanical stresses . Characteristics of the in vivo bone environment including the three dimensional (3-D) lacunocanalicular structure and extracellular matrix composition have previously been shown to play major roles in influencing mechanotransduction processes within bone cells . To more accurately model this phenomenon in vitro, we cultured human BMSCs on 3-D, partially demineralized bone scaffolds in the presence of four-point bending loads within a novel bioreactor . The effect of mechanical loading and dexamethasone concentration on BMSC osteogenic differentiation and mineralized matrix production was studied for 8 and 16 days of culture . Mechanical stimulation after 16 days with 10 nM dexamethasone promoted osteogenic differentiation of BMSCs by significantly elevating alkaline phosphatase activity as well as alkaline phosphatase and osteopontin transcript levels over static controls . Mineralized matrix production also increased under these culture conditions . Dexamethasone concentration had a dramatic effect on the ability of mechanical stimulation to modulate these phenotypic and genotypic responses . These results provide increased insight into the role of mechanical stimulation on osteogenic differentiation of human BMSCs in vitro and may lead to improved strategies in bone tissue engineering.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 368 - 76
Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger; Hrdlicka PJ et al.; Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal . We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A . niger grown in computer-controlled bioreactors . On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition . Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation . The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium . Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 361 - 7
Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production; Hagedorn A et al.; Fluorescence spectra of a 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) fermentation culture broth were combined with measurable process variables for off-line and on-line process monitoring . Culture broth fluorescence in UV and visible ranges was acquired by a fiber optic LCD array spectrometer . Process dynamics was followed on-line using a fiber optic probe attached to an external recirculation loop of the bioreactor . Partial least squares and stepwise regression methods were used to correlate measurable process parameters with the components of the fluorescence spectra . Both methods provided adequate approximation of yeast density, HEMF, glucose, and ethanol concentrations from fluorescence spectra . HEMF production was observed during the oxido-reductive growth phase when there was a lack of measurable oxygen in the culture broth and an excess of glucose . The addition of glucose resulted in the rapid production of HEMF and other metabolite intermediates such as ethanol, acetate, and glycerol.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 239 - 47
Evaluation of oxygen mass transfer in Aspergillus niger fermentation using data reconciliation; Patel N et al.; Fermentation experiments using Aspergillus niger result in a very viscous broth due to the growth of filamentous microorganism . For viscous fermentation processes, it is difficult to estimate with confidence the volumetric oxygen mass transfer coefficient (K(L)a), which can be used for scale-up or design of bioreactors . In the present study, four methods based on dynamic and stationary approaches were used to measure K(L)a throughout the fermentation . Data reconciliation was used to obtain a more reliable and consistent K(L)a . The K(L)a value obtained by a data reconciliation technique was found to be more reliable since it takes into consideration both the reliability of all measured variables and the accuracy of all mass balance equations.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 200 - 6
Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF; Cantarella M et al.; Steam-exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam-explosion pretreatment process . The SE temperature and time conditions were 214 degrees C and 6 min, resulting in a log R(0) of 4.13 . In enzymatic hydrolysis tests at 45 degrees C, the biomass loading in the bioreactor was 100 g(DW)/L (dry weight) and the enzyme-to-biomass ratio 0.06 g/g(DW) . The enzyme activities for endo-glucanase, exo-glucanase, and beta-glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively . The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5-hydroxymethyl furfural (5-HMF), syringaldehyde, 4-hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate . Acetic acid (2 g/L), furfural, 5-HMF, syringaldehyde, 4-hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase . Synergism and cumulative concentration effects of these compounds were not detected . SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used . The presence of acetic acid, vanillin, and 5-HMF (0.5 g/L) in SSF of 100 g(DW) /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 74 - 81
Dye decolorization by manganese peroxidase in an enzymatic membrane bioreactor; Lopez C et al.; In the present work an enzymatic membrane reactor (EMR) for the oxidation of azo dyes by manganese peroxidase (MnP) has been developed . The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane . The membrane allowed for most of the enzymatic activity to be recovered while both the parent dye and the degradation products could pass through . Different operational strategies (batch, fed-batch, and continuous) and parameters such as enzyme activity, H(2)O(2) feeding rate, hydraulic retention time (in continuous operation), and dye loading rate were studied . At best conditions, a continuous operation with a dye decolorization higher than 85% and minimal enzymatic deactivation was feasible for 18 days, attaining an efficiency of 42.5 mg Orange II oxidized/MnP unit consumed.

J Chromatogr A, 2004 Feb 13, 1026(1-2), 149 - 57
Purification of recombinant DNA-derived factor IX produced in transgenic pig milk and fractionation of active and inactive subpopulations; Lindsay M et al.; Transgenic animal bioreactors can be engineered to make gram per liter quantities of complex recombinant glycoproteins in milk . However, little is known about the limitations in post-translational processing that occurs for very complex proteins and how this impacts the task of purification . We report on the purification of recombinant factor IX (rFIX) from the milk of transgenic pigs having an expression level of 2-3 g rFIX/(l(-1) h(-1)), an expression level that is about 20-fold higher than previously reported . This purification process efficiently recovers highly active rFIX and shows that even complex mixtures like pig milk, which contains 60 g/l total endogenous milk protein and multiple subpopulations of rFIX, can be processed using conventional, non-immunoaffinity chromatographic methods . Without prior removal of caseins, heparin-affinity chromatography was used to first purify the total population of rFIX at greater than 90% yield . After the total population was isolated, the biologically active and inactive subpopulations were fractionated by high-resolution anion exchange chromatography using an ammonium acetate elution . Capillary isoelectric focusing of the active and inactive rFIX fractions demonstrated that the active subpopulations are the most acidic.

Trends Biotechnol, 2004 Feb, 22(2), 80 - 6
The role of bioreactors in tissue engineering; Martin I et al.; Ex vivo engineering of living tissues is a rapidly developing area with the potential to impact significantly on a wide-range of biomedical applications . Major obstacles to the generation of functional tissues and their widespread clinical use are related to a limited understanding of the regulatory role of specific physicochemical culture parameters on tissue development, and the high manufacturing costs of the few commercially available engineered tissue products . By enabling reproducible and controlled changes of specific environmental factors, bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues . In addition, by automating and standardizing tissue manufacture in controlled closed systems, bioreactors could reduce production costs, thus facilitating a wider use of engineered tissues.

Biotechnol Bioeng, 2004 Mar 5, 85(5), 475 - 81
Milking microalga Dunaliella salina for beta-carotene production in two-phase bioreactors; Hejazi MA et al.; A new method was developed for production of beta-carotene from Dunaliella salina . Cells were grown in low light intensity and then transferred to a production bioreactor illuminated at a higher light intensity . It was a two-phase bioreactor consisting of an aqueous and a biocompatible organic phase . Mixing of the cells and extraction were performed by recirculation of the organic phase . Two experiments were performed . In the first experiment, bioreactors were operated at two different solvent recirculation rates of 150 and 200 mL min(-1) . The beta-carotene extraction rate increased significantly at the higher recirculation rate, without exerting any influence on cell number and viability . A second experiment was carried out at a recirculation rate (200 mL min(-1)) appropriate for the study of long-term production of beta-carotene . The results show that D . salina at high light intensity remained viable for a long period (>47 days) in the presence of a biocompatible organic phase; however, cell growth was very slow . beta-Carotene could be continuously extracted to the organic phase; the cells continued to produce beta-carotene and the extracted molecules were continuously reproduced . As a result, beta-carotene was continuously removed ("milked") from the cells . beta-Carotene extraction efficiency in this system was >55%, and productivity was 2.45 mg m(-2) day(-1), much higher than that of commercial plants .

J Theor Biol, 2004 Feb 21, 226(4), 483 - 501
Modeling threshold phenomena, metabolic pathways switches and signals in chemostat-cultivated cells: the Crabtree effect in Saccharomyces cerevisiae; Thierie J; The long-term Crabtree effect in Saccharomyces cerevisiae cultivated in aerobic chemostat at steady state has been studied for three different substrate concentrations in the feed of the bioreactor (data: J . Gen . Microbiol., 129 (1983) 653) . We have shown that a model using two ways of transport/metabolization (T/M) of hyperbolic form, with high and low affinity for the substrate, allowed to represent correctly the main characteristics of the phenomenon . The model is based on an explicit form of the T/M kinetics when the bioreactor is considered as a polyphasic dispersed system (PDS) . Mass balances analysis also allows to quantify the critical dilution rate value (threshold), Dc, of the transition between respiratory and respirofermentative mode, for which ethanol is produced . A good approximation for the threshold is Dc = V(S)0 Y(Xc, S) where Y(Xc,S) is the average yield coefficient before transition and V(S)0, the maximum specific rate of high affinity T/M pathway . The theoretical value is 0.3 h(-1), and is equal to the experimental value . We thus show in a quantitative way that the transition depends both on culture conditions (global characteristic of the system) and on strain properties (intrinsic characteristic of the microorganism as well) . Using two different methods to calculate the residual substrate has carried out the comparison between the simulations end the experimental data . This allowed showing that the latter is not well represented by Monod's model and has confirmed that the affinity for the substrate varies according to the biomass . We have then shown how to calculate the most important specific rates (or metabolic flux) related to biomass, ethanol, oxygen, hydrogen, respiratory and fermentative CO(2) and H(2)O within the cellular phase . It has appeared that the oxygen uptake rate directly depends on high-affinity T/M pathway . This let us think that the regulation of the Crabtree effect in S . cerevisiae depends on the saturation of some glucose metabolization and transport pathways rather than on saturation of the respiratory chains . The specific rates analysis has also allowed us to show, at least in this case, that the metabolization rate (biosynthesis+fueling) had its maximum value on the whole dilution rates interval; metabolites excretion (ethanol and fermentative CO(2)) only intervenes to drain a "surplus" glucose flux . As a consequence, the transport capacity must be higher than the one of metabolization . Maximization of the metabolization specific rate could then be used as an optimization criterion in the stoichiometric calculation of metabolic flux (and not the specific growth rate maximization because growth is limited in a chemostat (mu = D)) . We have also shown that the mass balances based on the T/M processes are in agreement with molar and elementary balances of the general stoichiometric equation for glucose respiration and fermentation under aerobic conditions . Thanks to the specific rates calculating the stoichiometric coefficients has done this . The total mass balance difference does not exceed 4%, which is compatible with the experimental carbon balance . Finally, we have emphasized that the ratio of biosynthesis flux and metabolization flux is constant before and after transition . This observation could be applied as soon as the free substrate concentration in the cellular phase is low . The paper succinctly describes the former theoretical results on which the model is built and sufficiently explains the algorithm for straightforward implementation.

Biotechnol Bioeng, 2004 Feb 20, 85(4), 376 - 81
Microbioreactor arrays with parametric control for high-throughput experimentation; Maharbiz MM et al.; A scalable array technology for parametric control of high-throughput cell cultivations is demonstrated . The technology makes use of commercial printed circuit board (PCB) technology, integrated circuit sensors, and an electrochemical gas generation system . We present results for an array of eight 250 microl microbioreactors . Each bioreactor contains an independently addressable suite that provides closed-loop temperature control, generates feed gas electrochemically, and continuously monitors optical density . The PCB technology allows for the assembly of additional off-the-shelf components into the microbioreactor array; we demonstrate the use of a commercial ISFET chip to continuously monitor culture pH . The electrochemical dosing system provides a powerful paradigm for reproducible gas delivery to high-density arrays of microreactors . Growth data are presented for Escherichia coli cultured in the array with varying microaerobic conditions using electrochemically generated oxygen . Additionally, we present data on carbon dioxide generation for pH dosing .

Water Sci Technol, 2003, 48(11-12), 107 - 14
Cost-effective treatment solutions for rural areas: design and operation of a new package treatment plant for single households; Daude D et al.; The design approach and operation of a newly developed package plant treating domestic sewage from single households were evaluated . Combining submerged aerated filter (SAF) technology with jet aeration and incorporating both into a compact and shallow tank resulted in a cost-effective treatment solution . A trial unit was permanently installed at a rural site, serving a single household . Jet aeration proved to be the best aeration method for the shallow bioreactor design . Further trials revealed a 50% reduction in suspended solids (SS) through the use of a static effluent filter and found that annual plant maintenance was vital to sustain stable operating conditions . Despite high variations in influent conditions, the trial unit produced good effluent quality during steady-state operation . Average effluent BOD5, COD and SS values were 19.6 mg l(-1), 98 mg l(-1) and 32 mg l(-1) achieving overall removal efficiencies of 94.2%, 85.9% and 87.6% respectively . However, effluent ammonia nitrogen (NH4-N) levels were found to be inconsistent varying from 9 mg l(-1) to over 60 mg l(-1).

Water Sci Technol, 2003, 48(11-12), 85 - 91
Application of small community sewer system for improving the quality of the water resource in Korea; Myung GN et al.; An existing SBR plant in a rural area was retrofitted from a conventional fill-and-draw system to an intermittent-aeration system for additional nitrogen removal . This study indicated that organic and nitrogen removal efficiency was improved over that before the retrofitting . But effluent phosphorus concentration was increased gradually with the operating time . In the latter period of investigation, phosphorus concentration in effluent was higher than influent . It was regarded that an excessively accumulated phosphorus was released again under the anaerobic conditions of the sludge storage tank . The application of the electro-coagulation process was investigated as an alternative method in order to prevent phosphorus from re-releasing . A laboratory test for electro-coagulation indicated that T-P removal was more stable than the biological method only . In addition, it was confirmed that T-N and organic materials as well as T-P were removed simultaneously by the electrochemical reaction in the bioreactor combined with electrolysis by more than the bioreactor only.

Int J Phytoremediation, 2003, 5(4), 315 - 31
Non-significance of rhizosphere degradation during phytoremediation of MTBE; Ramaswami A et al.; Methyl tertiary butyl ether (MTBE) is a gasoline additive associated with groundwater pollution at gas station sites . Previous research on poplar trees in hydroponic systems suggests that phytovolatilization is an effective mechanism for phytoremediation of MTBE (Rubin and Ramaswami, 2001), but the potential for microbial degradation of MTBE in the rhizosphere of trees had not been assessed . MTBE had largely been considered recalcitrant to microbial processes, but recent fieldwork suggests rapid biodegradation may occur in certain cases . This paper investigates the potential for rhizosphere degradation of MTBE at time frames relevant for phytoremediation . Three experiments were conducted at different levels of aggregation to examine possible degradation of MTBE by rhizosphere microorganisms that had been acclimated to low levels of MTBE for 6 weeks . MTBE soil die-away studies, conducted with both poplar trees and fescue grass, found no significant differences between MTBE concentration in vegetated and unvegetated soils over a two-week attenuation period . Closed chamber tests comparing hydroponic and rhizospheric poplar tree systems also showed essentially complete recovery of MTBE mass in both systems, suggesting an absence of degradation . Finally, rhizosphere microbes tested in aerated bioreactors were found to be thriving and metabolizing root materials, but did not show measurable degradation of MTBE . In all tests, the MTBE degradation product, Tert Butyl Alcohol (TBA), was not detected . The insignificance of MTBE degradation by rhizosphere microorganisms suggests that plant processes be the primary focus of further research on MTBE phytoremediation.

Biochim Biophys Acta, 2004 Jan 20, 1676(2), 155 - 61
Calfection: a novel gene transfer method for suspension cells; Lindell J et al.; We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells . Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection . We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker . Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection . Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method . Calfection differs from the widely known calcium phosphate coprecipitation technique . The physico-chemical composition of the DNA interacting complexes is not yet known . The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C . In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh . Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter . High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.

Lett Appl Microbiol, 2004, 38(2), 176 - 9
Novel method for cell immobilization and its application for production of oligosaccharides from sucrose; Shin HT et al.; AIMS: The purpose of the present investigation was to develop a novel method for cell immobilization . METHODS AND RESULTS: Aureobasidium pullulans cells were mixed with an alginate solution, and the mixture was extruded to form small gel beads as hydrated-immobilized cells . The beads were then placed at -15 degrees C for 6-24 h to induce freeze-dehydration . The freeze-dehydration resulted in shrinkage of beads as a result of water removal reducing bead volume by 82% and bead weight by 85% . The dehydrated beads were successfully used for the production of fructo-oligosaccharides in a model reactor system . CONCLUSIONS: Dehydrated beads may provide some commercial advantages over conventional immobilized cells . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that bioreactor performance can be improved up to two times by the use of the dehydrated beads.

Crit Rev Biotechnol, 2003, 23(3), 175 - 94
Cartilage tissue engineering: controversy in the effect of oxygen; Malda J et al.; Articular cartilage lacks the ability to repair itself and consequently defects in this tissue do not heal . Tissue engineering approaches, employing a scaffold material and cartilage producing cells (chondrocytes), hold promise for the treatment of such defects . In these strategies the limitation of nutrients, such as oxygen, during in vitro culture are of major concern and will have implications for proper bioreactor design . We recently demonstrated that oxygen gradients are indeed present within tissue engineered cartilaginous constructs . Interestingly, oxygen, besides being an essential nutrient, is also a controlling agent of developmental processes including cartilage formation . However, the specific role of oxygen in these processes is still obscure despite the recent advances in the field . In particular, the outcome of published investigations is inconsistent regarding the effect of oxygen tension on chondrocytes . Therefore, this article describes the possible roles of oxygen gradients during embryonic cartilage development and reviews the data reported on the effect of oxygen tension on in vitro chondrocyte proliferation and differentiation from a tissue engineering perspective . Furthermore, possible causes for the variance in the data are discussed . Finally, recommendations are included that may reduce the variation, resulting in more reliable and comparable data.

Acta Microbiol Pol, 2003, 52(3), 301 - 13
Improvement of rifemycins production by Amycolatopsis mediterranei in batch and fed-batch cultures; El-Enshasy HA et al.; The production of rifamycins B and SV using glucose as main C-source by Amycolatopsis mediterranei in batch and fed-batch culture was investigated . Fed-batch culture using glucose as mono feeding substrate either in the form of pulse addition, in case of shake flask, or with constant feeding rate, in bioreactor level, proved to be an alternative production system with a significant increase in both volumetric and specific antibiotic production . The maximal concentrations of about 1146 mg/l and 2500 mg/l of rifamycins B and SV, respectively, was obtained in fed-batch culture in bioreactor level under non-oxygen limitation . On the other hand, the rate of rifamycins production was increased from 6.58 to 12.13 mg/l x h for rifamycin B and from 9.47 to 31.83 mg/l x h for rifamycin SV on the bioprocess transfer and improvement from the conventional batch cultivation in shake flask to fed-batch cultivation in stirred tank bioreactor.

Biochim Biophys Acta, 2004 Jan 30, 1608(1), 23 - 33
Heme protein films with polyamidoamine dendrimer: direct electrochemistry and electrocatalysis; Shen L et al.; Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes . Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples . While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about -0.34 V vs . saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of -0.47 V . The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior . The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (k(s)) and formal potentials (E (degrees ')) were estimated by square wave voltammetry with nonlinear regression analysis . UV-vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films . Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.

Mol Biotechnol, 2004 Jan, 26(1), 39 - 60
Generation and production of engineered antibodies; Kipriyanov SM et al.; Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes . This review specifically focuses on what is now called antibody engineering, and discusses the generation of chimeric, humanized, and fully human recombinant antibodies, immunoglobulin fragments, and artificial antigen-binding molecules . Since the production of recombinant antibodies is a limiting factor in their availability, and a shortage is expected in the future, different expression systems for recombinant antibodies and transgenic organisms as bioreactors are also discussed, along with their advantages and drawbacks.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2003 Dec, 21(6), 419 - 21
{Heterotopic osteogenesis of autogenous marrow stromal cells with recombinant human bone morphogenetic protein 2 gene transfection and porous calcium phosphate ceramic as a scaffold}; Chen X et al.; OBJECTIVE: To observe the heterotopic osteogenesis of autogenous marrow stromal cells loading on porous calcium phosphate ceramic scaffolds with rhBMP2 gene transfection in a Sprague-Dawley rat model . METHODS: Autogenous marrow stromal cells were obtained from left femurs and tibias of 20 male adult SD rats under general anesthesia and sterile condition and cultured in alpha-Minimal Essential Medium supplemented with 15% fetal bovine serum . RhBMP2 gene was transfected into stromal cells by means of LipofectAMINE 2000 reagent five days after primary culture . The stably gene expressive cells were selected with G-418 for 14 days and mixed with stromal cells without transfection . The mixture cells were seeded and subcultured for another 10 days in porous calcium phosphate bioceramic that had been subjected to surface-modification via soaking in human plasma fibronectin . The cell-ceramic compound was implanted subcutaneously and intramuscularly in the corresponding rat . Lab animals were sacrificed at two-week intervals till twenty weeks postoperatively and the involved samples were removed . RESULTS: Morphologic and histological study demonstrated that cell-ceramic compound had an ability of heterotopic osteogenesis, which was similar to that of autogenous osteoblasts in previous study . CONCLUSION: It seems that autogenous stromal cells with rhBMP2 transfection acts as a bioreactor promoting proliferation and differentiation of stem cells when they are replanted into the corresponding animals.

Water Res, 2004 Feb, 38(3), 663 - 72
Evaluation of natural and enhanced PCP biodegradation at a former pesticide manufacturing plant; Kao CM et al.; Pentachlorophenol (PCP) has been used in the past as a pesticide, herbicide, antifungal agent, bactericide, and wood preservative . Thus, PCP is among the most ubiquitous chlorinated compounds found in groundwater contamination . A former pesticide manufacturing plant located in southern Taiwan has been identified as a PCP spill site . In this study, groundwater samples collected from the PCP site were analyzed to assess the occurrence of natural PCP biodegradation . Microcosm experiments were conducted to (1) evaluate the feasibility of biodegrading PCP by indigenous microbial consortia under aerobic and cometabolic conditions, and (2) determine the potential of enhancing PCP biodegradation using cane molasses and biological sludge cake as the substitute primary substrates under cometabolic conditions . The inocula used in this microcosm study were aquifer sediments collected from the PCP site and activated sludges collected from the municipal and industrial wastewater treatment plants . Results from this field investigation indicate that the natural biodegradation of PCP is occurring and causing the decrease in PCP concentration . Microcosm results show that the indigenous microorganisms can biodegrade PCP under both aerobic and aerobic cometabolism conditions . A PCP-degrading bacterium was isolated from the collected aquifer sediments and identified as Pseudomonas mendocina NSYSU via some biochemical tests and further conformation of DNA sequencing . In batch cultures, P . mendocina NSYSU used PCP as its sole source of carbon and energy . The isolated bacterium, P . mendocina NSYSU, was capable of completely degrading PCP as indicated by the increase in biomass formation with the decrease in PCP concentrations occurred in the carbon-free medium simultaneously . Results indicate that the in situ or on-site aerobic bioremediation using indigenous microorganisms or inoculated bacteria would be a feasible technology to clean up the studied PCP-contaminated site . Results from this study will be useful in designing a scale-up in situ or on-site PCP bioremediation system (e.g., on-site bioreactor) for field application.

Artif Organs, 2004 Jan, 28(1), 83 - 98
Oxygen consumption in a hollow fiber bioartificial liver--revisited; Patzer JF 2nd; Oxygen consumption dynamics in a hollow fiber, hepatocyte-loaded bioartificial liver are investigated both theoretically and experimentally . The theoretical model is based upon the Krogh cylinder, which approximates the bioreactor as a collection of cylindrical elements comprised of an inner fiber lumen for media perfusion, the fiber wall through which oxygen can diffuse, and an annular region of hepatocytes surrounding the fiber . The primary non-dimensional parameters that describe the system are: (i) the Peclet number, Pe, which is the ratio of convective oxygen transport through the lumen to diffusive oxygen transport to the fiber walls; (ii) the hepatocyte saturation parameter, theta, which is the ratio of the inlet oxygen partial pressure to the Michaelis-Menten half-rate oxygen partial pressure; (iii) the Thiele modulus, phi2, which is the ratio of oxygen consumption rate to oxygen diffusion rate in the hepatocyte annulus; (iv) the hepatocyte permeability ratio, beta31, which is the ratio of oxygen permeability in the hepatocyte cell mass to oxygen permeability in the perfusing lumen medium; and (v) the hepatocyte annular thickness, rho3, which is the ratio of the exterior hepatocyte annular radius to the fiber lumen radius . Only Pe and theta are easily manipulated operating variables . phi2, beta31, and rho3 are engineering design parameters that are set when a bioreactor is fabricated . The model results are expressed as the effective hepatocyte utilization ratio, Vratio, which is the ratio of the observed oxygen consumption rate to the intrinsic hepatocyte oxygen consumption rate . Large regions of Vratio > 0.9, which is deemed an acceptable effective hepatocyte utilization are found for parameter values consistent with standard hollow fiber cartridges used in bioartificial liver fabrication . The extent of the Vratio > 0.9 region increases to a plateau with increasing Pe, increases with increasing theta, decreases with increasing phi2, increases with increasing beta31, and decreases with increasing rho3 . The theoretical results indicate that Vratio > 0.9 is found whenever the experimentally observed fractional oxygen consumption from the perfusing medium, is less than 0.25 . Combination of the theoretical and experimental results indicate that intrinsic, per cell oxygen consumption in the hollow fiber system may decrease as hepatocyte cell density increases and that this decrease may be due to lower intrinsic oxygen requirements in denser suspensions and not due to diffusion limitations in oxygen transport in the hollow fiber system as might be expected from two-dimensional, monolayer culture oxygen consumption measurements.

Anal Chem, 2003 Oct 1, 75(19), 5046 - 53
Quantitation of underivatized free amino acids in mammalian cell culture media using matrix assisted laser desorption ionization time-of-flight mass spectrometry; Dally JE et al.; In this investigation, a quantitative matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) method was developed for the analysis of underivatized free amino acids in mammalian cell culture media . Calibration curves were developed for 12 amino acids over the linear range of 1-100 microM with coefficients of determination ranging from r2 = 0.9220 to r2 = 0.9973 . An aerospray method was utilized for the sample deposition method, and the matrix, alpha-cyano-4-hydroxycinnamic acid, served as the internal standard . This assay was used to analyze bioreactor samples from five time points in the process . Concentrations determined through interpolation of the calibration curves were comparable to those obtained via reversed-phase HPLC based analysis with an average percent difference of 19.71% . Repeatability and intermediate precision studies were also performed, and the relative standard deviations ranged from 0.5943 to 21.41 and 3.157 to 18.97, respectively.

Biotechnol Bioeng, 2004 Jan 5, 85(1), 96 - 102
Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor; Cruz AJ et al.; The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors . Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer . In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process . Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle . The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water . A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed . To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted . The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0% . An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model . Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor . Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production . Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes . The model incorporated this improvement predicting higher cephalosporin C productivity .

Biotechnol Bioeng, 2004 Jan 5, 85(1), 34 - 46
Fluid mechanics of a spinner-flask bioreactor; Sucosky P et al.; Spinner-flask bioreactors have been used for the production of articular cartilage in vitro . The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue . The present research focuses on the experimental and numerical characterization of the flow field within a spinner flask operating under conditions used to produce cartilage . Laboratory experiments carried out in a scaled-up model bioreactor employ particle-image velocimetry (PIV) to determine velocity and shear-rate fields in the vicinity of the construct closest to the stir bar, in addition to turbulence properties . Numerical computations calculated using FLUENT, a commercial software package, simulate the flow field in the same model bioreactor under similar operating conditions . In the computations, scaffolds were modeled as both solid and porous media with different permeabilities and flow rates through various faces of the construct nearest the stir bar were examined .

Biotechnol Bioeng, 2004 Jan 5, 85(1), 20 - 8
Blocking caspase-3 activity with a U6 SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta; Lai D et al.; Apoptosis responding to various insults in bioreactors was observed to reduce cell viability and prevent cells from growing to high density . Inhibition of apoptosis in different ways has proved to be effective in keeping cells viable in high density and result in higher recombinant protein production . In apoptosis, death signals activate a family of proteinases, namely caspases, in a cascade and ultimately activate the final effector proteinase, caspase-3, which cleaves various substrates and drives the cells irreversibly to death . Caspase-3 is the executioner of an apoptotic cell and thus plays a central role in apoptosis . Therefore inhibition of caspase-3 may provide an effective way for apoptosis prevention . In this study, we designed a ribozyme targeted at the 451 nt of hamster caspase-3's open reading frame (ORF) and the ribozyme was proved to be effective in cleaving caspase-3 mRNA in vitro . Then, the ribozyme was cloned into a vector under the control of U6 snRNA promoter, an RNA polymerase III promoter, for high rate of transcription in vivo . The vector was transfected into an interferon-beta producing recombinant CHO cell line, and some clones were screened out that exhibited low caspase-3 production by Western blot analysis . One such clone was then further analyzed and it showed good anti-apoptosis property with respect to cell viability, cell density, and interferon-beta production .

Biotechnol Bioeng, 2004 Jan 20, 85(2), 222 - 33
Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability; Yoshimoto M et al.; Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared . The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA) . The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate . Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e) . Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30 . At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39) . At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value . This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate . Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation) . The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes . Inside the liposomes, the target substrate is then converted into the corresponding products .

Biotechnol Bioeng, 2004 Jan 20, 85(2), 138 - 46
Large-scale expansion of cytomegalovirus-specific cytotoxic T cells in suspension culture; Foster AE et al.; Clinical trials in recent years involving the adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) have shown promise in restoring immunity against viral infection and reducing tumor burden in patients with solid and hematological malignancies . However, the large cell number required to achieve efficacy, 10(9) to 10(11), makes routine application of adoptive immunotherapy impractical . Investigation into new methods of CTL expansion may be useful in addressing this problem . Use of stirred suspension bioreactors are one such method that may allow large-scale T-cell expansion . Suspension cultures offer advantages over conventional static culture methods, including providing a homogeneous culture environment, and the potential for optimization and control of culture conditions . We generated cytomegalovirus (CMV)-specific CTL and investigated the potential of stirred bioreactor systems for expansion of large cell numbers . We found that CTL can be readily expanded ( > 200-fold) from cryopreserved stocks by nonspecific stimulation in the presence of allogeneic feeder cells and interleukin-2 (IL-2) . Activated CTL inoculated into either suspension or static cultures could be subsequently expanded tenfold, and showed similar growth kinetics and metabolism independent of the culture vessel used . Furthermore, CTL remained specific for CMVpp65 peptide through the expansion phases, as demonstrated by pp65-tetramer staining ( > 95% tetramer(+)) and cytotoxicity assays . This study indicates that suspension reactor systems may be useful in large-scale expansion of antigen-specific CTL lines or clones, and may facilitate the advancement of routine adoptive immunotherapy .

Transplant Proc, 2003 Dec, 35(8), 2837 - 42
Cell therapy of renal failure; Fissell WH et al.; The kidney is unique in that it is the first organ for which long-term ex vivo substitutive therapy has been available . The first hemodialyzer was successfully applied to a human patient with acute renal failure in 1948, and the first successful allograft transplantation was performed with a kidney in 1951 . Both treatments are used today . There is ample evidence that the small solute clearance function provided by hemodialysis does not confer the same survival advantage as a functional kidney, both in acute and in chronic renal failure . To mimic the metabolic, endocrine, and immunologic functions of the kidney, our group has successfully engineered a bioartificial device that includes a conventional dialysis filter and a bioreactor containing 10(9) renal proximal tubule cells . We have demonstrated differentiated activity of these cells both in vitro and ex vivo in a large animal model . The bioreactor has been shown to confer a survival advantage in two large animal models of gram-negative sepsis, seemingly due to modulation of inflammatory mediators . This bioartificial kidney has now completed a Phase I clinical trial in acute renal failure.

J Biotechnol, 2004 Jan 8, 107(1), 55 - 64
Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity; Maranga L et al.; The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium . At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell . In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions . Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized . An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up . The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu . The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions . Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor . Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity . The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.

Water Sci Technol, 2003, 48(8), 203 - 10
A new method for the fast determination of kinetic parameters in anaerobic digestion processes and application to textile wet processing wastewater; Feitkenhauer H et al.; A new method for the fast and efficient determination of the anaerobic degradability and for the calculation of kinetic parameters is proposed . It is based on the idea that monitoring of the acidification step in the anaerobic degradation cascade is sufficient, as the subsequent steps (acetogenesis, methanogenesis) are well known processes dependent on the intermediate concentrations and not on the original substrate used . The investigation of the acidification is subdivided into two steps . In the first step, biomass is adapted to the new substrate in a continuously operated bioreactor . The second step, the only step monitored, is the acidification of the substrate (or wastewater) in a batch experiment . Three different methods to monitor the acidification were tested: gas chromatography (offline), on-line titration and monitoring of the base consumption (direct titration) . The results of these methods were compared to "traditional" batch tests . It is shown that the simplest method (direct titration) revealed the most important information . The investigated substrates were selected pollutants of textile wet processing wastewater.

Water Sci Technol, 2003, 48(8), 159 - 67
The enzymology of sludge solubilisation utilising sulphate reducing systems: the role of lipases; Whiteley CG et al.; The first stage in the degradation and recycling of particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the sulphidogenic environment . An investigation into the enzymology of these processes has shown that lipase enzyme activities were found predominantly associated with the organic particulate matter of the sewage sludge . Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant . pH and temperature optimisation studies showed optima at between 6.5 and 8 and 50-60 degrees C, respectively . All the lipase enzymes from the methanogenic bioreactors indicated extensive stability for at least an hour at their respective optimum temperatures and pH; sulphidogenic lipases reflected limited stability at these temperatures and pH during this time period . Though sulphate showed inhibitory properties towards lipases both sulphide and sulphite appeared to enhance the activity of the enzymes . It is argued that these sulphur species, liberated at different times during the sulphate reduction process, disrupt the integrity of the organic particulate floc by neutralising acidic components on the surface . The release of further entrapped enzymes from the organic particulate matter results in a subsequent enhancement of hydrolysis of polymeric material.

Water Sci Technol, 2003, 48(8), 127 - 33
Evaluation of biomass production in unleaded gasoline and BTEX-fed batch reactors; Acuna-Askar K et al.; BTEX removal under aerobic conditions by unleaded gasoline acclimated biomass and BTEX acclimated biomass, and the effect of surfactant on BTEX biodegradation were evaluated . The effect of BTEX concentration as the sole source of carbon for biomass acclimation and the effect of yeast extract on cell growth in unleaded gasoline-fed reactors were also evaluated . For the unleaded gasoline acclimated biomass, benzene was shown the most recalcitrant among all BTEX, followed by o-xylene and toluene with 16-23%, 35-41% and 57-69% biodegradation, respectively . Ethylbenzene was consistently the fastest BTEX chemical removed with 99% biodegradation for the four bioreactor acclimated biomasses tested . For the 1,200 ppm BTEX acclimated biomass, benzene showed the highest removal efficiency (99%) among the four biomass environmental conditions tested, along with 99% toluene and 99% ethylbenzene biodegradation . O-xylene showed 92-94% removal . In all bioassays tested Tergitol NP-10 was fully removed, and did not have a substantial effect on BTEX biodegradation at the end of a 10-day evaluation.

Biotechnol Lett, 2003 Nov, 25(21), 1853 - 6
Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors; Du M et al.; Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d . Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g(-1) astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l(-1), astragaloside IV 0.9 mg g(-1)) . Polysaccharide yields were similar amongst the different bioreactors (range 25-32 mg g(-1)) . The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A . membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.

Tissue Eng, 2003 Dec, 9(6), 1243 - 53
Cultivation in rotating bioreactors promotes maintenance of cardiac myocyte electrophysiology and molecular properties; Bursac N et al.; We tested the hypothesis that cardiomyocytes maintained their phenotype better if cultured as three-dimensional tissue constructs than if cultured as confluent monolayers . Neonatal rat cardiomyocytes were cultured on biomaterial scaffolds in rotating bioreactors for 1 week, and resulting tissue constructs were compared with confluent monolayers and slices of native ventricular tissue with respect to proteins involved in cell metabolism (creatine kinase isoform MM), contractile function (sarcomeric myosin heavy chain), and intercellular communication (connexin 43), as well as action potential characteristics (e.g., membrane resting potential, maximum depolarization slope, and action potential duration), and macroscopic electrophysiological properties (maximum capture rate) . The molecular and electrophysiological properties of cardiomyocytes cultured in tissue constructs, although inferior to those of native neonatal ventricles, were superior to those of the same cells cultured as monolayers . Construct levels of creatine kinase, myosin heavy chain, and connexin 43 were 40-60% as high as ventricle levels, whereas monolayer levels of the same proteins were only 11-20% as high . Construct action potential durations were 1.8-fold higher than those in ventricles, whereas monolayer action potential durations were 2.4-fold higher . Pharmacological studies using 4-aminopyridine showed that prolonged action potential duration and reduced maximum capture rate in tissue constructs as compared with native ventricles could be explained by decreased transient outward potassium current.

Tissue Eng, 2003 Dec, 9(6), 1215 - 26
Chondrogenesis of aged human articular cartilage in a scaffold-free bioreactor; Marlovits S et al.; Chondrogenesis of aged human articular chondrocytes was evaluated under controlled in vitro conditions, using a rotating bioreactor vessel . Articular chondrocytes isolated from 10 aged patients (median age, 84 years) were increased in monolayer culture . A single-cell suspension of dedifferentiated chondrocytes was inoculated in a rotating wall vessel, without the use of any scaffold or supporting gel material . After 90 days of cultivation, a three-dimensional cartilage-like tissue was formed, encapsulated by fibrous tissue resembling a perichondrial membrane . Morphological examination revealed differentiated chondrocytes ordered in clusters within a continuous dense cartilaginous matrix demonstrating a strong positive staining with monoclonal antibodies against collagen type II and articular proteoglycan . The surrounding fibrous membrane consisted of fibroblast-like cells, and showed a clear distinction from the cartilaginous areas when stained against collagen type I . Transmission electron microscopy revealed differentiated and highly metabolically active chondrocytes, producing an extracellular matrix consisting of a fine network of randomly distributed cross-banded collagen fibrils . Chondrogenesis of aged human articular chondrocytes can be induced in vitro in a rotating bioreactor vessel using low shear and efficient mass transfer . Moreover, the tissue-engineered constructs may be used for further in vitro studies of differentiation, aging, and regeneration of human articular cartilage.

Biodegradation, 2003 Dec, 14(6), 423 - 36
Advances in biotreatment of acid mine drainage and biorecovery of metals: 1 . Metal precipitation for recovery and recycle; Tabak HH et al.; Acid mine drainage (AMD), an acidic metal-bearing wastewater, poses a severe pollution problem attributed to post mining activities . The metals usually encountered in AMD and considered of concern for risk assessment are arsenic, cadmium, iron, lead, manganese, zinc, copper and sulfate . The pollution generated by abandoned mining activities in the area of Butte, Montana has resulted in the designation of the Silver Bow Creek-Butte Area as the largest Superfund (National Priorities List) site in the U.S . This paper reports the results of bench-scale studies conducted to develop a resource recovery based remediation process for the clean up of the Berkeley Pit . The process utilizes selective, sequential precipitation (SSP) of metals as hydroxides and sulfides, such as copper, zinc, aluminum, iron and manganese, from the Berkeley Pit AMD for their removal from the water in a form suitable for additional processing into marketable precipitates and pigments . The metal biorecovery and recycle process is based on complete separation of the biological sulfate reduction step and the metal precipitation step . Hydrogen sulfide produced in the SRB bioreactor systems is used in the precipitation step to form insoluble metal sulfides . The average metal recoveries using the SSP process were as follows: aluminum (as hydroxide) 99.8%, cadmium (as sulfide) 99.7%, cobalt (as sulfide) 99.1% copper (as sulfide) 99.8%, ferrous iron (sulfide) 97.1%, manganese (as sulfide) 87.4%, nickel (as sulfide) 47.8%, and zinc (as sulfide) 100% . The average precipitate purity for metals, copper sulfide, ferric hydroxide, zinc sulfide, aluminum hydroxide and manganese sulfide were: 92.4, 81.5, 97.8, 95.6, 92.1 and 75.0%, respectively . The final produced water contained only calcium and magnesium and both sulfate and sulfide concentrations were below usable water limits . Water quality of this agriculturally usable water met the EPA's gold standard criterion.

Biodegradation, 2003 Dec, 14(6), 407 - 14
Effects of oxygen and nitrogen conditions on the transformation kinetics of 1,2-dichloroethenes by Methylosinus trichosporium OB3b and its sMMOC mutant; Kim HJ et al.; Transformation kinetics of trans- and cis-dichloroethylenes (DCE) by Methylosinus trichosporium OB3b wild type (WT) and PP319, a mutant that expresses soluble methane monooxygenase at copper levels up to micro 12 microM Cu (sMMOC), were determined to assess the effects of O2 level and N2-fixation on degradation capabilities . Two issues were examined: (1) the influence of O2 level and nitrogen-limitation on DCE degradation kinetics and toxicity in both organisms, and (2) the relative utility of PP319 for contaminant degradation in bioreactors . When both organisms were grown under high O2 conditions (80% saturation in air), maximum transformation rates (Vmax) and apparent first-order rate constants (Vmax/KM) were lower compared with organisms grown under low O2 conditions (10% saturation in air) regardless of nitrogen level . Further, Vmax values were near zero in nitrogen-limited WT cultures when O2 was high (as expected), whereas PP319 retained moderate Vmax levels even at high O2 levels . In general, elevated O2 conditions reduced DCE degradation rates in OB3b, although the negative effects of O2 were less in PP319 than in the WT . Given that PP319 retained moderate DCE degradation rates under most O2 and copper conditions, the mutant appears to have some utility for biodegradation applications.

Environ Technol, 2003 Oct, 24(10), 1235 - 42
Substrate concentration-independent aerobic granulation in sequential aerobic sludge blanket reactor; Liu QS et al.; The development of aerobic granules was studied in four column-type sequential aerobic sludge blanket reactors fed with different substrate concentrations ranging from 500 to 3000 mg l(-1) COD . Results showed that aerobic granules successfully formed in all reactors fed with different substrate concentrations, indicating that the formation of aerobic granules is independent of the substrate concentration . The granule size, roundness, compactness, physical strength, as well as cell surface hydrophobicity and cell polysaccharides contents of the cultivated aerobic granules were investigated . It was shown that aerobic granules formed with different substrate concentrations had similar roundness and compactness . However, the size of aerobic granules slightly increased with an increase in substrate concentration, while granule strength decreased with substrate concentration . It was found that there was a significant increase in cell surface hydrophobicity and cell polysaccharides of the aerobic granules compared to that of seed sludge . The high cell surface hydrophobicity and high cell polysaccharides contents were believed to play an important role in the formation of aerobic granules . However, substrate concentration seems not to be a governing factor for the formation of aerobic granules . The results of this study would be useful for developing aerobic granules-based bioreactor and for better understanding of the mechanism of aerobic granulation . It was also clearly demonstrated that aerobic granules-based bioreactor would have great potential in the treatment of high-strength wastewater.

Biotechnol Appl Biochem . 2003 Dec 10; {Epub ahead of print}
Effect of pH on the production of the antitumor antibiotic retamycin by Streptomyces olindensis; Guimaraes LM et al.; The effect of pH on cell growth and retamycin production in batch bioreactor cultures of Streptomyces olindensis ICB20 was investigated . In controlled pH fermentations, in the range from 6.0 to 8.0, the best results regarding retamycin production were achieved at pH 7.0 and its maximum concentration, around 1.36 AU (AU = absorbance units), was about 430037;, 580037; and 2320037; higher than the values obtained at pH 7.5, 6.0 and 8.0, respectively.

Chemosphere, 2004 Mar, 54(10), 1573 - 80
Acclimation of anaerobic sludge degrading chlorophenols and the biodegradation kinetics during acclimation period; Ye FX et al.; The acclimation of sludge from Hangzhou citrate factory and Hangzhou municipal wastewater treatment plant for degradation dechlorination of chlorophenols (CPs) compounds, and its biodegradation kinetics were studied in batch process with or without addition of sucrose . Three monochlorophenols (2-CP; 3-CP; 4-CP) and pentachlorophenol (PCP) were concurrently fed to different bioreactors . The parameters that were monitored included biogas production, biogas composition and chemical oxygen demand (COD) . The results showed that acclimation with chlorophenol can increase the degradation activity of anaerobic sludge and degradation rate of chlorophenolic compounds, and reduce the lag time . Degradation dechlorination activity of the acclimated sludge strongly depended on sludge source, microorganism population and chlorophenol congener . 2-CP was more easily acclimated than 3-CP and 4-CP . Among the four tested compounds, 4-CP was the most difficult to be acclimated . The observed degradation rate with presence of sucrose was higher than that with absence of sucrose, suggesting that addition of the external carbon source can stimulate the formation of acclimated sludge which could effectively degrade chlorophenols . Kinetic equations of biodegradation of chlorophenols were also presented in this paper.

Cardiovasc Res, 2003 Dec 1, 60(3), 497 - 509
Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study; Schenke-Layland K et al.; OBJECTIVE: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes . The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves . METHODS: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries . Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days . Controls were grown over an equivalent time period under static conditions . Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin-eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength) . RESULTS: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix . Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions . Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls . CONCLUSION: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 14683 - 8 Epub 2003 Dec 01.
Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces; Sikavitsas VI et al.; In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells . Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant . This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant . Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype . Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds . The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1842 - 6
Scale-up of a liquid static culture process for hyperproduction of ganoderic acid by the medicinal mushroom Ganoderma lucidum; Tang YJ et al.; Scale-up of a liquid static culture process was studied for hyperproduction of ganoderic acid (GA) by a famous Chinese traditional medicinal mushroom, Ganoderma lucidum . Initial volumetric oxygen transfer coefficient (K(L)a) and area of liquid surface per liquid volume (A(s)) were identified as key factors affecting cell growth and GA accumulation in liquid static cultures of G . lucidum, on the basis of which a multilayer static bioreactor was designed . At a low initial K(L)a level of 2.1 h(-1), a thick layer of white mycelia was formed on the liquid surface, and an optimal production of total GA (i.e., GA production in the liquid and on the liquid surface) was obtained . Both the formation of white mycelia and production of GA on the liquid surface were enhanced with an increase of A(s) within the range as investigated (0.24-1.53 cm(2)/mL) . At an A(s) value of 0.90 cm(2)/mL, the total GA production reached maximum . A successful scale-up from a 20-mL static T-flask to a 7.5-L three-layer static bioreactor was achieved based on initial K(L)a . The maximum biomass (20.8 +/- 0.1 g DW/L), GA content (4.96 +/- 0.13 mg/100 mg DW), and total GA production (976 +/- 35 mg/L) were attained in static bioreactors . Not only GA content but also its production obtained in this work were the highest ever reported.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1828 - 36
Neural-network-based identification of tissue-type plasminogen activator protein production and glycosylation in CHO cell culture under shear environment; Senger RS et al.; An artificial neural network (ANN) modeling scheme has been constructed for the identification of both recombinant tissue-type plasminogen activator (r-tPA) protein production and glycosylation from Chinese hamster ovary (CHO) cell culture, cultivated in a stirred bioreactor . A series of hybrid feed-forward backpropagation neural networks were constructed to function as a software sensor . This enabled predictions of viable cell density, r-tPA content, and r-tPA glycosylation . The sensor was based on an initial input vector space consisting of simple metabolite concentrations, batch cultivation time, and a description of shear stress applied to the culture . Metabolite concentrations of the culture supernatant, included in the input vector space, were obtained from a single isocratic HPLC measurement . The shear stress component of the input space enabled accurate culture state prediction over a wide range of agitation rates . Coefficient of determination (r(2)) values between ANN predicted and experimental measurements of 0.945, 0.943, 0.956, and 0.990 were calculated to validate individual ANN prediction accuracy for total ammonia, apparent viable cell density, total r-tPA, and Type II glycoform concentrations, respectively.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1762 - 6
Optimal NS0 cell growth in a hollow fiber bioreactor requires increased serum concentration or a cholesterol supplement on the cell side of the fiber; Gramer MJ et al.; An NSO/GS cell line secreting a humanized antibody was routinely propagated in a T-flask using 2% serum . For scale-up of antibody production, this cell line was inoculated into a hollow fiber system using the same serum concentration . The metabolic activity increased for a few days in the hollow fiber system, but invariably the activity dropped dramatically as the cells died by day 7 . A hollow fiber micro-bioreactor was used as a screening tool to examine possible reasons for cell death in the large-scale system . As seen in the hollow fiber system, cells died when 2% serum was used either on the cell side only or on both sides of the fiber in the micro-bioreactor . In contrast, the use of 20% serum on the cell side of the fiber and basal medium on the non-cell side resulted in good cell expansion at high viability . Regardless of the cell side serum concentration, no further growth enhancements were seen when up to 20% serum was placed on the non-cell side of the fiber . These results suggest that a serum component that does not readily cross the fiber is limiting cell growth in the hollow fiber bioreactors . The addition of a cholesterol-rich lipid supplement resulted in better cell growth in the micro-bioreactor, while the addition of other non-cholesterol lipid supplements resulted in no growth enhancement . The growth-enhancing properties of the cholesterol supplement were more pronounced at lower serum concentrations, suggesting that poor growth at low serum concentration was due to suboptimal cholesterol levels . When the cell side serum concentration was increased to 20% in the hollow fiber system, cells grew and filled the bioreactor, allowing a 39-day production run . These results demonstrate that this NSO cell line requires an increased cell side serum concentration for optimal growth and that this requirement is likely due to the inherent cholesterol dependency of this cell line.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1643 - 7
Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor; Ryu SA et al.; D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor . Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively . Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w) . Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

Biotechnol Prog, 2003 Nov-Dec, 19(6), 1632 - 8
Enzymatic synthesis of 3'-hydroxyacetaminophen catalyzed by tyrosinase; Valero E et al.; 3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase . Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available . This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative . A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol . This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.

Cell Transplant, 2003, 12(7), 779 - 85
The effects of poly(ethyleneimine) (PEI) molecular weight on reinforcement of alginate hydrogels; Kong HJ et al.; Alginate hydrogels are widely used for cell encapsulation and transplantation, and they are frequently surface reinforced with secondary polymers to enhance their mechanical rigidity and stability . We hypothesized that the molecular weight (MW) of the polymer utilized to reinforce alginate would be an important factor in their stability, particularly when the gel network was homogeneously reinforced with the polymer . This hypothesis was investigated with alginate hydrogels cross-linked with Ca2+, and reinforced throughout the bulk of the gel with poly(ethyleneimine) (PEI) having different MWs . Interactions between the two polymers became significant following gelation, leading to higher elastic moduli (E) than gels with no PEI . The decrease in E of gels incubated in isotonic salt solutions over time, utilized as an indication of gel break down, was ameliorated with an increase in the MW of the PEI . In addition, the dependencies of the moduli and viscoelasticity on the temperature also became smaller with the use of high MW PEI . This is likely due to the limited mobility of high MW PEI, leading to a higher energy for dissociation . The stable interactions between the alginate and PEI prevented alterations of the pore structure in the gels, and slowed the deterioration of gel properties even under continuous agitation in a bioreactor . The results of this study will likely be useful in designing alginate encapsulation strategies for various applications.

Cell Transplant, 2003, 12(7), 717 - 31
Generation of 3D retina-like structures from a human retinal cell line in a NASA bioreactor; Dutt K et al.; Replacement of damaged cells is a promising approach for treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP); however, availability of donor tissue for transplantation remains a major obstacle . Key factors for successful engineering of a tissue include the identification of a neural cell line that is: homogeneous but can be expanded to give rise to multiple cells types; is nontumorigenic, yet capable of secreting neurotrophic factors; and is able to form three-dimensional (3D), differentiated structures . The goal of this study was to test the feasibility of tissue engineering from a multipotential human retinal cell line using a NASA-developed bioreactor . A multipotential human retinal precursor cell line was used to generate 3D structures . In addition, retinal pigment epithelium (RPE) cells were cocultured with neural cells to determine if 3D retinal structures could be generated in the bioreactor with cells grown on laminin-coated cytodex 3 beads . Cell growth, morphology, and differentiation were monitored by light and scanning electron microscopy, Western blot analysis, and analysis of glucose use and lactate production . The neuronal retinal precursor cell line cultured in a bioreactor gave rise to most retinal cell types seen in monolayer culture . They formed composite structures with cell-covered beads associated with one another in a tissue-like array . The beginning of layering and/or separation of cell types was observed . The neuronal cell types previously seen in monolayer cultures were also seen in the bioreactor . Some of the retinal cells differentiate into photoreceptors in the bioreactor with well-developed outer segment-like structures, a process that is critical for retinal function . Moreover, the neuronal cells that were generated resembled their in vivo phenotype more closely than those grown under other conditions . Outer segments were almost never seen in the monolayer cultures, even in the presence of photoreceptor-inducing growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor (TGF-alpha) . Muller cells were occasionally seen when retinal, RPE cells were cocultured with retinal cells in the bioreactor . These have never been seen in this retinal cell line before . Cells grown in the bioreactor expressed several proteins specific for the retinal cell types: opsin, protein kinase C-alpha, dopamine receptor D4, tyrosine hydroxylase, and calbindin.

J Extra Corpor Technol, 2003 Sep, 35(3), 200 - 2
Bioreactors for tissue engineering--a new role for perfusionists?
Sistino JJ.
Tissue engineering is an exciting new area of medicine with rapid growth and expansion over the last decade . It has the potential to have a profound impact on the practice of medicine and influence the economic development in the industry of biotechnology . In almost every specialty of medicine, the ability to generate replacement cells and develop tissues will change the focus from artificial organs and transplantation to growing replacement organs from the patient's own stem cells . Once these organs are at a size that requires perfusion to maintain oxygen and nutrient delivery, then automated perfusion systems termed "bioreactors" will be necessary to sustain the organ until harvesting . The design of these "bioreactors" will have a crucial role in the maintenance of cellular function throughout the growth period . The perfusion schemes necessary to determine the optimal conditions have not been well elucidated and will undergo extensive research over the next decade . The key to progress in this endeavor will development of long-term perfusion techniques and identifying the ideal pressures, flow rates, type of flow (pulsatile/nonpulsatile), and perfusate solution . Perfusionists are considered experts in the field of whole body perfusion, and it is possible that they can participate in the development and operation of these "bioreactors." Additional education of perfusionists in the area of tissue engineering is necessary in order for them to become integral parts of this exciting new area of medicine.

Bioresour Technol, 2004 Mar, 92(1), 103 - 9
An economical bioreactor for evaluating biogas potential of particulate biomass; Wilkie AC et al.; An economical bioreactor designed for evaluating the biogas potential of particulate biomass is described . The bioreactor uses a simple stirring apparatus, called the Bordeaux stirrer, to enable gas-tight mixing of fermentation cultures . The apparatus consists of a low-rpm motor connected to a bent steel stir rod, which is placed in a length of flexible plastic tubing inserted through a rubber stopper in a gas-tight manner . This stirrer is suitable for providing intermittent or continuous mixing in bench-scale anaerobic cultures containing particulate biomass . The reactor system may be operated as a batch-fed or semi-continuously fed digester . This communication documents the advantages of the stirring apparatus, describes the details of reactor fabrication and operation, and outlines the type of experimental work for which the bioreactor is suitable.

Eur J Cardiothorac Surg, 2003 Dec, 24(6), 906 - 11
Bioartificial grafts for transmural myocardial restoration: a new cardiovascular tissue culture concept; Kofidis T et al.; OBJECTIVE: Survival of bioartificial grafts that are destined to restore cardiac function stands and falls with their nutrient supply . Engineering of myocardial tissue is limited because of lack of vascularization . We introduce a new concept to obtain bioartificial myocardial grafts in which perfusion by a macroscopic core vessel is simulated . METHODS: We have designed an experimental reactor with multiple chambers for the production of bioartificial tissue or tissue precursors . By introduction of in- and output lines of distinct diameter and insertion of a core vessel into each chamber, we established pulsatile, continuous flow through the embodied three-dimensional tissue culture . In the present study, collagen components served as the ground matrix wherein neonatal rat cardiomyocytes were inoculated . For the assessment of cellular viability and distribution in comparison to static, non-perfused culture, fluor-desoxy-glucose-positron-emission-tomography and life/dead assays were employed . RESULTS: We obtained 3D constructs of 8-mm thickness, which display high viability and metabolism (6.0+/-1.3(e-03) in the perfused vs . 4.0+/-0.3(e-03) in the unperfused chambers) . The core vessel has the size of a human coronary and remained patent during the entire culture process . We observed centripetal migration of the embedded cardiomyocytes to the site of the core vessel . Cardiomyocytes partially resumed a spindle like form without additional stretch . CONCLUSIONS: The present dynamic tissue culture concept is highly effective in manufacturing thick, viable grafts for cardiac muscle restoration, which could be surgically anastomosable . The bioreactor may accommodate multiple types of cells and tissues for innumerable in vitro and in vivo applications.

J Air Waste Manag Assoc, 2003 Nov, 53(11), 1391 - 400
Partitioning gas tracer tests for measurement of water in municipal solid waste; Imhoff PT et al.; A key component in the operation of almost all bioreactor landfills is the addition of water to maintain optimal moisture conditions . To determine how much water is needed and where to add it, in situ methods are required to measure water within solid waste . Existing technologies often result in measurements of unknown accuracy, because of the variability of solid waste materials and time-dependent changes in packing density, both of which influence most measurement methods . To overcome these problems, a new technology recently developed by hydrologists for measuring water in the vadose zone--the partitioning gas tracer test--was tested . In this technology, the transport behavior of two gas tracers within solid waste is used to measure the fraction of the void space filled with water . One tracer is conservative and does not react with solids or liquids, while a second tracer partitions into the water and is separated from the conservative tracer during transport . This technology was tested in four different solid waste packings and was capable of determining the volumetric water content to within 48% of actual values, with most measurement errors less than 15% . This technology and the factors that affect its applicability to landfills are discussed in this paper.

Water Sci Technol, 2003, 48(6), 23 - 31
Influence of multiple substrates on anaerobic protein degradation in a packed-bed bioreactor; Tommaso G et al.; Data on the influence of substrate composition on the anaerobic degradation of bovine serum albumin (BSA) in a bench-scale packed-bed reactor are presented and discussed from the standpoint of substrate consumption kinetics . The experiments were carried out in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor fed with BSA based substrates . BSA was the sole carbon source in the first one, while the others were composed of BSA, carbohydrates and lipids . In all the experiments, the HAIB reactor was operated at the hydraulic detention time of 4 hours . The reactor's performance was evaluated based on physicochemical and chromatographic analyses and also on microscopy techniques . A kinetic model of irreversible first-order series-parallel reactions with two intermediate products was proposed, allowing evaluation of the microbial consortium's affinity with the substrates and the metabolic compounds formed . As the first-order kinetic model adhered quite well to the experimental data, the initial protein degradation rates (k) were estimated . The presence of carbohydrates and lipids led the initial protein degradation rate to be reduced . However, the system fed with protein and carbohydrates showed higher process stability.

Water Sci Technol, 2003, 48(6), 9 - 16
Enrichment and immobilization of quinone-respiring bacteria in anaerobic granular sludge; Cervantes FJ et al.; The capacity of an anaerobic granular sludge for serving as an immobilizing mechanism for quinone-respiring bacteria was evaluated . The inoculum was continuously fed with a basal medium containing the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as a terminal electron acceptor . Complete reduction of AQDS was achieved by the granular sludge for a prolonged period in an anaerobic bioreactor provided with a mixture of volatile fatty acids as a substrate . Phylogenetic analysis revealed the enrichment and immobilization of AQDS-respiring bacteria appearing as dominant organisms in the microbial population of the AQDS-supplemented reactor, compared to a reactor control operated under methanogenic conditions . The consistent quinone-reducing capacity observed in the consortium indicates that it is feasible to apply quinone-reducing microorganisms in continuous bioreactors and this ability can potentially be important in wastewaters rich in humic substances . The quinone reducing activity could also be applied to accelerate the conversion of xenobiotics susceptible to reductive biotransformations such as azo dyes and polychlorinated compounds in continuous bioreactors.

Tissue Eng, 2003 Oct, 9(5), 967 - 79
Novel system for engineering bioartificial tendons and application of mechanical load; Garvin J et al.; Cells cultured in three-dimensional collagen gels express a more native state phenotype because they form a syncytial network that can be mechanically loaded . Moreover, cells remodel their matrix by eliminating water, and by reorganizing and aligning the collagen fibrils . Last, the ability to subject cells to mechanical loading in a native matrix is desirable because cells, in tissues as well as the matrix, bear strains and alter their expression profile consistent with either immobilization, moderate activity, or repetitive loading . This is the first report of a model bioreactor system to fabricate and culture tendon cell-populated, linear, tethered matrix constructs that can be mechanically loaded by a computer-driven, pressure-controlled system . Bioartificial tissues (BATs) as tendon constructs were molded in a novel, rubber bottom Tissue Train culture plate bearing nonwoven nylon mesh anchors at the east and west poles of each culture well . Mechanical loading was achieved by placing an Arctangle loading post (an Arctangle is a rectangle with curved short ends) beneath each well of the six-well culture plate and using vacuum to displace the flexible membrane downward, resulting in uniaxial strain on the BAT . BATs populated with avian flexor tendon cells expressed collagen genes I, III, and XII as well as aggrecan, fibronectin, prolyl hydroxylase, and tenascin, consistent with expression levels of cells grown on collagen-bonded two-dimensional surfaces or in native, whole, avian flexor tendon . Likewise, cells in BATs established a morphology of linearly arranged cells aligned with the principal strain direction as in fasicles of whole tendons . Last, BATs that were mechanically loaded had an ultimate tensile strength that was nearly 3-fold greater than that of nonloaded BATs in the first week of culture . Taken together, these results indicate that tendon cells fabricated in a mechanically loaded, linear collagen gel construct assume a phenotype that is similar to that of a native tendon in terms of appearance and expression and are stronger than nonexercised counterparts yet far weaker than native adult tendons . This technique represents a novel approach to culturing cells in a mechanically active, three-dimensional culture environment that can be readily used for the fabrication of tissue simulates for drug testing or tissue engineering.

Tissue Eng, 2003 Oct, 9(5), 893 - 908
Three-dimensional model of angiogenesis: coculture of human retinal cells with bovine aortic endothelial cells in the NASA bioreactor; Dutt K et al.; Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration . We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis . Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days . Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated . However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor . In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts . The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells . This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin) . Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation . Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors.

Habitation (Elmsford), 2003, 9(1-2), 1 - 8
The effect of drying and size reduction pretreatments on recovery of inorganic crop nutrients from inedible wheat residues; Strayer RF et al.; Inorganic nutrients can be easily recovered from ALS crop residue solid wastes by aqueous leaching . However, oven drying and milling pretreatment of these residues has been frequently required to accommodate crop scientists and facility storage limitations . As part of a research study that will compare three different bioreactor technologies for processing these wastes, we realized that different drying and size-reduction pretreatments had been utilized for each technology . This paper compares the effects of residue pretreatment on recovery of nutrients by leaching . Pretreatments included three drying methods {fresh, oven-dried (70 degrees C overnight), and freeze-dried} and two size reduction methods {chopped (2 cm length) and milled (2 mm diameter)} . Determination of mass balances (dry weight and ash content of solids) before and after leaching indicated solubilization was least for fresh residues (23% dry weight loss and 50% for ash loss), and most for freeze-dried residues (41-47% dry weight loss and nearly 100% for ash loss) . Mineral recovery of major elements (NO3, PO4, K, Ca, and Mg) in leachates was poorest for fresh residues . P and K recovery in leachates were best for oven-dried residues and Ca, Mg, and N recovery best for freeze-dried residues . The differences in recovery for N, P, and K in leachates were minimal between chopping and milling and slightly better for Ca and Mg from milled residues.

Adv Space Biol Med, 2003, 9, 25 - 39
Bdelloid rotifers as model system to study developmental biology in space; Ricci C et al.; Bdelloid rotifers are suitable model systems for space experiments . Due to their developmental pattern they appear adequate to investigate the role of the cytoskeleton during oogenesis and during early developmental stages, and to reflect the effects of disturbances in the spatial arrangement of cytoskeletal components . The effect of weightlessness on the developmental pattern of a bdelloid rotifer will be studied in the International Space Station: in preparation for it we are performing ground-based experiments on the development of rotifer embryos under either increased or decreased gravity . The model studied is Macrotrachela quadricornifera, a species of rotifers belonging to the Bdelloidea class . Samples exposed to gravity disturbance were analyzed for morphology and fitness-related parameters . Rotifers were exposed over several days to altered gravity conditions and the morphology of eggs laid during this period were investigated using a confocal laser microscope . A subset of eggs was allowed to hatch to determine newborn developmental time and age at maturity . High (up to 20 g) gravity was obtained in a slow centrifuge suitable for animal cultivation over several days . To produce low (simulated 0.0001 g) gravity a Random Positioning Machine equipped with a 'rotifer bioreactor' was used . Under all conditions the rotifer retained normal life-history traits, and did not show permanent changes in embryo morphology, regardless to the stresses to which it was exposed . Only some modification of the shape of early embryos, experiencing 20 g, has been noted, but later developmental stages appeared unaffected, and normal juveniles hatched . Whether this result indicates any capacity to repair damage during embryogenesis of these Spiralia experiencing 20 g is an open question . The significance of the result as well as the use of instruments to simulate gravity perturbations are discussed.

Water Res, 2004 Jan, 38(1), 47 - 60
Perchlorate removal in sand and plastic media bioreactors; Min B et al.; The treatment of perchlorate-contaminated groundwater was examined using two side-by-side pilot-scale fixed-bed bioreactors packed with sand or plastic media, and bioaugmented with the perchlorate-degrading bacterium Dechlorosoma sp . KJ . Groundwater containing perchlorate (77microg/L), nitrate (4mg-NO(3)/L), and dissolved oxygen (7.5mg/L) was amended with a carbon source (acetic acid) and nutrients (ammonium phosphate) . Perchlorate was completely removed (<4microg/L) in the sand medium bioreactor at flow rates of 0.063-0.126L/s (1-2gpm or hydraulic loading rate of 0.34-0.68L/m(2)s) and in the plastic medium reactor at flow rates of <0.063L/s . Acetate in the sand reactor was removed from 43+/-8 to 13+/-8mg/L (after day 100), and nitrate was completely removed in the reactor (except day 159) . A regular (weekly) backwashing cycle was necessary to achieve consistent reactor performance and avoid short-circuiting in the reactors . For example, the sand reactor detention time was 18min (hydraulic loading rate of 0.68L/m(2)s) immediately after backwashing, but it decreased to only 10min 1 week later . In the plastic medium bioreactor, the relative changes in detention time due to backwashing were smaller, typically changing from 60min before backwashing to 70min after backwashing . We found that detention times necessary for complete perchlorate removal were more typical of those expected for mixed cultures (10-18min) than those for the pure culture (<1min) reported in our previous laboratory studies . Analysis of intra-column perchlorate profiles revealed that there was simultaneous removal of dissolved oxygen, nitrate, and perchlorate, and that oxygen and nitrate removal was always complete prior to complete perchlorate removal . This study demonstrated for the first time in a pilot-scale system, that with regular backwashing cycles, fixed-bed bioreactors could be used to remove perchlorate in groundwater to a suitable level for drinking water.

In Vitro Cell Dev Biol Anim, 2003 Sep-Oct, 39(8-9), 368 - 74
In situ collagen gelation: a new method for constructing large tissue in rotary culture vessels; Su GN et al.; In situ collagen gelation is a method that combines a static three-dimensional culture technique with rotating bioreactors . This method was designed for large dense tissue engineering ex vivo . To challenge the current limitations on size, we combined the static collagen gel embedding method with high-aspect ratio rotating bioreactors . Rat calvarial cells in gelated collagens were cultured in rotating vessels with 5 mM beta-glycerophosphate-containing medium for 1, 2, or 3 wk and then analyzed for cell morphology, cell distribution, and viability, as well as for contraction of the collagen gel . The size of collagen gels with rat calvarial cells averaged 2.8 cm in diameter x 0.25 cm in thickness at the end of 3 wk . Scanning electron microscopy and laser scanning confocal microscopy of collagen gels revealed a homogeneous distribution of living cells . Despite the barrier effects from induced calcification, in collagen gels, cell metabolic activity (alkaline phosphatase assay and 3-{4,5-dimethyl-2-thiazolyl}-2,5-diphenyl-2H-tetrazolium bromide assay) increased over the 3 wk, and cell viability (trypan blue exclusion and flow cytometry analysis) remained at about 90% at the end of 3 wk . Based on our results, we determined that in situ collagen gelation provides a feasible method for engineering large dense tissue ex vivo.

Clin Plast Surg, 2003 Oct, 30(4), 507 - 17
Tissue engineered small-diameter vascular grafts; Schmedlen RH et al.; Arterial occlusive disease remains the leading cause of death in western countries and often requires vascular reconstructive surgery . The limited supply of suitable small-diameter vascular grafts has led to the development of tissue engineered blood vessel substitutes . Many different approaches have been examined, including natural scaffolds containing one or more ECM proteins and degradable polymeric scaffolds . For optimal graft development, many efforts have modified the culture environment to enhance ECM synthesis and organization using bioreactors under physiologic conditions and biochemical supplements . In the past couple of decades, a great deal of progress on TEVGs has been made . Many challenges remain and are being addressed, particularly with regard to the prevention of thrombosis and the improvement of graft mechanical properties . To develop a patent TEVG that grossly resembles native tissue, required culture times in most studies exceed 8 weeks . Even with further advances in the field, TEVGs will likely not be used in emergency situations because of the time necessary to allow for cell expansion, ECM production and organization, and attainment of desired mechanical strength . Furthermore, TEVGs will probably require the use of autologous tissue to prevent an immunogenic response, unless advances in immune acceptance render allogenic and xenogenic tissue use feasible . TEVGs have not yet been subjected to clinical trials, which will determine the efficacy of such grafts in the long term . Finally, off-the-shelf availability and cost will become the biggest hurdles in the development of a feasible TEVG product . Although many obstacles exist in the effort to develop a small-diameter TEVG, the potential benefits of such an achievement are exciting . In the near future, a nonthrombogenic TEVG with sufficient mechanical strength may be developed for clinical trials . Such a graft will have the minimum characteristics of biological tissue necessary to remain patent over a period comparable to current vein graft therapies . As science and technology advance, TEVGs may evolve into complex blood vessel substitutes . TEVGs may become living grafts, capable of growing, remodeling, and responding to mechanical and biochemical stimuli in the surrounding environment . These blood vessel substitutes will closely resemble native vessels in almost every way, including structure, composition, mechanical properties, and function . They will possess vasoactive properties and be able to dilate and constrict in response to stimuli . Close mimicry of native blood vessels may aid in the engineering of other tissues dependent upon vasculature to sustain function . With further understanding of the factors involved in cardiovascular development and function combined with the foundation of knowledge already in place, the development of TEVGs should one day lead to improved quality of life for those with vascular disease and other life-threatening conditions.

Environ Sci Technol, 2003 Nov 1, 37(21), 4925 - 33
Post-depositional behavior of Cu in a metal-mining polishing pond (East Lake, Canada); Martin AJ et al.; The post-depositional behavior of Cu in a gold-mining polishing pond (East Lake, Canada) was assessed after mine closure by examination of porewater chemistry and mineralogy . The near-surface (upper 1.5 cm) sediments are enriched in Cu, with values ranging from 0.4 to 2 wt % . Mineralogical examination revealed that the bulk of the Cu inventory is present as authigenic copper sulfides . Optical microscopy, energy-dispersion spectra, and X-ray data indicate that the main Cu sulfide is covellite (CuS) . The formation of authigenic Cu-S phases is supported by the porewater data, which demonstrate that the sediments are serving as a sink for dissolved Cu below sub-bottom depths of 1-2 cm . The zone of Cu removal is consistent with the occurrence of detectable sulfide and the consumption of sulfate . The sediments can be viewed as a passive bioreactorthat permanently removes Cu as insoluble copper sulfides . This process is not unlike that which occurs in other forms of bioremediation, such as wetlands and permeable reactive barriers . Above the zone of Cu removal, dissolved Cu maxima in the interfacial porewaters range from 150 to 450 microg L(-1) and reflect the dissolution of a Cu-bearing phase in the surface sediments . The reactive phase is thought to be a component of treatment sludges delivered to the lake as part of cyanide treatment . Flux calculations indicate that the efflux of dissolved Cu from the sediments to the water column (14-51 microg cm(-2) yr(-1)) can account for the elevated levels of dissolved Cu in lake waters (approximately 50 microg L(-1)) . Implications for lake recovery are discussed.

Artif Organs, 2003 Nov, 27(11), 1041 - 9
Convective flow through a hollow fiber bioartificial liver; Moussy Y; A study of open shell flow through a hollow fiber bioartificial liver with a cell packed shell region (the extracapillary space between the fibers) was performed . Previous analyses of flow through a hollow fiber bioartificial liver have ignored the porous nature of the shell region . Existing analytic expressions were used to describe the flow through the porous hollow fibers, and the flow through the shell region was described using Darcy's law . The operational parameters were taken from clinical trials of the Hepatix extracorporeal liver assist device (ELAD) . From the experimental parameters, the permeability value of the cell packed shell region of the ELAD was determined . The results suggest the possible formation of preferential flow channels and hypoperfusion of many cells leading to an inadequately functioning bioreactor . Some simple design changes may improve the performance of the ELAD.

Bioprocess Biosyst Eng, 2003 Dec, 26(2), 93 - 101 Epub 2003 Nov 13.
Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor; Fan Z et al.; Conversion of paper sludge to ethanol was investigated with the objective of operating under conditions approaching those expected of an industrial process . Major components of the bleached Kraft sludge studied were glucan (62 wt.%, dry basis), xylan (11.5%), and minerals (17%) . Complete recovery of glucose during compositional analysis required two acid hydrolysis treatments rather than one . To avoid the difficulty of mixing unreacted paper sludge, a semicontinuous solids-fed laboratory bioreactor system was developed . The system featured feeding at 12-h intervals, a residence time of 4 days, and cellulase loading of 15 to 20 FPU/g cellulose . Sludge was converted to ethanol using simultaneous saccharification and fermentation (SSF) featuring a beta-glucosidase-supplemented commercial cellulase preparation and glucose fermentation by Saccharomyces cerevisiea . SSF was carried out for a period of 4 months in a first-generation system, resulting in an average ethanol concentration of 35 g/L . However, steady state was not achieved and operational difficulties were encountered . These difficulties were avoided in a retrofitted design that was operated for two 1-month runs, achieving steady state with good material balance closure . Run 1 with the retrofitted reactor produced 50 g/L ethanol at a cellulose conversion of 74% . Run 2 produced 42 g/L ethanol at a conversion of 92% . For run 2, the ethanol yield was 0.466 g ethanol/g glucose equivalent fermented and >94% of the xylan fed to the reactor was solubilized to a mixture of xylan oligomers and xylose.

Math Biosci, 2004 Jan, 187(1), 53 - 91
A survey of mathematical models of competition with an inhibitor; Hsu SB et al.; Mathematical models of the effect of inhibitors on microbial competition are surveyed . The term inhibitor is used in a broad sense and includes toxins, contaminants, allelopathic agents, etc . This includes both detoxification where the inhibitor is viewed as a pollutant and control where the inhibitor is viewed as an aid to controlling a bioreactor . The inhibitor may be supplied externally or may be created as an anti-competitor toxin . This includes plasmid-bearing, plasmid-free competition . The literature is spread across journals in different disciplines and with different notation . The survey attempts to present the mathematical models and the results of the corresponding analysis within a common framework and notation . Detailed mathematical proofs are not given but the methods of proof are indicated, references cited, and the results presented in tables . Open problems are indicated where there is a gap in the theory.

Water Res, 2003 Dec, 37(20), 4981 - 91
Determination of biodegradable dissolved organic carbon using entrapped mixed microbial cells; Khan E et al.; Several methods for determining biodegradable dissolved organic carbon (BDOC) in water have been developed within the last two decades . However, the problem with most of these methods is the length of time required for the start-up (colonization) and/or determination from days to weeks . In this study, a simple and rapid continuous bioreactor procedure using immobilized cells was developed for BDOC determination . In the first stage of the development, the bioreactors were aerated to ensure that dissolved oxygen was not rate limiting . However, BDOC results obtained from the aerated bioreactors suffered from a large error caused by the release of background dissolved organic carbon (DOC) . As a consequence, a different bioreactor scheme, in which the feed (sample) instead of the bioreactor was aerated, was tested . Results show that the feed aerated (FA) bioreactor is a better tool for BDOC determination especially for waters with low initial organic concentrations because of less background DOC released by the immobilized cell systems . Using the FA bioreactor, the accurate and reproducible measurement of BDOC can be achieved within a hydraulic retention time of 3 h, and no start-up period is required.

J Pharm Pharmacol, 2003 Sep, 55(9), 1181 - 98
Hepatocyte hollow-fibre bioreactors: design, set-up, validation and applications; Planchamp C et al.; Hepatocytes carry out many vital biological functions, such as synthetic and catabolic reactions, detoxification and excretion . Due to their ability to restore a tissue-like environment, hollow-fibre bioreactors (HFBs) show great potential among the different systems used to culture hepatocytes . Several designs of HFBs have been proposed in which hepatocytes or hepatocyte-derived cell lines can be cultured in suspensions or on a solid support . Currently the major use of hepatocyte HFBs is as bioartificial livers to sustain patients suffering from acute liver failure, but they can also be used to synthesize cell products and as cellular models for drug metabolism and transport studies . Here, we present an overview of the set-up of hepatocyte HFBs and aim to provide potential users with the basic knowledge necessary to develop their own system . First, general information on HFBs is given, including basic principles, transport phenomena, designs and cell culture conditions . The importance of the tests necessary to assess the performance of the HFBs, i.e . the viability and functionality of hepatocytes, is underlined . Special attention is paid to drug metabolism studies and to adequate analytical methods . Finally, the potential uses of hepatocyte HFBs are described.

Appl Environ Microbiol, 2003 Nov, 69(11), 6644 - 9
Diffusional properties of methanogenic granular sludge: 1H NMR characterization; Lens PN et al.; The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging . NMR spectroscopy measurements were performed at 22 degrees C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons) . Self-diffusion coefficients of H(2)O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water . Interestingly, self-diffusion coefficients of H(2)O were independent of the aggregate size for the size fractions investigated . Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55 degrees C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10 degrees C) temperatures . Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90 degrees C for 30 min) modified the diffusional transport up to 20% . In contrast, deactivation of aggregates by HgCl(2) did not affect the H(2)O self-diffusion coefficient in aggregates . Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.

Appl Environ Microbiol, 2003 Nov, 69(11), 6577 - 86
Microaerophilic cooperation of reductive and oxidative pathways allows maximal photosynthetic membrane biosynthesis in Rhodospirillum rubrum; Grammel H et al.; The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions) . R . rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose . Employing a unique partial O(2) pressure (pO(2)) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect . A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions . Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose . Fructose was utilized via the Embden-Meyerhof-Parnas pathway . High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures . No glucose-6-phosphate dehydrogenase activity was detected under any conditions . We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions . The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions . Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions . We propose a model by which the primary "switching" of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R . rubrum.

Transgenic Res, 2003 Oct, 12(5), 569 - 75
Biologically active human interferon alpha-2b produced in the egg white of transgenic hens; Rapp JC et al.; We have previously described the expression of a bacterial protein in the egg white of transgenic chickens using a replication-deficient retroviral vector . Here we report the expression of a glycosylated human protein, interferon alpha-2b (hIFN), in the egg white of transgenic hens . The hIFN secreted into the egg white was biologically active as determined by a viral inhibition assay . Purification and carbohydrate analysis of the hIFN expressed in egg white revealed that two of the six major glycosylated hIFN species match the naturally occurring human hIFN glycovariants . These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active, and glycosylated proteins in egg white.

Transgenic Res, 2003 Oct, 12(5), 541 - 53
The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins; Bosze Z et al.; Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells . Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis . Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale . This review summarizes recent research based on the transgenic rabbit model.

Bioprocess Biosyst Eng, 2003 Dec, 26(2), 109 - 16 Epub 2003 Nov 04.
Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans; Hekmat D et al.; An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented . Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process . A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells . On the basis of experimentally validated model calculations, a threshold value of about 60 kg x m(-3) DHA was obtained . The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column . In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material . Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg x m(-3) h(-1) . However, it was observed that the maximum achievable productivity had not been reached yet.

J Artif Organs, 2003, 6(2), 130 - 7
Effect of pore diameter of a porous membrane on progenitor cell content during membrane-separated coculture of hematopoietic cells and a stromal cell line; Takagi M et al.; The effect of pore diameter of a porous membrane on the progenitor cell content during a membrane-separated coculture of murine bone marrow hematopoietic cells and a murine stromal cell line, in which stromal cells adhered onto the lower surface of the membrane and hematopoietic cells were incubated on the upper surface of the membrane, was investigated in order to design a membrane bioreactor for ex vivo expansion of hematopoietic primitive cells employing exogeneic stromal cells . The hematopoietic progenitor cell {colony forming unit (CFU) Mix} content at 1 week in the membrane-separated coculture increased as the pore diameter of the membrane decreased from 12.0 to 0.4 microm . However, a further decrease in pore diameter from 0.4 to 0.1 microm did not affect the CFU-Mix content . Observation of stromal cells that adhered on the lower surface of the porous membranes (0.4 and 3.0 microm pore diameters) under a confocal scanning microscope after staining with rhodamine phalloidin suggested that stromal cells can migrate to the membrane's upper surface through pores with a diameter greater than 3.0 microm . Consequently, membrane having small (< or = 0.4 microm) pores that stromal cells could not pass through prevented direct contact between stromal and hematopoietic cells, which resulted in a higher content of hematopoietic progenitor cells (CFU-Mix) during the membrane-separated coculture.

Bioresour Technol, 2004 Jan, 91(2), 135 - 40
Reuse of microbially treated olive mill wastewater as fertiliser for wheat (Triticum durum Desf.); Fausto Cereti C et al.; Free cells of Aspergillus niger were grown on olive mill wastewater (OMW) supplemented with rock phosphate (RP) in an air-lift bioreactor in batch and repeated-batch processes . The fungus grew well and reduced the chemical oxygen demand of the waste by 35% and 64% in the batch and repeated-batch (fourth batch) processes, respectively . Total sugar content was consistently reduced (ca . 60%) in both processes while reduction of total phenols was minimal . RP was solubilised and maximum soluble P was 0.63 and 0.75 gl(-1) in the batch and repeated-batch (third batch), respectively . Several types of OMW+/-RP, microbially-treated or not, were tested in a greenhouse for their fertilising ability on a soil-wheat (Triticum durum Desf.) model system . Beneficial effects were highest using OMW treated by the repeated-batch process . The treated plants showed an increase in seed biomass, spike number, and kernel weight . Harvest index was highest (0.49+/-0.04) after treatment with OMW from the repeated-batch process.

Biotechnol Appl Biochem . 2003 Oct 31; {Epub ahead of print}
Shear stress preconditioning and tissue engineering based paradigms for generating arterial substitutes; Baguneid M et al.; To evaluate the impact shear stress preconditioning and surface modification, using adhesive moieties, have on a novel tissue engineered graft (TEG) . Preconditioning of these constructs within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), was performed to establish whether low shear stress conditions promoted endothelial cell (EC) retention and differentiation . TEGs (n=24) were generated by the contraction and compaction of collagen (I) by porcine aortic smooth muscle cells (SMCs) onto a compliant polyester graft scaffold . EC were radiolabeled with 111Indium-tropolonate and seeded onto the luminal surface of the TEGs . Following organ culture in a bioreactor (7 days), TEGs were split into 4 groups (n=6 per each group): Group A acted as controls with TEGs unmodified and seeded with radiolabeled ECs; Group B underwent luminal pre-coating with fibronectin (FN) (75mg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 1-2 dyn/cm 2 for 7 days prior to EC seeding and Group D TEGs were preconditioned for 7 days at 1-2 dyn/cm 2 followed by luminal pre-coating with FN prior to EC seeding . The resistance to physiological shear stress of the seeded ECs was assessed using a gamma counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 9.32 dynes/cm 2 . Environmental scanning electron microscopy (ESEM) was used to determine distribution and degree of differentiation of the attached ECs and tissue Plasminogen Activator (tPA) assays provided a measure of function and viability.

Biomech Model Mechanobiol, 2002 Jun, 1(1), 69 - 82
Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment; Raimondi MT et al.; Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage . Fluid-induced shear is known to enhance chondrogenesis on animal cells . A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems . We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure . A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described . The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 x 10(-3) Pa (0.03 dyn/cm(2)) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 mum/s . Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure.

Biotechnol Lett, 2003 Oct, 25(19), 1581 - 90
Replacement of immobilised cell bioreactors by smaller immobilised enzyme bioreactors: unique-outcome predictability for cytochromes P450 isoforms?
Wiseman A.
Both immobilized enzymes (IME) and immobilized cells (IMC) are acceptable as the biocatalysts essential for the attainment of rapid rates of bioconversion in bioreactors . IMC can display higher than expected cellular permeability whilst IME can exhibit high catalytic constant (kcat/Km) despite limitations on substrate utilisation due to an unstired diffusion layer of solvent . Scale-down switching from IMC to IME involves the replacement of high-volume biotechnology by low-volume biotechnology, sometimes using IME mimics in partially non-aqueous solvent systems . Highly purified IME systems covalently immobilised to particles of, for instance, microcrystalline cellulose or porous glass, can retain both the hydrophilic and hydrophobic intermediate products in situ of the chosen sequence of enzyme reactions . These bioconversions, therefore, are as efficient as those with IMC where enzymes are often particle- or membrane-bound so that even hydrophilic intermediates are not released rapidly into solution . This mimicry of in vivo biosynthetic pathways that are compartmentalised in vivo (e.g . of lysosomes, mitochondria and endoplasmic reticulum) can replace larger IMC by IME especially in application of up to 2700 cytochromes P450 isoforms in bioprocessing . In silico investigation of appropriate model IME systems, in comparison with IMC systems, will be needed to define the optimal bioreactor configuration and parameters of operation, such as pH, T and oxygen mass transfer rate (OTR) . The application solely of hazop (applied hazard and operability concepts) may, nevertheless, not be recommended to replace fully the in silico and real-lab pilot-scale and scale studies . Here, food-safe bioprocessing has to be achieved without incorporation of recognised biohazards; especially in the form of unacceptable levels of toxic metals that promote a risk-analysis uncertainty.

Ann Biomed Eng, 2003 Oct, 31(9), 1017 - 30
Bioreactors for cardiovascular cell and tissue growth: a review; Barron V et al.; Heart disease is a major cause of death in the Western world . In the past three decades there has been a number of improvements in artificial devices and surgical techniques for cardiovascular disease; however, there is still a need for novel devices, especially for those individuals who cannot receive conventional therapy . The major disadvantage of current artificial devices lies in the fact that they cannot grow, remodel, or repair in vivo . Tissue engineering offers the possibility of developing a biological substitute material in vitro with the inherent mechanical, chemical, biological, and morphological properties required in vivo, on an individual patient basis . In order to develop a true biological cardiovascular device a dynamic physiological environment needs to be created . One approach that employs the use of a simulated biological environment is a bioreactor in which the in vivo biomechanical and biochemical conditions are created in vitro for functional tissue development . A review of the current state of the art bioreactors for the generation of tissue engineered cardiovascular devices is presented in this study . The effect of the simulated physiological environment of the bioreactor on tissue development is examined with respect to the materials properties of vascular grafts, heart valves, and cardiac muscles developed in these bioreactors.

J Gen Appl Microbiol, 2003 Aug, 49(4), 235 - 43
Effect of support materials on antibiotic MSW2000 production by immobilized Streptomyces violatus; el-Naggar MY et al.; The production of an antibiotic by free and immobilized cells of Streptomyces violatus through entrapment or adsorption on different materials was investigated . S . violatus entrapped in Ca-alginate beads gave low antibiotic activity compared to the free cell or adsorbed cell, while the adsorption of S . violatus on sponge cubes yielded the highest antibiotic concentration after 4 days of incubation in static cultures . A starch concentration of 10 g/L was optimum for the production of the antibiotic by adsorbed cells . The weight and size of the sponge cubes used for immobilization influenced production of the antibiotic and the optimum weight and size of the sponge were 0.8 g and 1.0 cm(3), respectively, yielding a maximum antibiotic production of 280 mg/ml . Maximum antibiotic production was obtained at an initial pH value of 7.5 and in an inoculum size of 3 ml (spore suspension) per 50 ml . The production of the antibiotic in a fixed-bed bioreactor reached a maximum value after 2 days of incubation at a circulation rate of 30 ml/h . The immobilized cells in the bioreactor were reused seven successive times over a period of 14 days.

J Gen Appl Microbiol, 2003 Aug, 49(4), 219 - 33
Theoretical and methodological studies of continuous microbial bioreactors; Toda K; This article reviews most of the author's studies on process development and reactor design for continuous microbial reactions . (1) Enzyme reactions of growing and non-growing microbial cells immobilized in agar gel beads were analyzed pertaining to the effects of external and internal diffusion of substrate on reaction kinetics . (2) Experimental correlations of production rates of beta-fructosidase and acid phosphatase with dilution rate of continuous culture were simulated based on an operon model for enzyme regulation . (3) Population dynamics of an amylase-producing bacteria and their mutant were discussed in relation to enzyme productivity in a continuous culture of spore-forming bacteria . (4) Plasmid mobilization in a mixed population of donor, recipient, and helper cells was investigated in a continuous culture as a model study of accidental release of a genetically modified plasmid into a natural environment . (5) A production rate increase of up to 100-fold was achieved by cell-recycle culturing of continuous acetic acid fermentation using a filter module with a hollow fiber membrane . (6) The feasibility of a continuous surface culture for the biooxidation of organic substances was ascribed to an enhanced oxygen absorption rate in the presence of a microbial film on a liquid surface . (7) Simultaneous separation of inhibitory products using an electrodialysis module during some organic acid fermentations was effective for increasing production in a continuous culture.

J Biotechnol, 2003 Nov 6, 105(3), 205 - 13
Production of supercoiled multimeric plasmid DNA for biopharmaceutical application; Voss C et al.; Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance . Non-viral vectors like plasmid DNA are currently investigated in various clinical trials . Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors . A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented . Cultivation of these clones on semi-defined glycerol medium in a 7 l bioreactor shows structural stability of dimers and trimers during the whole cultivation process . Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer . Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers.

Curr Opin Biotechnol, 2003 Oct, 14(5), 468 - 74
Bioreactor state estimation and control; Komives C et al.; Advanced control methods have been effectively employed for industrial chemical processing for decades . Only recently, however, have model-based strategies been implemented for biological processes . Some notable advances include the enhancement of metabolic flux models to describe the dynamic behavior observed in biochemical reactors . The combination of more than one type of model in a hybrid form was shown to perform well for bioprocess control applications.

Biotechnol Appl Biochem . 2003 Oct 28; {Epub ahead of print}
A convenient method for identification and expression of an eukaryotic gene; Hu R et al.; In order to identify whether the product of a genetically modified or newly isolated mammalian gene has biological activity, the gene of interest is usually subcloned into a mammalian expression vector and then expressed in an in vitro system such as in tissue culture . In this study an efficient in vivo system has been developed by employing a mammary gland specific vector and expressing the targeted protein in the lactating goat mammary glands . In this system, the synthesized lumbrokinase cDNA variant (LK-m) and the tissue-type plasminogen activator (tPA) cDNA were selected as genes of interest and cloned downstream the goat beta-casein regulatory sequence . The LK-m and tPA expressing plasmids were prepared in high purity and injected into lactating goat mammary gland tissues in amount of 100-800mug . High level expression of the lumbrokinase and tPA was detected as indicated in fibrin lysis activity by fibrin agarose plate assay . Dynamic study showed that the specific expression starts immediately after injection, generally reaches peak in 6-9 hours, persists for 20-24 hours at peak and the expression lasts for four days with gradual decline in quantities . The potential use of this system as bioreactor for production of biological proteins in substitution of construction of transgenic animals is implicated from this study.

Z Naturforsch {C}, 2003 Sep-Oct, 58(9-10), 726 - 31
Biodegradation of naphthalene by free and alginate entrapped Pseudomonas sp; Abou Seoud M et al.; Naphthalene degradation by freely suspended and immobilized cells of Pseudomonas sp . isolated from contaminated effluents has been investigated in batch cultures and continuously in a packed bed reactor . Naphthalene concentration was varied from 25 mM to 75 mM, the temperature (30 degrees C) and pH (7.0) were kept constant . The results showed good acclimation of the strain to carbon source and degradation rate was highly affected by initial concentration . Alginate-entrapped cells have given good yields although initial rates were not as high as those encountered with free cells . A first order exponential decay kinetic model was proposed with values of parameters for each initial concentration . A laboratory scale packed-bed bioreactor was designed using parameters calculated above and continuous experiments were realized at different flow rates (100 to 200 ml/h), with different feed concentrations and operating during 30 days . The conversion at low feed concentrations and low flow rates was complete whereas at high flow rates and high concentrations it was less efficient because of diffusional limitations and short residence time.

Chemosphere, 2004 Jan, 54(3), 283 - 9
Bioleaching of heavy metals from livestock sludge by indigenous sulfur-oxidizing bacteria: effects of sludge solids concentration; Chen SY et al.; A technologically and economically feasible process called bioleaching was used for the removal of heavy metals from livestock sludge with indigenous sulfur-oxidizing bacteria in this study . The effects of sludge solids concentration on the bioleaching process were examined in a batch bioreactor . Due to the buffering capacity of sludge solids, the rates of pH reduction, ORP rise and metal solubilization were reduced with the increase of the solids concentration . No apparent influence of solids concentration on sulfate produced by sulfur-oxidizing bacteria was observed when the solids concentration was less than 4% (w/v) . A Michaelis-Menten type of equation was able to well describe the relationship between solids concentration and rate of metal solubilization . Besides, high efficiencies of metal solubilization were achieved after 16 d of bioleaching . Therefore, the bioleaching process used in this study could be applied to remove heavy metals effectively from the livestock sludge.

Biotechnol Bioeng, 2003 Dec 5, 84(5), 591 - 6
Effect of mixing rate on beta-carotene production and extraction by dunaliella salina in two-phase bioreactors; Hejazi MA et al.; beta-Carotene has many applications in the food, cosmetic, and pharmaceutical industries; Dunaliella salina is currently the main source for natural beta-carotene . We have investigated the effect of mixing rate and whether it leads to the facilitated release of beta-carotene from the cells of Dunaliella salina in two-phase bioreactors . Three pairs of bioreactors were inoculated at the same time, operated at 100, 150, and 170 rounds per minute, respectively, and illuminated with a light intensity of 700 micromol m(-2) s(-1) . Each pair consisted of one bioreactor containing only aqueous phase for the blank and one containing the water phase together with dodecane, which is biocompatible with the cells . Comparison of the viability and growth of the cells grown under different agitation rates shows that 170 rpm and 150 rpm are just as good as 100 rpm . The presence and absence of the organic phase also has no influence on the viability and growth of the cells . In contrast to the growth rate, the extraction rate of beta-carotene is influenced by the stirrer speed . The extraction rate increases at a higher stirring rate . The effectiveness of extraction with respect to power input is comparable for all the applied mixing rates, even though it is slightly lower for 100 rpm than the others . The chlorophyll concentration in the organic phase remained very low during the experiment, although at higher mixing rates, chlorophyll impurity increased up to 3% (w/w) of the total extracted pigments . At 170 rpm carotenoid and chlorophyll undergo the highest extraction rate for both pigments-0.5% of the chlorophyll and 6% of the carotenoid is extracted .

Biotechnol Bioeng, 2003 Dec 5, 84(5), 583 - 90
Primmorphs from archaeocytes-dominant cell population of the sponge hymeniacidon perleve: improved cell proliferation and spiculogenesis; Zhang X et al.; Marine sponges (Porifera) possess an extraordinary diversity of bioactive metabolites for new drug discovery and development . In vitro cultivation of sponge cells in a bioreactor system is very attractive for the sustainable production of sponge-derived bioactive metabolites; however, it is still a challenging task . The recent establishment of sponge primmorphs, multicellular aggregates from dissociated mixed-cell population (MCP), has been widely acknowledged to hold great promise for cultivation in vitro . Here we present a new method to establish an in vitro sponge primmorph culture from archaeocyte-dominant cell population (ADCP) enriched by a Ficoll gradient, rather than a mixed-cell population (MCP) . Our rationale is based upon the totipotency (the ability of a cell to differentiate into other cell types) of archaeocyte cells and the different biological functions of various sponge cell types . A sponge, Hymeniacidon perleve collected from the China Yellow Sea was used as a model system for this investigation . Distinct dynamics of primmorph formation were observed while significant increases in DNA synthesis, cell proliferation (up to threefold), and cell growth (up to fourfold) were achieved . Furthermore, a time-dependent spiculogenesis was clearly demonstrated in our longterm culture, indicating high metabolic activity of primmorphs from the ADCP . This new method represents an important step forward to advance sponge cell culture in vitro that may lead to commercial exploitation of sponge-derived drugs .

Biotechnol Bioeng, 2003 Dec 5, 84(5), 518 - 32
Organic tissues in rotating bioreactors: fluid-mechanical aspects, dynamic growth models, and morphological evolution; Lappa M; This analysis deals with advances in tissue-engineering models and computational methods as well as with novel results on the relative importance of "controlling forces" in the growth of organic constructs . Specifically, attention is focused on the rotary culture system, because this technique has proven to be the most practical solution for providing a suitable culture environment supporting three-dimensional tissue assemblies . From a numerical point of view, the growing biological specimen gives rise to a moving boundary problem . A "volume-of-fraction" method is specifically and carefully developed according to the complex properties and mechanisms of organic tissue growth and, in particular, taking into account the sensitivity of the construct/liquid interface to the effect of the fluid-dynamic shear stress (it induces changes in tissue metabolism and function that elicit a physiological response from the biological cells) . The present study uses available data to introduce a set of growth models . The surface conditions are coupled to the transfer of mass and momentum at the specimen/culture-medium interface and lead to the introduction of a group of differential equations for the nutrient concentration around the sample and for the evolution of tissue mass displacement . The models are then used to show how the proposed surface kinetic laws can predict (through sophisticated numerical simulations) many of the known characteristics of biological tissues grown using rotating-wall perfused vessel bioreactors . This procedure provides a validation of the models and associated numerical method and also gives insight into the mechanisms of the phenomena . The interplay between the increasing size of the tissue and the structure of the convective field is investigated . It is shown that this interaction is essential in determining the time evolution of the tissue shape . The size of the growing specimen plays a critical role with regard to the intensity of convection and the related shear stresses . Convective effects, in turn, are found to impact growth rates, tissue size, and morphology, as well as the mechanisms driving growth . The method exhibits novel capabilities to predict and elucidate experimental observations and to identify cause-and-effect relationships .

Chin Med J (Engl), 2003 Oct, 116(10), 1471 - 4
Metabolic effects of a novel bioartificial liver on serum from severe hepatitis patients: an in vitro study; Yan F et al.; OBJECTIVE: To establish a novel bioartificial liver (BAL) consisting of spheroids of porcine hepatocytes in a hollow-fiber bioreactor, and to perform an in vitro study on its metabolic effects on the serum from severe hepatitis B patients . METHODS: Hepatocytes were isolated from pup pigs and cultured as aggregate spheroids through rotation and vibration . Phase-contrast microscopy, transmission electron microscopy, and scanning electron microscopy were used for morphological detection of hepatocyte spheroids . The hepatocyte spheroids were then transferred into the shell of a polysulfone hollow-fiber bioreactor, creating a novel BAL . Diluted serum samples of severe hepatitis B patients were circulated for 3 hours each into the bioreactor, by using an extracorporeal circulatory system . Every half hour, including both before and after perfusion, serum samples were collected to assay total bilirubin (TBIL), total protein (TP), albumin (ALB), and globulin (GLB) concentrations in order to judge the metabolic effects of this novel BAL . RESULTS: Most hepatocytes had formed spheroids with high viability after 24 hours in culture . After 3 hours of perfusion, when compared with the control group, the serum concentration of TBIL in the treatment group decreased significantly (P < 0.01), but the serum concentrations of TP and ALB increased significantly (P < 0.05) . CONCLUSIONS: Hepatocytes can be conveniently cultured as aggregate spheroids through a rotation and vibration method . The novel BAL is efficient in removing bilirubin from the serum of severe hepatitis B patients, and in supplying the serum with ALB . Thus, the BAL might provide effective therapy for patients with severe hepatitis B.

Cancer Immunol Immunother, 2004 Feb, 53(2), 92 - 9 Epub 2003 Oct 18.
Immunization with a plant-produced colorectal cancer antigen; Verch T et al.; Cancer vaccination has become an important focus of oncology in recent years . Active immunization with tumor-associated antigens such as colorectal cancer antigen GA733-2 is thought to potentially overcome the reoccurrence of metastasis . As recombinant protein production in bioreactors is costly and subject to growing safety concerns, we tested plants as an alternative for the expression of a potential colorectal cancer vaccine . Comparing colorectal cancer antigen GA733-2 produced in tobacco plants with the same antigen produced in insect cell culture, we found a similar humoral immune response to injection of either of the two antigen preparations into mice . Some minor differences were observed in the cellular response that might be due to impurities . Our studies compare for the first time, immunization with the same antigen expressed in either plants or insect cell culture . This will provide important data for use of plants as production systems of therapeutics.

Eur Cell Mater, 2003 Oct 01, 6, 1 - 10; discussion 10-1
A bone fluid flow hypothesis for muscle pump-driven capillary filtration: II . Proposed role for exercise in erodible scaffold implant incorporation; Winet H; A model is presented for enhancement of fluid flow through bone matrix and any porous tissue engineering scaffold implanted within it . The mechanism of enhancement is the skeletal muscle pump in compartments adjacent to the bone . Pressure waves from muscle pump contractions aided by increased blood pressure during exercise coupled with temporary occlusion of arteries leading to and veins from the bone, increase hydraulic pressure in cortical bone capillaries so as to amplify capillary filtration . It is proposed that capillary filtration increase is sufficiently convective to contribute to bone fluid flow and associated percolation through tissue engineered scaffold matrix implants . Importance of this contribution is its relative role in maintaining seeded cells in bioreactor scaffolds . Validation of the hypothesis starts at a minimum level of demonstrating that capillary filtration is convective . At a maximum level confirmation of the hypothesis requires demonstration that capillary filtration-based interstitial flow is sufficient to stimulate not only host bone cells (as proposed in part I of the hypothesis) but bioreactor-seeded cells as well . Preliminary data is presented supporting the prediction that skeletal muscle contraction generates convective capillary filtration.

Biomaterials, 2003 Dec, 24(27), 5009 - 14
Pulsatile perfusion and cardiomyocyte viability in a solid three-dimensional matrix; Kofidis T et al.; BACKGROUND: The manufacture of full thickness three-dimensional myocardial grafts by means of tissue engineering is limited by the impeded cellular viability in unperfused in vitro systems . We introduce a novel concept of pulsatile tissue culture perfusion to promote ubiquitous cellular viability and metabolism . METHODS: In a novel bioreactor we established pulsatile flow through the embedded three-dimensional tissue culture . Fibrin glue served as the ground matrix wherein neonatal rat cardiomyocytes were inoculated . Fluor-Deoxy-Glucose-Positron-Emission-Tomography (FDG-PET) and life/dead assays were employed for comparative studies of glucose uptake resp . cell viability . RESULTS: A solid 8 mm thick structure resulted . Cellular viability significantly increased in the perfused chambers . We observed centripetal migration of the embedded cardiomyocytes to the site of the core vessel . However, cellular viability was high in the periphery of the tissue block too . FDG-PET revealed enhanced metabolic activity in perfused chambers . CONCLUSIONS: The present concept is highly effective in enhancing cellular viability and metabolism in a three-dimensional tissue culture environment . It could be utilized for various co-culture systems and the generation of viable tissue grafts.

Appl Microbiol Biotechnol, 2004 Apr, 64(2), 192 - 8 Epub 2003 Oct 11.
Homogeneous batch cultures of Aspergillus oryzae by elimination of wall growth in the Variomixing bioreactor; Larsen B et al.; A novel principle for mixing and aeration in stirred bioreactors, named Variomixing, was developed . Four baffles are rotated intermittently at a rotational speed slower or similar to the speed of a centrally placed axial flow impeller . Rotational speeds of the baffles and impeller of 5-10 and 500-600 rpm, respectively, results in the highly turbulent flow regime characteristic of conventional bioreactors with high mixing and mass transfer capacities . Stagnant zones around crevices and crannies in which wall growth may commence are avoided since the baffles are never completely at rest . Increasing the rotational speed of the baffles (5 s every 5 min), so that it follows the speed of the impeller (500-600 rpm), cancels the effect of the baffles and a deep vortex and high peripheral liquid flow rates at the reactor wall develop . The vortex ensures that also the head-space of the reactor wall is flushed and any deposits removed . The filamentous fungus Aspergillus oryzae has been grown in batch cultures in the Variomixing bioreactor . Compared to conventional laboratory-scale bioreactors, in which more than 30% of all biomass was found attached to walls, less than 2% of the total A . oryzae biomass was found on the walls in the Variomixing bioreactor.

Environ Toxicol Chem, 2003 Oct, 22(10), 2280 - 92
Comparison of synthetic surfactants and biosurfactants in enhancing biodegradation of polycyclic aromatic hydrocarbons; Makkar RS et al.; Polycyclic aromatic hydrocarbon (PAH) contamination of the environment represents a serious threat to the health of humans and ecosystems . Given the human health effects of PAHs, effective and cost-competitive remediation technologies are required . Bioremediation has shown promise as a potentially effective and low-cost treatment option, but concerns about the slow process rate and bioavailability limitations have hampered more widespread use of this technology . An option to enhance the bioavailability of PAHs is to add surfactants directly to soil in situ or ex situ in bioreactors . Surfactants increase the apparent solubility and desorption rate of the PAH to the aqueous phase . However, the results with some synthetic surfactants have shown that surfactant addition can actually inhibit PAH biodegradation via toxic interactions, stimulation of surfactant degraders, or sequestration of PAHs into surfactant micelles . Biosurfactants have been shown to have many of the positive effects of synthetic surfactants but without the drawbacks . They are biodegradable and nontoxic, and many biosurfactants do not produce true micelles, thus facilitating direct transfer of the surfactant-associated PAH to bacteria . The results with biosurfactants to date are promising, but further research to elucidate surfactant-PAH interactions in aqueous environments is needed to lead to predictive, mechanistic models of biosurfactant-enhanced PAH bioavailability and thus better bioremediation design.

Biotechnol Adv, 1991, 9(3), 411 - 24
Immobilization of animal cells in fixed bed bioreactors; Lazar A; Immobilization of animal cells has become a highly popular means of achieving high-density animal cell cultures . The advantages of immobilization are that it stabilizes cells in culture and enables long-term culture periods to be achieved . Immobilization increases cell productivity by increasing the usable substrate surface area for anchorage-dependent cells, or by facilitating perfusion of anchorage-independent cells . A method for production of secreted biological products from anchorage-dependent and independent cells is described . The method is based on immobilization of animal cells within the polymeric matrix of polyurethane foam, packed in a fixed bed bioreactor.

Biotechnol Adv, 1994, 12(3), 467 - 87
High density cell culture by membrane-based cell recycle; Chang HN et al.; Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture . Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors . This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation . A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented . Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations.

Biotechnol Adv, 1994, 12(1), 1 - 39
Rheology of filamentous fermentations; Olsvik E et al.; The performance of a bioreactor containing a filamentous fermentation broth is greatly influenced by the rheological properties of the broth . These properties are determined mainly by the concentration of biomass, its growth rate and morphology . Included in the morphology are such factors as the geometry of hyphae (length, diameter, branching frequency), hyphal flexibility and hyphal-hyphal interactions, which can all be affected by the operational design of the reactor . Thus, correlations describing viscosity as a function of biomass only are of limited value . A better understanding of the relations between morphology and rheology may be achieved by a combination of rheological and morphological studies.Rheological properties are normally determined using off-line measurements in-spite of associated problems with sample treatment influencing the results . Equipment for dynamic, on-line, measurement of morphology and rheology is available, but little used in filamentous fermentations . Controlling the rheological properties of mycelial fermentations may be difficult because of the great number of factors influencing mycelial development and/or hyphal-hyphal interactions.Polymer solutions are often used to simulate flow behaviour of filamentous fermentations and scale-up and mass transfer considerations are based on these studies . Although much information has been gained this way, the predictions developed do not include the effect of an active biomass on the mass transfer and flow properties of the culture . It is important to carry out studies on the non-homogeneous fermentation fluids, and develop correlations based on these studies.

Biotechnol Adv, 1993, 11(3), 611 - 20
Gas concentration and temperature gradients in a packed bed solid-state fermentor; Gowthaman MK et al.; In solid-state fermentation (SSF), interaction of heat and mass transfer with biochemical reaction (growth associated enzyme production) affects the bioreactor performance . This interaction was earlier observed to cause temperature and gaseous concentration gradients which reduced the effective bed height of the bioreactor . Since forced aeration is known to alleviate this problem, a packed column bioreactor with forced aeration was employed in the present study . Using wheat bran and Aspergillus niger CFTRI 1105, experiments were conducted for the production of the enzyme amyloglucosidase at various air flow rates . Temperatures and gas concentrations were recorded and enzyme activities estimated at different bed heights during the course of SSF . Gas concentration and temperature gradients decreased with increasing air flow rate . The packed column allowed the use of larger bed heights and yielded higher enzyme activities (6,260 Units/gDMB) than trays (345 Units/gDMB) . Enzyme activity was affected more by temperature than concentration gradients, and increased with air flow rates.

Biotechnol Adv, 1993, 11(3), 599 - 610
Operational parameters for packed beds in solid-state cultivation; Gumbira-Sa'id E et al.; Packed bed cultivation systems have potential for widespread application in solid-state cultivation (SSC), but they are poorly characterized . The effects of particle size and substrate loading on the growth of Rhizopus oligosporus on sago-beads in packed bed bioreactors were investigated . Pressure drop and protein were monitored as indicators of fungal growth in cultivations performed in a large column (4.9 cm internal diameter and 60 cm height) and a system of small columns (4.2 cm internal diameter and 5.2 cm height) . The differential pressure drop increased to a maximum between 34 and 44 h and then decreased again . The maximum differential pressure drop attained was greatest for the smallest particle size and for the lower substrate loadings . However, since the protein content continued to increase throughout the cultivation, pressure drop could not be used to monitor growth directly.

Biotechnol Adv, 1993, 11(2), 279 - 99
Animal cells in turbulent fluids: details of the physical stimulus and the biological response; Cherry RS; Animal cells in large scale bioreactors are subjected to a variety of fluid forces for which they are not adapted by evolution . In severe cases the result is cell death, but under more modest agitation conditions an increasing number of nonlethal responses affecting growth rate, metabolism, and product formation have been reported . The forces causing these responses have not been characterized because particle-turbulence interactions are extremely complex . The current understanding of the microscopic structure of turbulence in an infinite liquid and in boundary layers shows that an average shear stress alone is not likely to be adequate to describe the bioreactor environment . Combining knowledge of the physical stimuli and the biological responses will lead to better ways of limiting cell damage and possibly to using physical stresses as a means of specifically modifying cell behavior.

Biotechnol Adv, 1983, 1(1), 59 - 71
Biotechnology developments for the treatment of toxic and inhibitory wastewaters; Hall ER et al.; The cost-effectiveness of biological processes has encouraged many researchers to consider biotreatment for the stabilization of toxic or recalcitrant wastewaters . However, to ensure adequate removal of trace contaminants and satisfactory performance with high strength inhibitory industrial wastewaters, conventional biotechnology is being re-evaluated . This review summarizes selected recent contributions to the development of appropriate biotechnology for toxic wastewater treatment . Microbiological constraints and potential solutions are examined . Assessments of conventional biological processes for contaminant control are reviewed, and several new developments in bioreactor design for inhibitory wastes are presented.

Biotechnol Adv, 1991, 9(1), 51 - 68
Production scale insect cell culture; Agathos SN; Insect cells in culture are currently commanding great interest as superior hosts for the efficient production of biologicals with applications in health care and in agriculture . Insect cell culture is ripe for scale-up technologies, in order to meet future projected production requirements of (a) insect viruses used as bioinsecticides and (b) recombinant proteins of therapeutic potential for humans and animals . The single most prominent system used in research-based and in commercial insect cell culture today involves lepidopteran cells transfected with baculovirus expression vectors for abundant formation of recombinant biologicals . However, dipteran insect cell lines also are beginning to emerge as useful tools in biotechnology . Current practices in bioprocess development using insect cell culture, advances in media formulation and in insect cell bioreactor design, and emerging trends are presented and critically evaluated.

Biotechnol Adv, 1990, 8(4), 763 - 83
Immobilized mammalian cell cultivation in hollow fiber bioreactors; Piret JM et al.; Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing . One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs) . These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs . Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter . Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.

Biotechnol Adv, 1987, 5(1), 129 - 45
Membrane bioreactors: Engineering aspects; Chang HN; Membrane bioreactors have in-situ separation capability lacking in other types of immobilized cell reactors . This makes them very useful for certain systems . Enzyme reactions utilizing cofactors and hydrolysis of macromolecules are advantageous in membrane reactors . Anaerobic cell culture may be efficiently carried out in membrane cell recycle systems, while aerobic cultures work well in dual hollow fiber reactors . Animal and plant cells have much a better chance of success in membrane reactors because of the protective environment of the reactor and the small oxygen uptake rate of these cells.

Biotechnol Adv, 1989, 7(4), 527 - 75
Substrate inactivation of enzymes in vitro and in vivo; Gray MR; Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction . These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process . The first type is enzymes which use molecular oxygen as a substrate . The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity . The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins . The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups . Sub classes are defined within each broad classification based on kinetics and stoichiometry . Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes . The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type . The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.

Biotechnol Adv, 1986, 4(2), 207 - 18
Fed-batch culture technology; Minihane BJ et al.; The use of fed-batch procedures offers distinct advantages over other modes of operation of bioreactors, and is a widely researched technique . These advantages are discussed; some uses of fed-batch procedures and the associated methods of modelling and control are reviewed.

Biotechnol Adv, 1985, 3(1), 39 - 63
Fuels and industrial chemicals through biotechnology . 2; Linko P; An overview of research on the biotechnical production of fuels and industrial chemicals during the two-year period of 1983-1984 is presented . Ethanol fermentation has continued to be the subject of major interest . A considerable amount of work has been directed to alternative feedstocks such as pentose sugars and lactose, and to bacterial fermentations . Reports on extrusion cooking as a continuous pretreatment method for subsequent ethanol fermentation, and on novel alternative downstream processing techniques have been published . In addition to ethanol fermentation, much attention has been paid to the biotechnical production of 2,3-butanediol, and of a number of organic and amino acids . In general, there appears to be a growing interest in the application of biocatalysis for the production of specialty chemicals, although only a few examples will be discussed in this paper . The construction of a demonstration plant to produce ethanol from molasses by a two 10 kL bed-volume immobilized yeast bioreactors at the Kyowa Hakko Kogyo Company Hofu plant, the announcement by Nitto Chemical Industries Company to begin the biotechnical production of acrylamide, and the French decision to construct pilot plants for the biotechnical production of acetone-butanol-ethanol cosolvent and of ethanol from renewable resources represent major scale-up developments.

Biotechnol Adv, 2000 Jul, 18(4), 303 - 19
Microencapsulation of microbial cells; Park JK et al.; The high level of biocatalysts such as microbial cells and enzymes plays an important role in increasing the productivity of a bioreactor . The beads entrapped with microbial cells are not strong enough for long-term use . The small void space of polymer matrix and the leakage of cells limit a final cell loading in the beads . The recent success of encapsulating microbial cells makes it possible to prepare dense biocatalyst composed of recombinant microbial cells . In addition to encapsulating microbial cells, immobilization of animal and plant cells in capsules is also briefly described.

Biotechnol Adv, 2001 Nov, 19(7), 525 - 38
Two-phase partitioning bioreactors in fermentation technology; Malinowski JJ; The two-phase partitioning bioreactor concept appears to have a great potential in enhancing the productivity of many bioprocesses . The proper selection of an organic solvent is the key to successful application of this approach in industrial practice . The integration of fermentation and a primary product separation step has a positive impact on the productivity of many fermentation processes . The controlled substrate delivery from the organic to the aqueous phase opens a new area of application of this strategy to biodegradation of xenobiotics . In this review, the most recent advances in the application of two-liquid phase partitioning bioreactors for product or substrate partitioning are discussed . Modeling and performance optimization studies related to those bioreactor systems are also reviewed.

J Environ Qual, 2003 Sep-Oct, 32(5), 1650 - 7
Removal of selenate in simulated agricultural drainage water by a rice straw bioreactor channel system; Zhang Y et al.; Removal of selenium (Se) from agricultural drainage water is important in protecting wetland wildlife . Three flow-through bioreactor channel systems (BCSs), each with three channels filled with rice (Oryza sativa L.) straw, were set in the laboratory to determine removal of selenate {Se(VI)} (1020 microg L(-1)) from drainage water with a salinity of 10.4 dS m(-1), a pH of 8.1, and a nitrate (NO3-) range of 0 to 100 mg L(-1) . Results showed that the rice straw effectively reduced Se(VI) during 122 to 165 d of the experiments . Calculation of Se mass in the three BCSs showed that 89.5 to 91.9% of the input Se(VI) was reduced to red elemental Se {Se(0)}, where 96.6 to 98.2% was trapped in the BCSs . Losses of each gram of rice straw were almost equal to the removal of 1.66 mg of Se from the drainage water as a form of red Se(0), indicating that rice straw is a very effective organic source for removing Se(VI) from drainage water.

Water Sci Technol, 2003, 48(4), 169 - 74
Semi-continuous anaerobic digestion of food waste using a hybrid anaerobic solid-liquid bioreactor; Wang JY et al.; A hybrid anaerobic solid-liquid (HASL) bioreactor was developed to enhance food waste conversion . The HASL bioreactor is a modified two-phase anaerobic digestion system with a solid waste reactor and a high-rate anaerobic wastewater reactor, which was a UASB reactor . In this study, the methanogenesis in the UASB reactor was investigated under a semi-continuous operation of the food waste digestion system . The HASL process, including 7 days of start-up and 23 days of semi-continuous operation followed by 6 days of batch operation, was commenced by loading 2.8 kg of shredded food waste . During the semi-continuous operation, 0.2 kg of the same waste was loaded daily . The leachate from acidification phase, i.e., solid waste reactor, remained acidic (pH 4.9-5.5) and high in total VFA (TVFA), 9,500-11,500 mg/L, and COD (8,000-11,800 mg/L) levels . In the methanogenesis phase, i.e., UASB reactor, effective TVFA and COD removals (88 +/- 5% and 85 +/- 7%, respectively) were obtained, while the methane content was 71% . At the end of operation, about 78% of VS added in the HASL bioreactor was removed, while TOC and total COD reductions were 78% and 79%, respectively . The results showed that the use of UASB reactor in the semi-continuous HASL system can enhance the methanogenesis process and increase the methane content in biogas production.

Water Sci Technol, 2003, 48(4), 129 - 38
Co-digestion of primary sewage sludge and industrial wastewater under anaerobic sulphate reducing conditions: enzymatic profiles in a recycling sludge bed reactor; Whiteley CG et al.; The first stage in the degradation and recycling of primary sewage sludge and particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the anaerobic sulphidogenic environment . Solubilization of complex carbon substrates provides the primary reaction in the BioSURE Process, and is effected in the recycling sludge bed reactor (RSBR) . During the process of anaerobic digestion, macromolecules are broken down into simpler low molecular weight compounds in the presence of extracellular enzymes . Though hydrolysis of the complex carbon sources was enhanced under biosulphidogenic conditions, no studies have examined the role of enzymes and the enzymatic profiles in the RSBR . To investigate the overall enzymology in the RSBR variations in COD, pH, sulphate, sulphite and sulphide concentrations, carbohydrates, protein and activities of glucosidases, proteases and lipases were studied over 50 days in the reactor at three different depths: 0-16 cm; 16-32 cm; 32-50 cm . While the pH profile remained fairly constant between 6.9 and 7.3 the sulphate and sulphide levels, as expected, changed dramatically as sulphate reduction took place . Proteases, lipases and glucosidases all showed enhanced activity with depth in the bioreactor . It is suggested that the increased sulphide concentration generated during the sulphate reduction process stimulates the enzymes, leading to enhanced solubilisation of primary sewage sludge.

Water Sci Technol, 2003, 48(4), 29 - 36
Process enhancement in anaerobic bioreactor landfills; Kim J et al.; The development of bioreactor landfills originated with the recognition that the sequential acid and methane fermentation phases of anaerobic waste stabilization could be accelerated by leachate recirculation . Original studies suggested the potential for such a change in design and management of landfills receiving both municipal and industrial solid wastes, followed by both pilot- and full-scale bioreactor landfill demonstrations . The enhancement of waste transformation within controlled bioreactor landfills is addressed in terms of the sequential phases of waste stabilization, temporal and spatial distribution of leachate and gas generation patterns, mechanisms of conversion, and proposed process and operational modifications . Selected results from investigations on bioreactor landfills include co-treatment of organic and inorganic constituents and the use of dedicated treatment zones.

Analyst, 2003 Sep, 128(9), 1150 - 6
A universal temperature controlled membrane interface for the analysis of volatile and semi-volatile organic compounds; Creaser CS et al.; A universal temperature controlled membrane interface (TCMI) has been constructed for hollow-fibre membranes . The membrane temperature is controllable in the range -70 to 250 degrees C using an electric heater and a flow of cooled nitrogen or helium gas . Volatile and semi-volatile organic compounds may be detected either by continuous diffusion across the membrane or by in-membrane pre-concentration followed by thermal desorption into the detector . The TCMI interface is demonstrated in combination with mass spectrometry and GC-MS, for the determination of VOCs and SVOCs in aqueous and air samples and for the on-line monitoring of a bioreactor.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1574 - 82
Effects of three-dimensional culturing on osteosarcoma cells grown in a fibrous matrix: analyses of cell morphology, cell cycle, and apoptosis; Chen C et al.; Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis . Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy . The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion) . The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells . Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture . In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days . Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at approximately 15% . These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1498 - 504
Design of a tubular loop bioreactor for scale-up and scale-down of fermentation processes; Papagianni M et al.; Microorganisms traveling through circulation loops in large-scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients . The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another . One approach to study this problem is to look at the circulation loop itself . The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose . The reactor carries a number of ports and probes along its length for the determination of concentration gradients within . The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (t(c), s) is used as a measure of simulated reactor size . The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process . Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison . From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1480 - 6
Using computational fluid dynamics software to estimate circulation time distributions in bioreactors; Davidson KM et al.; Nonideal mixing in many fermentation processes can lead to concentration gradients in nutrients, oxygen, and pH, among others . These gradients are likely to influence cellular behavior, growth, or yield of the fermentation process . Frequency of exposure to these gradients can be defined by the circulation time distribution (CTD) . There are few examples of CTDs in the literature, and experimental determination of CTD is at best a challenging task . The goal in this study was to determine whether computational fluid dynamics (CFD) software (FLUENT 4 and MixSim) could be used to characterize the CTD in a single-impeller mixing tank . To accomplish this, CFD software was used to simulate flow fields in three different mixing tanks by meshing the tanks with a grid of elements and solving the Navier-Stokes equations using the kappa-epsilon turbulence model . Tracer particles were released from a reference zone within the simulated flow fields, particle trajectories were simulated for 30 s, and the time taken for these tracer particles to return to the reference zone was calculated . CTDs determined by experimental measurement, which showed distinct features (log-normal, bimodal, and unimodal), were compared with CTDs determined using CFD simulation . Reproducing the signal processing procedures used in each of the experiments, CFD simulations captured the characteristic features of the experimentally measured CTDs . The CFD data suggests new signal processing procedures that predict unimodal CTDs for all three tanks.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1417 - 26
Production of podophyllotoxin using cross-species coculture of Linum flavum hairy roots and Podophyllum hexandrum cell suspensions; Lin HW et al.; Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin . The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors . In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P . hexandrum suspensions . Because roots of L . flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P . hexandrum to provide an in situ supply of coniferin . Compared with P . hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P . hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively . The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture . Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.

Biotechnol Prog, 2003 Sep-Oct, 19(5), 1391 - 402
Perspectives for the industrial enzymatic production of glycosides; de Roode BM et al.; Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry . Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry . Thus, enzyme-catalyzed reactions are a good alternative . However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale . Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product . Unfortunately, a bioreactor for the commercial scale production of glycosides is not available . The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.

J Pediatr Surg, 2003 Sep, 38(9), 1348 - 53
Fetal tissue engineering: in vitro analysis of muscle constructs; Fuchs JR et al.; BACKGROUND/PURPOSE: This study was aimed at examining the impact of different tissue engineering techniques on fetal muscle construct architecture . METHODS: Myoblasts from ovine specimens of fetal skeletal muscle were expanded in culture and their growth rates determined . Cells were seeded at different densities onto 3 scaffold types, namely polyglycolic acid (PGA) treated with poly-l-lactic acid (PLLA), a composite of PGA with poly-4-hydroxybutyrate (P4HB), and a collagen hydrogel . Constructs were maintained in a bioreactor and submitted to histologic, scanning electron microscopy, and DNA analyses at different time-points . Statistical analysis was by the likelihood ratio and paired Student's t tests (P <.05) . RESULTS: Fetal myoblasts proliferated at faster rates than expected from neonatal cells . Cell attachment was enhanced in the PGA/PLLA matrix and collagen hydrogel when compared with the PGA/P4HB composite . Necrosis was observed at the center of all constructs, directly proportional to cell seeding density and time in the bioreactor . CONCLUSIONS: Fetal myoblasts can be expanded rapidly in culture and attach well to PGA/PLLA, as well as collagen hydrogel but less optimally to PGA/P4HB . Excessive cell seeding density and bioreactor time may worsen final construct architecture . These findings should be considered during in vivo trials of muscle replacement by engineered fetal constructs.

Waste Manag, 2003, 23(8), 703 - 8
Recycling of spent nickel-cadmium batteries based on bioleaching process; Zhu N et al.; Only 1-2 percent of discarded dry batteries are recovered in China . It is necessary to find an economic and environmentally friendly process to recycle dry batteries in this developing country . Bioleaching is one of the few techniques applicable for the recovery of the toxic metals from hazardous spent batteries . Its principle is the microbial production of sulphuric acid and simultaneous leaching of metals . In this study, a system consisting of a bioreactor, settling tank and leaching reactor was developed to leach metals from nickel-cadmium batteries . Indigenous thiobacilli, proliferated by using nutritive elements in sewage sludge and elemental sulphur as substrates, was employed in the bioreactor to produce sulphuric acid . The overflow from the bioreactor was conducted into the settling tank . The supernatant in the settling tank was conducted into the leaching reactor, which contained the anode and cathodic electrodes obtained from nickel-cadmium batteries . The results showed that this system was valid to leach metals from nickel-cadmium batteries, and that the sludge drained from the bottom of the settling tank could satisfy the requirements of environmental protection agencies regarding agricultural use.

Int J Artif Organs, 2003 Aug, 26(8), 735 - 42
A novel bioartificial liver containing small tissue fragments: efficiency in the treatment of acute hepatic failure induced by carbon tetrachloride in rats; Soloviev V et al.; The efficiency of a new bioartificial liver (BAL) containing small tissue fragments in the treatment of acute hepatic failure induced by carbon tetrachloride in rats was evaluated . A day after injection (i.p.) of CCl4 the animals were connected to a BAL containing liver fragments (fragment BAL) and a BAL containing no liver fragments (no-fragment BAL), and extracorporeal hemoperfusion was carried out for 4 h . The activities of alanine transaminase and lactate dehydrogenase as well as the concentrations of ammonia, glucose, urea, and amino acids in plasma were measured . A tendency to the stabilisation of ammonia, glucose, phenylalanine, tyrosine, and other amino acids was revealed at the end of hemoperfusion in poisoned rats connected to the fragment BAL . A statistically significant difference in survival between the animals connected to the fragment BAL and no-fragment BAL was found . The results obtained indicate that the bioreactor containing small liver fragments is effective in the treatment of acute hepatic failure in animals.

Water Sci Technol, 2003, 48(3), 127 - 34
Occurrence and removal of endocrine disrupters in landfill leachate treatment plants; Wintgens T et al.; Endocrine disrupting compounds can affect the hormone system in organisms . Industrial chemicals with estrogenic effects were detected in large quantities in landfill leachates . Membrane technology has proven to be an effective barrier to these substances and thus widely applied in the treatment of landfill leachate . The removal techniques under investigation are membrane bioreactors, nanofiltration, activated carbon adsorption, ozonation as well as reverse osmosis . Investigations were conducted at two different landfill leachate treatment plants with a variety of process configurations . The xenoestrogenic substances nonylphenol and bisphenol A were detected in high microg/L-ranges in raw landfill leachate . Membrane bioreactors (MBRs) were capable of removing more than 80% of the nonylphenol load . Final effluent concentrations range between 1-12 microg/L nonylphenol and 3-30 microg/L bisphenol A respectively . Reverse osmosis treatment proved to be less effective in nonylphenol and bisphenol A removal than MBR with further polishing stages like nanofiltration and activated carbon adsorption.

Water Sci Technol, 2003, 48(3), 113 - 9
Fluid hydrodynamics in submerged and sidestream membrane bioreactors; Le-Clech P et al.; As with all membrane processes, turbulence, as promoted by aeration in submerged membrane bioreactors (MBRs) or pumping in sidestream (SS) systems to produce somewhat higher effective cross-flow velocities, increases mass transfer and reduces fouling . This is manifested in an elevated critical flux, the flux at which the membrane permeability is sustained . However, the non-Newtonian nature of the sludge makes precise rheological characterisation difficult . In this study, a calculation of the appropriate hydrodynamics parameters for a SS MBR configuration is presented . Optimisation of the aeration in a submerged MBR system has been attained by defining the minimum air velocity required for Taylor bubble formation.

Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 665 - 8
{Use of normal human hepatocytes in a hybrid organ system}; Auth MK et al.; Using the bioreactor model developed by J . Gerlach, we examined the potential of normal human hepatocytes for application in bioartificial liver devices . From normal human donor livers 1.5 x 10(8) hepatocytes were isolated . Hepatocytes were perfused in a woven multi-compartment capillary system in serum-free culture medium containing ammoniachloride over a period of 2 weeks . These cells demonstrated a well differentiated ultrastructure with formation of junctional complexes and bile canaliculi between adjacent cells . During reactor run, a constant albumin synthesis with levels above 11 mg/ml and maintenance of urea production and lignocaine metabolism (MEGX-test) were detected . These initial results indicate that normal human hepatocytes express typical morphology and ultrastructure and are able to keep differentiated functions in suitable perfusion models . Combination of the distinct human liver cell populations might enable promotion of further specific functions (clotting factors) and induction of liver cell proliferation.

J Biomed Mater Res A, 2003 Oct 1, 67(1), 87 - 95
Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds; Gomes ME et al.; This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures . Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture . SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process . For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days . The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions . However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion . This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence . Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions . In the scaffolds cultured under static conditions, only a surface layer of cells was observed . These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds . They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue .

J Biomed Mater Res A, 2003 Oct 1, 67(1), 54 - 60
Cementum engineering with three-dimensional polymer scaffolds; Jin QM et al.; Cloned cementoblasts (OCCMs), periodontal ligament fibroblasts (SV-PDLs), and dental follicle (SV-F) cells obtained from mice were used as a tool to study periodontal tissue engineering . OCCM, SV-PDL, and SV-F cells were seeded onto three-dimensional poly lactic-co-glycolic acid (PLGA) scaffolds and cultured with the use of bioreactors or implanted subcutaneously in severe combined immune deficiency (SCID) mice for up to 6 weeks . We explored the behavior of these cells in porous PLGA sponges by cell growth, expression of mineral-associated genes using reverse transcriptase polymerase chain reaction, and mineralization by histologic analysis in vitro and in vivo . Results indicated that cells attached to PLGA scaffolds under either static or dynamic conditions in vitro . Only OCCM implants, retrieved from both in vitro bioreactors and SCID mice at 3-and 6-weeks post-cell implantation exhibited mineral formation . Types I and XII collagens, osteocalcin, and bone sialoprotein genes were detected in all implants retrieved from SCID mice . These results suggest that delivery of selected cells via PLGA scaffolds may serve as a viable approach for promoting periodontal tissue regeneration .

Virology, 2003 Sep 15, 314(1), 16 - 25
Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor; Aizaki H et al.; Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research . Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier . Subsequent detection of increased HCV RNA suggested viral replication . Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant . Infectivity was shown by successful secondary passage to a new culture . Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible . The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection . It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Biotechnol Lett, 2003 Aug, 25(15), 1271 - 4
Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity; Foster LJ et al.; Microgravity can influence cell growth and function . A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNFa monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor . Microgravity significantly decreased cell growth (from 1.7 x 10(6) to 7.9 x 10(5) cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 microg of anti-TNFalpha hmAb per 10(6) viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively) . The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.

Biotechnol Lett, 2003 Aug, 25(16), 1363 - 7
Suspension culture of anchorage-dependent animal cells using nanospheres of the biodegradable polymer, poly(lactic-co-glycolic acid); Ryu JH et al.; A novel method was developed for suspension culture of anchorage-dependent animal cells using biodegradable polymer nanospheres . The addition of poly(lactic-co-glycolic acid) nanospheres (907 nm average diam.) to the culture of human embryonic kidney 293 cells in stirred suspension bioreactors promoted the aggregate formation and cell growth (4.4-fold versus 2.2-fold growth for 7 d), compared to culture without nanospheres . This method may be useful for the culture of various types of anchorage-dependent animal cells in large-scale suspension bioreactors.

Biotechnol Lett, 2003 Sep, 25(17), 1441 - 4
Microbial degradation of high and low molecular weight polyaromatic hydrocarbons in a two-phase partitioning bioreactor by two strains of Sphingomonas sp; Daugulis AJ et al.; A mixture of six polyaromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene, pyrene, chyrysene and benzo{a}pyrene), varying in size from 2 to 5 rings, was dissolved in dodecane, and used as the delivery phase of a partitioning bioreactor . Two species of Sphingomonas were then used individually, and as a consortium, to determine which of the PAHs were degraded . Only low molecular weight PAHs (naphthalene, phenanthrene and fluoranthene) were degraded by the individual strains, but the consortium degraded all substrates either to completion or near completion.

J Biotechnol, 2003 Oct 9, 105(1-2), 189 - 98
Reproducing shake flasks performance in stirred fermentors: production of alginates by Azotobacter vinelandii; Reyes C et al.; Keeping equal the initial power drawn (0.27 W l(-1)) in shake flasks and in a stirred fermentor did not reproduce the behaviour of alginate production by Azotobacter vinelandii . A lower mean molecular weight (1.1x10(6) Da) of the polymer was obtained in the bioreactor as compared to that obtained in shake flasks (1.9x10(6) Da) . The reasons for this can reside in the fact that the evolution of the power drawn in the shake flasks could be considerably different to that observed in the stirred bioreactor . A drastic drop in the specific power drawn is expected in the shake flasks as a consequence of the increased viscosity, which caused the liquid not following the movement of the shaker . This was supported by the fact that cultures developed in the fermentor at lower initial power drawn (as low as 0.027-0.056 W l(-1)) or in a culture in which the power drawn was deliberately reduced along cultivation, produced alginates with similar molecular characteristics as that obtained in shake flasks.

Tissue Eng, 2003, 9 Suppl 1, S65 - 75
Use of ultrathin shell microcapsules of hepatocytes in bioartificial liver-assist device; Sun T et al.; We previously encapsulated hepatocytes in ultrathin shell microcapsules and showed them to have enhanced differentiated functions over cells cultured in monolayer . Here we have used these microencapsulated hepatocytes in a bioartificial liver-assisted device (BLAD) with a rat hepatectomy model . Primary rat hepatocytes were encapsulated in 150- to 200-microm microcapsules, using an electrostatic droplet generator . The microencapsulated hepatocytes exhibited good in vitro urea synthesis activity in plasma from rats with fulminant hepatic failure (FHF) . The ex vivo hemoperfusion was conducted in FHF rats by perfusing plasma at a rate of 1-2 mL/min through 1.5-2 x 10(8) encapsulated hepatocytes packed into a packed-bed bioreactor . Hemoperfusion with the bioreactor was initiated 5 h after operative induction of liver failure and continued for 7 h . The BLAD-treated rats showed improvements over the control groups in survival time and metabolic indicators, including ammonia and total bilirubin levels . Furthermore, expanded bed adsorption (EBA) detoxification technology using Streamline-SP resin was explored to complement the bioreactor with microencapsulated hepatocytes . In vitro experiments indicated that serum ammonia could be specifically removed in dose-dependent manner, whereas the total serum proteins were unaffected by the resin . In ex vivo experiments, hemoperfusion with the resin was initiated 3 h after operative induction of liver failure and continued for 7 h . The resin-treated rats showed obvious serum ammonia removal with no observable total blood protein and blood cell adsorption . Therefore, Streamline-SP resin can potentially be integrated into a BLAD for improved efficacy.

Water Res, 2003 Jul, 37(13), 3098 - 109
The contribution of biotic and abiotic processes during azo dye reduction in anaerobic sludge; van der Zee FP et al.; Azo dye reduction results from a combination of biotic and abiotic processes during the anaerobic treatment of dye containing effluents . Biotic processes are due to enzymatic reactions whereas the chemical reaction is due to sulfide . In this research, the relative impact of the different azo dye reduction mechanisms was determined by investigating the reduction of Acid Orange 7 (AO7) and Reactive Red 2 (RR2) under different conditions . Reduction rates of two azo dyes were compared in batch assays over a range of sulphide concentrations in the presence of living or inactivated anaerobic granular sludge . Biological dye reduction followed zero order kinetics and chemical dye reduction followed second-order rate kinetics as a function of sulfide and dye concentration . Chemical reduction of the dyes was greatly stimulated in the presence of autoclaved sludge: whereas chemical dye reduction was not affected by living or gamma-irradiated-sludge . Presumably redox-mediating enzyme cofactors released by cell lysis contributed to the stimulatory effect . This hypothesis was confirmed in assays evaluating the chemical reduction of AO7 utilizing riboflavin, representative of the heat stable redox-mediating moieties of common occurring flavin enzyme cofactors . Sulfate influenced dye reduction in accordance to biogenic sulfide formation from sulfate reduction . In assays lacking sulfur compounds, dye reduction only readily occurred in the presence of living granular sludge, demonstrating the importance of enzymatic mechanisms . Both chemical and biological mechanisms of dye reduction were greatly stimulated by the addition of the redox-mediating compound, anthraquinone-disulfonate . Based on an analysis of the kinetics and demonstration in lab-scale upward-flow anaerobic sludge bed reactors, the relative importance of chemical dye reduction mechanisms in high rate anaerobic bioreactors was shown to be small due to the high biomass levels in the reactors.

Cardiovasc Pathol, 2003 Sep-Oct, 12(5), 271 - 6
Advances in vascular tissue engineering; Thomas AC et al.; Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the supply of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials . Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries . Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture . Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties . Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells . Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery . "Tissue-engineered blood vessels" may also be formed from layers of human vascular cells grown in culture . These engineered vessels have many of the characteristics of arteries formed in vivo . "Artificial arteries" may be also be derived from peritoneal granulation tissue in body "bioreactors" by adapting the body's natural wound healing response to produce a hollow tube.

Bioprocess Biosyst Eng, 2003 Mar, 25(5), 323 - 9 Epub 2003 Jan 29.
Output regulation of a class of unstructured models of continuous bioreactors: steady-state approaches; Wu W et al.; This article deals with the output regulation of continuous bioreactors in the face of constant disturbances and inverse dynamics . Nonlinear controllers developed on the basis of approximate equilibrium manifolds can almost attenuate measurable or unmeasurable disturbances on the output . This nonlinear feed-forward/feedback control framework without any tuning parameters can be directly implemented to strictly nonlinear systems . Under dynamic actuator constraints and the availability of only output signals for use in the control law, closed-loop simulations demonstrate that the proposed control techniques are superior to a nonlinear PI control scheme based on the identified Hammerstein model.

Bioprocess Biosyst Eng, 2003 Mar, 25(5), 315 - 21 Epub 2003 Jan 15.
Effect of fluid dispersion on cybernetic control of microbial growth on substitutable substrates; Patnaik PR; Many fermentation media contain two or more substrates, which a microorganism utilizes for similar purposes . Depending on the conditions prior to and during a fermentation, the substrates may be utilized in succession or simultaneously . Since it is difficult to portray this behavior through mechanistic models, a cybernetic method was proposed earlier . Here the microorganism chooses the mode of substrate utilization that maximizes its own survival, usually expressed by the growth rate . In a fully dispersed bioreactor, simultaneous utilization generates higher growth rates but leads to low biomass concentrations since this utilization pattern is preferred at low concentrations of the substrates . In this study it has been shown that by allowing less than complete dispersion in the broth it is possible to shift from sequential to simultaneous utilization at high concentrations, thereby enabling both high growth rates and large biomass concentrations . This strategy thus allows the natural incomplete dispersion in large bioreactors to be gainfully exploited.

Bioprocess Biosyst Eng, 2002 Apr, 25(1), 43 - 52 Epub 2002 Mar 07.
Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction; Gerigk MR et al.; A fully integrated process for the microbial production and recovery of the aromatic amino acid L-phenylalanine is presented . Using a recombinant L-tyrosine (L-Tyr) auxotrophic Escherichia coli production strain, a fed-batch fermentation process was developed in a 20-l-scale bioreactor . Concentrations of glucose and L-Tyr were closed-loop-controlled in a fed-batch process . After achieving final L-phenylalanine (L-Phe) titres >30 g/l the process strategy was scaled up to 300-l pilot scale . In technical scale fermentation L-phenylalanine was continuously recovered via a fully integrated reactive extraction system achieving a maximum extraction rate of 110 g/h (final purity >99%) . It was thus possible to increase L-Phe/glucose selectivity from 15 mol% without to 20.3 mol% with integrated product separation.

Bioprocess Biosyst Eng, 2002 Apr, 25(1), 21 - 7 Epub 2002 Feb 08.
Static and dynamic behavior of a class of unstructured models of continuous bioreactors with growth associated product; Ajbar A et al.; The static and dynamic behavior of a class of unstructured models of continuous bioprocesses, for which the product is growth associated, are analyzed using elementary concepts of singularity theory and continuation techniques . The class consists of models for which both the rates of utilization of limiting substrate and product formation are linearly proportional to the specific cell growth rate . The kinetic expressions are allowed to assume general forms of substrate and nonbiomass product . The steady-state analysis allows the derivation of analytical results and the construction of a useful picture in the models' parameter space delineating the different static behavior these models can predict, including unique steady states and bistability . The analysis of the dynamic behavior allows the derivation of general analytical conditions for the occurrence of periodic behavior in the models . It is also shown that the subclass of these models for which the specific cell growth rate expression is monotonic with respect to the nonbiomass product is unable to predict a stable oscillatory behavior regardless of the expression of the growth rate . These results illustrate the fundamental weakness of this class of unstructured models in predicting transient behavior in continuous cultures . The effect of kinetic and operating parameters on the stability characteristics of these models is also investigated.

Bioprocess Biosyst Eng, 2002 Apr, 25(1), 1 - 12 Epub 2002 Feb 09.
The tuning of a model-based estimator for the specific growth rate of Candida utilis; November EJ et al.; Control of microbial conversion processes is frequently inhibited by the infeasibility of measuring important process variables . In order to circumvent this lack of measurements, an accurate or valuable and conveniently measurable on-line hardware measurement can be combined with the balance equations describing the process to obtain estimates of less easily measurable variables . In this article the on-line estimation of the specific growth rate of Candida utilis is evaluated . The observer-based estimator requires a hardware measurement of the biomass during fermentations in conjuction with a model of the process; therefore the Biomass Monitor, giving an on-line measurement of viable biomass, is used in the bioreactor experiments described . The optimal tuning of the estimation for the experimental conditions is described and several alternative adaptations of the design of the estimator are presented . The influence of implemented time intervals for discretization of the estimator on the reliability of the estimated growth rate values is discussed . Additionally, the necessary choice of an initial value of the estimated specific growth rate has proven to be of great importance in practice.

Bioprocess Biosyst Eng, 2002 Jun, 25(2), 85 - 96 Epub 2002 Mar 26.
Integrated L-phenylalanine separation in an E . coli fed-batch process: from laboratory to pilot scale; Maass D et al.; Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation . An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient . Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach . Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase . Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation . A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.

Adv Space Res, 2003, 31(7), 1791 - 7
Waste system implications for Mars missions; Drysdale AE et al.; Waste technologies for Mars missions have been analyzed, considering equivalent system mass and interface loads . Storage or dumping seems most appropriate for early missions with low food closure . Composting or other treatment of inedible biomass in a bioreactor seems most attractive for moderate food closure (50-75%) . Some form of physicochemical oxidation of the composted residue might be needed for increased food closure, but oxidation of all waste does not seem appropriate due to excess of production of carbon dioxide over demand . More comprehensive analysis considering interfaces with other mission systems is needed . In particular, in-situ resource utilization is not considered, and might provide resources more cheaply than waste processing . c2003 COSPAR . Published by Elsevier Science Ltd . All rights reserved.

Adv Space Res, 2003, 31(7), 1683 - 91
Aquatic modules for bioregenerative life support systems: developmental aspects based on the space flight results of the C.E.B.A.S . MIN-MODULE; Blum V; The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) is an artificial aquatic ecosystem which contains teleost fishes, water snails, ammonia oxidizing bacteria and edible non-gravitropic water plants . It serves as a model for aquatic food production modules which are not seriously affected by microgravity and other space conditions . Its space flight version, the so-called C.E.B.A.S . MINI-MODULE was already successfully tested in the STS-89 and STS-90 (NEUROLAB) missions . It will be flown a third time in space with the STS-107 mission in January 2003 . All results obtained so far in space indicate that the basic concept of the system is more than suitable to drive forward its development . The C.E.B.A.S . MINI-MODULE is located within a middeck locker with limited space for additional components . These technical limitations allow only some modifications which lead to a maximum experiment time span of 120 days which is not long enough for scientifically essential multi-generation-experiments . The first necessary step is the development of "harvesting devices" for the different organisms . In the limited space of the plant bioreactor a high biomass production leads to self-shadowing effects which results in an uncontrolled degradation and increased oxygen consumption by microorganisms which will endanger the fishes and snails . It was shown already that the latter reproduce excellently in space and that the reproductive functions of the fish species are not affected . Although the parent-offspring-cannibalism of the ovoviviparous fish species (Xiphophorus helleri) serves as a regulating factor in population dynamics an uncontrolled snail reproduction will also induce an increased oxygen consumption per se and a high ammonia concentration in the water . If harvesting locks can be handled by astronauts in, e . g., 4-week intervals their construction is not very difficult and basic technical solutions are already developed . The second problem is the feeding of the animals . Although C.E.B.A.S.-based aquaculture modules are designed to be closed food loop systems (edible herbivorous fish species and edible water plants) which are already verified on Earth this will not be possible in space without devices in which the animals are fed from a food storage . This has to be done at least once daily which would waste too much crew time when done by astronauts . So, the development of a reliable automated food dispenser has highest priority . Also in this case basic technical solutions are already elaborated . The paper gives a comprehensive overview of the proposed further C.E.B.A.S.-based development of longer-term duration aquatic food production modules . c2003 COSPAR . Published by Elsevier Science Ltd . All rights reserved.

Transplantation, 2003 Sep 15, 76(5), 781 - 6
Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study; Gerlach JC et al.; INTRODUCTION: The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support . METHODS: Primary liver cells were isolated from 54 discarded organs (donor age 56.7+/-13.2 years) . Between 2.8x10(10) and 6.4x10(10) parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36) . The metabolic activity of the cells was regularly determined during culture . The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks . RESULTS: Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days . Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures . Vascular cavities (CD 31 immunoreactivity {IR}) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed . Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver . Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected . CONCLUSION: The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy . In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

Biotechnol Adv, 2003 Apr, 21(2), 109 - 22
Polysaccharopeptides of Coriolus versicolor: physiological activity, uses, and production; Cui J et al.; The protein-bound polysaccharides or polysaccharopeptides produced by Coriolus versicolor are effective immunopotentiators, which are used to supplement the chemotherapy and radiotherapy of cancers and various infectious diseases . Antitumor activity of polysaccharopeptides has been documented . Several kinds of protein-bound polysaccharides have been shown to be produced by the white rot fungus, C . versicolor . Although some of these polymers are structurally distinct, they are not distinguishable in terms of their physiological activity . This review focuses on the physiologically active polysaccharopeptides of C . versicolor . In nature, C . versicolor occurs as a mushroom body, but the fungus can be grown as mycelial biomass in submerged culture in bioreactors . Mushrooms gathered in the wild, cultivated mushrooms, and the mycelial biomass of submerged culture are used to produce the polysaccharopeptides . Submerged cultures are typically carried out in batches lasting 5-7 days and at 25-27 degrees C . Hot water extraction of the biomass is used to recover the thermostable polysaccharopeptides that are concentrated, purified, and dried into a powder for medicinal use . In view of the documented physiological benefits of these compounds, extensive research is underway on the structure, composition, production methods, and use of new C . versicolor strains for producing the therapeutic biopolymers . Properties, physiological activity, recovery, and purification of the bioactive polysaccharopeptides are discussed.

Biotechnol Adv, 2003 Aug, 21(5), 407 - 16
Challenges for mass production of nematodes in submerged culture; de la Torre M; Nematodes of Steinernema and Heterorhabditis genera are used as agents in insect biocontrol programs . They are associated with specific bacteria which are also involved in the mechanism of pathogenicity and which are consumed by nematodes as living food . S . feltiae has various developmental stages in its life cycle, including four juvenile stages, adults and the free living form . During mating, males coil themselves around the female, which is around 1 cm long . Successful commercialization of nematode-bacteria biocontrol products depends on the ability to produce sufficient quantities of these products at competitive prices for a full pest control program . This could be feasible if high cell density submerged cultures are designed and implemented; however, major problems related to nematodes mass production in a bioreactor remain unsolved due to the lack of knowledge about the physiological aspects of the nematode, bacteria and nematode-bacteria association, interaction between the three phases present in the bioreactor (liquid, gas, nematodes-bacteria), possibility of mating under hydrodynamic stress conditions, etc . We have found that the two most important engineering aspects to take into account the mass propagation of nematodes are oxygen transfer rate and hydrodynamics to allow mating and to avoid mechanical damage of juveniles in stage 2.This article focuses on several aspects related to the fermentation system such as kinetics of growth, shear stress, hydrodynamics fields in the bioreactor and oxygen demand . Also, results published by other groups, together with those of our own, will be discussed in relation to the main challenges found during the fermentation process.

Phytochemistry, 2003 Oct, 64(3), 717 - 24
High isoflavone content and estrogenic activity of 25 year-old Glycine max tissue cultures; Federici E et al.; Soy isoflavones are phytoestrogens which have been associated with several health benefits . In the present study, we report the production of isoflavones in a collection of 40 strains of soya cell cultures established in 1975 . A large variability in the isoflavone composition was observed and high-producing strains, with an isoflavone content of up to 46.3 mg g(-1) dry wt., were found . In comparison with soybeans, many callus strains had a higher isoflavone concentration (10-40 times) and a different ratio of genistin to daidzin forms . The highest producing strain was transferred to liquid medium in an Erlenmeyer flask and in a 10 l stirred-tank bioreactor where high isoflavone content (7% dry wt.), concentration (880 mg l(-1)) and a maximum productivity estimated to 60 mg l(-1) d(-1) were obtained . We further studied the estrogenic activity of pure compounds compared to plant cell culture extracts in the estrogen-responsive human endometrial Ishikawa cell line . Estrogen was confirmed to be 1000-10,000 times more active than isoflavones . The estrogenic activity of the extracts correlated to their isoflavone content . The activity of the malonyl isoflavones, assessed here for the first time, was lower than the aglycones . Taken together, these results suggest that soya cell cultures can be used as an alternative source to soybeans to provide high concentrations of bioactive isoflavones.

J Environ Sci (China), 2003 Jul, 15(4), 449 - 55
A pilot scale anoxic/oxic membrane bioreactor (A/O MBR) for woolen mill dyeing wastewater treatment; Zheng X et al.; A pilot-scale (10 m3/d) anoxic/oxic membrane bioreactor (A/O MBR) was tested for dyeing wastewater treatment of woolen mill without wasting sludge in 125 days operation . Results showed that the effluent quality was excellent, i.e . effluent COD less than 25 mg/L, BOD5 under 5 mg/L, turbidity lower than 0.65 NTU, and colour less than 30 DT, and met with the reuse water standard of China . The removal rates of COD, BOD5, colour, and turbidity were 92.4%, 98.4%, 74% and 98.9%, respectively . Constant-flux operation mode was carried out in this study, and backwash was effective for reducing membrane fouling and maintaining constant flux . Membrane fouling had heavy impact on energy consumption . More attention should be paid on pipe selection and design for the sidestream MBR system, too.

Cell Biol Int, 2003, 27(9), 755 - 60
Plasticity of the response of fetal mouse fibroblast to lactation hormones; Wang S et al.; In the work described in this article, mouse fetal fibroblasts were treated with three lactation hormones (insulin, progesterone and oxytocin) and the cellular changes were analyzed by RT-PCR-Southern hybridization . A gene-expression pattern characteristic of mammary epithelioid cells was induced by the hormones, as indicated by expression of the marker genes alpha-casein and beta-casein . Two mammary epithelial cell-specific gene expression vectors were constructed with bovine alpha-s1-casein or ovine beta-casein gene promoters directing an EGFP reporter gene . Transient expression of the EGFP gene was observed in cells treated by the hormones but not in control cells . Cell morphology also changed after insulin and oxytocin treatments; the cells resembled epithelial cells rather than fibroblasts . Our results suggest that mouse fetus fibroblasts can be partially induced by lactation hormones to resemble mammary epithelial cells . This procedure might help to increase the efficiency of gene targeting in studies of mammary gland bioreactors.

J Hepatol, 2003 Oct, 39(4), 552 - 8
Liver micro-organs transcribe albumin and clotting factors and increase survival of 92% hepatectomized rats; Grad-Itach E et al.; BACKGROUND/AIMS: Currently there is no effective non-surgical therapy for most patients with fulminant or end stage chronic liver disease . METHODS: We have prepared rat liver micro-organs (LMOs), which preserve the liver micro-architecture and ensure that no cell is more than 150 microm away from a source of nutrients and gases . The function of LMOs has been evaluated in vitro and in a new extra-corporeal liver device termed aLIVE in which LMOs are exposed to liver-like hemodynamic conditions . RESULTS: In vitro LMOs maintain normal physiological and biochemical functions including oxygen consumption, glucose metabolism, conversion of ammonia to urea, secretion of albumin and de novo transcription of genes coding for albumin and clotting factors . Inside the aLIVE bioreactor, LMOs also display sustained oxygen consumption, glucose metabolism and transcription of albumin and clotting factors IX and X, when connected both to normal and to 92% hepatectomized rats . Survival of 92% hepatectomized rats was 40% longer following a single 4-h treatment with aLIVE, compared to untreated animals . CONCLUSIONS: An extra-corporeal liver device, aLIVE, which provides key liver functions, has been developed . When tested in 92% hepatectomized rats, aLIVE improved the clinical condition and significantly increased survival time of the treated rats.

Biotechnol Bioeng, 2003 Nov 5, 84(3), 382 - 9
On the dynamics of a spherical scaffold in rotating bioreactors; Ramirez LE et al.; We analyze the dynamics of a spherical scaffold in rotating bioreactors (or clinostats) . The idealized clinostat environment consists of a purely rotational flow that is perpendicular to a gravitational field . We confirm through a detailed analytical study that lift effects considerably alter the position of the equilibrium point reached by the scaffolds in the (vertical) direction collinear to the gravitational field . This result holds for small particle and shear Reynolds numbers . Our analysis shows that the inertial lift effect is negligible in the horizontal direction . We show that for all rotations of practical interest, and for the range of particle Reynolds number smaller than unity, the vertical coordinate of the equilibrium point is strongly affected by consideration of lift effects . For light (heavy) particles, inclusion of lift in the formation forces the equilibrium position to be below (above) the horizontal plane that contains the axis of rotation . The equilibrium point for light particles is stable and therefore is observable experimentally . The equilibrium point for heavy particles is unstable . We also estimate the stress level applied to the scaffold and derive an algebraic expression that indicates that the stress level acting on the scaffold decreases with increasing shear Reynolds number .

Biotechnol Bioeng, 2003 Nov 5, 84(3), 332 - 42
Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency; Pham PL et al.; Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins) . As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required . In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes . The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids . Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold . Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1) . To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system . As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors . Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step . In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient . Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium . This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection .

Biotechnol Bioeng, 2003 Nov 5, 84(3), 265 - 73
Use of a plant-derived enzyme template for the production of the green-note volatile hexanal; Schade F et al.; Hexanal is a key organoleptic element of green-note that is found in both fragrances and flavors . We report a novel process for the production of hexanal using immobilized enzyme templates extracted from different plant sources in combination with hollow-fiber ultrafiltration for in situ separation . Enzyme templates, known to be responsible for the synthesis of hexanal from linoleic acid (18:2), were isolated from naturally enriched tissues including carnation petals, strawberry and tomato leaves . These templates were immobilized in an alginate matrix and used as a biocatalyst in a packed-bed bioreactor . Continuous product recovery was achieved using a hollow-fiber ultrafiltration unit . The effects of pH, reaction temperature, and substrate and enzyme concentrations were studied and their effects on hexanal generation identified and optimized . Utilizing optimized conditions, hexanal production 112-fold higher than endogenous steady-state levels in a corresponding amount of plant tissue could be achieved over a 30-minute period . Based on the reactor studies, product inhibition also appears to be an important factor for bioreactor-based hexanal production .

Stem Cells, 2003, 21(5), 536 - 45
Modeling stem cell population growth: incorporating terms for proliferative heterogeneity; Deasy BM et al.; Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research . Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions . A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models . This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations . The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death . Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss . We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation . We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed . Finally, we demonstrate an improved estimation of the kinetic parameters . This study contributes to both the biological and mathematical understanding of stem cell dynamics . Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Biotechnol Bioeng, 2003 Oct 20, 84(2), 240 - 4
Development of foamed emulsion bioreactor for air pollution control; Kan E et al.; A new type of bioreactor for air pollution control has been developed . The new process relies on an organic-phase emulsion and actively growing pollutant-degrading microorganisms, made into a foam with the air being treated . This new reactor is referred to as a foamed emulsion bioreactor (FEBR) . As there is no packing in the reactor, the FEBR is not subject to clogging . Mathematical modeling of the process and proof of concept using a laboratory prototype revealed that the foamed emulsion bioreactor greatly surpasses the performance of existing gas-phase bioreactors . Experimental results showed a toluene elimination capacity as high as 285 g(toluene) m(-3) (reactor) h(-1) with a removal efficiency of 95% at a gas residence time of 15 s and a toluene inlet concentration of 1-1.3 g x m(-3) . Oxygen limited the reactor performance at toluene concentration above about 0.7-1.0 g x m(-3); consequently, performance was significantly improved when pure oxygen was added to the contaminated air . The elimination capacity increased from 204 to 408 g x m(-3) h(-1) with >77% toluene removal at toluene inlet concentrations of 2-2.2 g x m(-3) . Overall, the results show that the performance of the FEBR far exceeds that of currently used bioreactors for air pollution control .

Biotechnol Bioeng, 2003 Oct 20, 84(2), 224 - 32
High cell density induces spontaneous bifurcations of dissolved oxygen controllers during CHO cell fermentations; Chung JD et al.; High cell density cultures of CHO cells growing in a bioreactor under dissolved oxygen control were found to undergo spontaneous bifurcations and a subsequent loss of stability some time into the fermentation . This loss of stability was manifested by sustained and amplified oscillations in the bioreactor dissolved oxygen concentration and in the oxygen gas flow rate to the reactor . To identify potential biological and operational causes for the phenomenon, linear stability analysis was applied in a neighborhood of the experimentally observed bifurcation point . The analysis revealed that two steady state process gains, K(P1) and K(P2), regulated k(l)a and gas phase oxygen concentration inputs, respectively, and the magnitude of K(P1) was found to determine system stability about the bifurcation point . The magnitude of K(P1), and hence the corresponding open-loop steady state gain K(OL1), scaled linearly with the bioreactor cell density, increasing with increasing cell density . These results allowed the generation of a fermentation stability diagram, which partitioned K(C)-N operating space into stable and unstable regions separated by the loci of predicted critically stable controller constants, K(C,critical), as a function of bioreactor cell density . This consistency of this operating diagram with experimentally observed changes in system stability was demonstrated . We conclude that time-dependent increases in cell density are the cause of the observed instabilities and that cell density is the critical bifurcation parameter . The results of this study should be readily applicable to the design of a more robust controller .

Biotechnol Bioeng, 2003 Oct 20, 84(2), 205 - 14
Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity; Wendt D et al.; We developed a bioreactor for automated cell seeding of three-dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions . Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods . Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types . Perfusion seeding of chondrocytes into Polyactive foams resulted in "viable cell seeding efficiencies," defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 +/- 6%) than those by static (57% +/- 5%) and spinner flask seeding (55% +/- 8%) . In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6-fold and 3.8-fold more uniformly distributed and formed more homogeneously sized cell clusters . Chondrocytes seeded by perfusion into Hyaff-11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding . Bone marrow stromal cells seeded by perfusion into ChronOS porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic . In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types .

Protein Expr Purif, 2003 Sep, 31(1), 34 - 41
Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system; Ding H et al.; We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3 . cDNA was constructed from extracted total RNA of Jurkat cells . Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses . For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant . Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein . N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing . The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells . The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli . The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.

Drug Metab Rev, 2003 May-Aug, 35(2-3), 145 - 213
New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures; Gebhardt R et al.; Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration . Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes . In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors . From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances . Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone . A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr . This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism . To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used . In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors . The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2 . This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested . A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner . The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions . This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization . A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora . It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions . Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side . Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction . A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices . For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes . Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

Appl Environ Microbiol, 2003 Sep, 69(9), 5472 - 82
Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor; Girguis PR et al.; Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture . Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments . To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea . We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system . Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments . Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners . Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia . Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments . The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.

J Clin Virol, 2003 Oct, 28(2), 141 - 54
Porcine endogenous retroviruses: no infection in patients treated with a bioreactor based on porcine liver cells; Irgang M et al.; BACKGROUND: Acute liver failure (ALF) remains a disease with high mortality . Bioartificial liver support systems, which combine living cells of the liver in an extracorporeal circuit, have been successfully used in first clinical trials . The shortage of human organs to be used for bioreactors and the lack of safe and effective human liver cell lines have resulted in pigs becoming an important hepatic cell source . However, using these cells may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) . PERVs are present in the genome of all pigs and are able to infect human cells in vitro . However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic . OBJECTIVES: To detect antibodies directed against specific epitopes from PERVs in seven individuals who were treated with porcine liver cell bioreactor therapy prior to liver transplantation . METHODS: Sera from seven patients treated with a hybrid liver support system based on porcine liver cells for ALF who survived the treatment and were discharged from hospital were investigated for antibodies against PERV . For this in addition to methods already reported (Xenotransplantation (2001) 125), new immunological detection methods were developed . RESULTS: PERV-specific antibodies were found in none of the patients using Western blot assays based on purified virus or recombinant viral core and envelope proteins or ELISA based on synthetic diagnostic peptides . CONCLUSION: The assays used are specific and sensitive, and correlated in their diagnostic value . The data indicate that no PERV infection had occurred in none of the patients treated with the CellModule bioreactor containing porcine cells.

Waste Manag, 2003, 23(7), 667 - 74
Municipal solid waste in situ moisture content measurement using an electrical resistance sensor; Gawande NA et al.; Moisture content (MC) is a crucial parameter for degradation of solid waste in landfills . Present MC measurement techniques suffer from several drawbacks . A moisture sensor for measurement of in situ moisture content of solid waste in landfills was developed . The sensor measures the electrical resistance across the granular matrix of the sensor, which in turn can be correlated to moisture content . The sensor was also equipped with a thermocouple and tubing that permits simultaneous measurement of temperature and gas sampling . The electrical conductivity of the surrounding moisture and the temperature in the matrix both affect the resistance measurements . This paper describes the results of laboratory experiments designed to select the appropriate granular media particle size, measure the influence of moisture electrical conductivity and temperature, and develop calibration relationships between measured resistance and gravimetrically determined moisture content . With a few limitations, the sensor is able to detect MC of solid waste under conditions allowing moisture movement into the sensor . The application of this technique shows promise for use in bioreactor landfills where high moisture contents are expected and desired.

Adv Space Biol Med, 2002, 8, 197 - 213
Space bioreactors and their applications; Walther I; Space biology is a young and rapidly developing discipline comprising basic research and biotechnology . With the prospect of longer space missions and the construction of the International Space Station several aspects of biotechnology will play a prominent role in space . In fact, biotechnological processes allowing the recycling of vital elements, such as oxygen or water, and the in-flight production of food becomes essential when considering the financial and logistic standpoint . Every kilogram which, having been recycled or produced in space, does not have to be uploaded will drastically reduce the cost of space missions . In addition, the scientific community is offered a better opportunity to investigate long-term biotechnological processes performing experiments with a duration ranging from weeks to months . Therefore, there is an increasing demand for sophisticated instrumentation to satisfy the requirements of future projects in space biology . The carryover of knowledge from conventional bioreactor technology to miniature space bioreactors for a monitored and controlled cell culturing is one of the key elements for this new dimension in space life science . The first space bioreactors were developed and flown at the end of the last century . It has been demonstrated that cells of different types, from bacteria to mammalian cells, can be successfully grown in this type of culture vessel . This chapter presents different generations of bioreactors developed so far, their performances in space and their potential for the future, as well as the activities of the European Space Agency (ESA) in this domain . A dedicated chapter by Lisa Freed on the rotating wall vessel reactor and the latest NASA bioreactor research is also part of this volume.

Adv Space Biol Med, 2002, 8, 177 - 95
Spaceflight bioreactor studies of cells and tissues; Freed LE et al.; Studies of the fundamental role of gravity in the development and function of biological organisms are a central component of the human exploration of space . Microgravity affects numerous physical phenomena relevant to biological research, including the hydrostatic pressure in fluid filled vesicles, sedimentation of organelles, and buoyancy-driven convection of flow and heat . These physical phenomena can in turn directly and indirectly affect cellular morphology, metabolism, locomotion, secretion of extracellular matrix and soluble signals, and assembly into functional tissues . Studies aimed at distinguishing specific effects of gravity on biological systems require the ability to: (i) control and systematically vary gravity, e.g . by utilizing the microgravity environment of space in conjunction with an in-flight centrifuge; and (ii) maintain constant all other factors in the immediate environment, including in particular concentrations and exchange rates of biochemical species and hydrodynamic shear . The latter criteria imply the need for gravity-independent mechanisms to provide for mass transport between the cells and their environment . Available flight hardware has largely determined the experimental design and scientific objectives of spaceflight cell and tissue culture studies carried out to date . Simple culture vessels have yielded important quantitative data, and helped establish in vitro models of cell locomotion, growth and differentiation in various mammalian cell types including embryonic lung cells {6}, lymphocytes {2,8}, and renal cells {7,31} . Studies done using bacterial cells established the first correlations between gravity-dependent factors such as cell settling velocity and diffusional distance and the respective cell responses {12} . The development of advanced bioreactors for microgravity cell and tissue culture and for tissue engineering has benefited both research areas and provided relevant in vitro model systems for studies of astronaut well-being (loss of muscle and skeletal tissues {15-17}) and gene- and cell-level responses to the mechanical environment {13,14,18} . All five of the spaceflight bioreactor studies described above utilized three-dimensional cell culture systems in which the cells were associated with biodegradable polymer scaffolds {17}, collagen gel {16}, or microcarrier beads {13-15,18} in order to promote the expression of differentiated cell function . In four of the five spaceflight bioreactor studies {15-18}, cells were cultured in perfused vessels (cartridges or rotating bioreactors) within recirculating loops designed to maintain medium composition within target ranges by a combination of gas exchange and fresh medium supply . Future spaceflight studies of cells and tissues are likely to involve a three-dimensional culture system, to promote cellular differentiation, and perfusion with or without rotation, to provide a gravity-independent mechanism for fluid mixing and mass transport . Previous spaceflight studies have guided the ongoing development of NASA flight hardware for the ISS (e.g . the EDU-2 and the CCU) . This next generation of hardware will have extended operational capabilities including on-line microscopy, in-line sensors for the monitoring and control of metabolic parameters, modular design for replicate cultures, and, perhaps most importantly of all, compatibility with the ISS centrifuge . The latter will permit in-flight, 1 g control cultures, and thereby allow the experimental variable to be gravity itself rather than the more general "spaceflight environment" . Technical limitations of spaceflight studies (e.g . allowable size, mass, and power) continue to motivate a creative approach to system design and to result in "spin-off" technologies (e.g . the STLV) for ground-based cell and tissue culture research . The increasing scientific and medical relevance of this work is evidenced by the growing number of publications in which advanced bioreactors are used for in vitro studies in physiologically relevant cell and tissue models.

Pharmacol Ther, 2003 Sep, 99(3), 261 - 82
Transgenic rabbits as therapeutic protein bioreactors and human disease models; Fan J et al.; Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to directly examine structure-function and cause-and-effect relationships in pathophysiological processes . Today, transgenic mice are available as a research tool in almost every research institution . On the other hand, the development of a relatively large mammalian transgenic model, transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also provided an alternative way to produce therapeutic proteins to treat human diseases . Transgenic rabbits expressing human genes have been used as a model for cardiovascular disease, AIDS, and cancer research . The recombinant proteins can be produced from the milk of transgenic rabbits not only at lower cost but also on a relatively large scale . One of the most promising and attractive recombinant proteins derived from transgenic rabbit milk, human alpha-glucosidase, has been successfully used to treat the patients who are genetically deficient in this enzyme . Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic cell nuclear transfer in the future . Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbitsPublication Types:

	Review
	Review, Tutorial

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