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 c