What Is Bioreactor?

A bioreactor is a vessel in which is carried out a chemical process which involves organisms or biochemically active substances derived from such organisms.

Bioreactors are commonly cylindrical, ranging in size from some liter to cube meters,and are often made of stainless steel.

Bioreactor design is quite a complex engineering task. Under optimum conditions the microorganisms or cells will reproduce at an astounding rate. The vessel's environmental conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide) flowrates, temperature, pH and dissolved oxygen levels, and agitation speed need to be closely monitored and controlled. One bioreactor manufacturer, Broadley-James Corporation, uses vessels, sensors, controllers, and a control system, digitally networked together for their bioreactor system.

Continuous flow stirred tank reactors (chemostat) In the continuous flow, stirred tank reactor (CSTR or chemostat) fresh medium is fed into the bioreactor at a constant rate, and medium mixed with cells leaves the bioreactor at the same rate. A fixed bioreactor volume is maintained and ideally, the effluent stream should have the same composition as the bioreactor contents. The culture is fed with fresh medium containing one and sometimes two growth-limiting nutrients such as glucose. The concentration of the cells in the bioreactor is controlled by the concentration of the growth-limiting nutrient. A steady state cell concentration is reached where the cell density and substrate concentration are constant. The cell growth rate (µ) is controlled by the dilution rate (D) of growth limiting nutrient.

Cell culture bioreactors are categorized into two types: 1. Those that are used for cultivation of anchorage dependent cells (e.g. primary cultures derived from normal tissues and diploid cell lines. 2. Those that are used for the cultivation of suspended mammalian cells (e.g. cell lines derived from cancerous tissues and tumors, transformed diploid cell lines, hybridomas). In some cases the bioreactor may be modified to grow both anchorage dependent and suspended cells. Ideally any cell culture bioreactor must maintain a sterile culture of cells in medium conditions which maximize cell growth and productivity.

Fouling can harm the overall sterility and efficiency of the bioreactor, especially the heat exchangers. To avoid it the bioreactor must be easily cleanable and must be as smooth as possible (therefore the round shape).

Heat exchange is needed to maintain the bioprocess at a constant temperature. Biological fermentation is a major source of heat, therefore in most cases bioreactors need water refrigeration. They can be refrigerated with an external jacket or, for very large vessels, with internal coils.

Optimal oxygen transfer is perhaps the most difficult task to accomplish. Oxygen is poorly soluble in water -and even less in fermentation broths- and is relatively scarce in air (20.8%). Oxygen transfer is usually helped by agitation, that is also needed to mix nutrients and to keep the fermentation homogeneous. There are however limits to the speed of agitation, due both to high power consumption (that's proportional to the cube of the speed) and the damage to organisms due to excessive tip speed.

Bioreactor treatment may be performed using microorganisms growing in suspension in the fluid or attached on a solid growth support medium. In suspended growth systems, such as fluidized beds or sequencing batch reactors, contaminated groundwater is circulated in an aeration basin where a microbial population aerobically degrades organic matter and produces carbon dioxide, water, and biomass. The biomass is settled out in a clarifier, then either recycled back to the aeration basin or disposed of as sludge. In attached growth systems, such as upflow fixed film bioreactors, rotating biological contactors (RBCs), and trickling filters, microorganisms are grown as a biofilm on a solid growth support matrix and water contaminants are degraded as they diffuse into the biofilm. Support media include solids that have a large surface area for bacterial attachment.

A bioreactor landfill operates to rapidly transform and degrade organic waste. The increase in waste degradation and stabilization is accomplished through the addition of liquid and air to enhance microbial processes. This bioreactor concept differs from the traditional “dry tomb” municipal landfill approach.

A bioreactor landfill is not just a single design and will correspond to the operational process invoked. There are three different general types of bioreactor landfill configurations:

Aerobic - In an aerobic bioreactor landfill, leachate is removed from the bottom layer, piped to liquids storage tanks, and re-circulated into the landfill in a controlled manner. Air is injected into the waste mass, using vertical or horizontal wells, to promote aerobic activity and accelerate waste stabilization. Anaerobic - In an anaerobic bioreactor landfill, moisture is added to the waste mass in the form of re-circulated leachate and other sources to obtain optimal moisture levels. Biodegradation occurs in the absence of oxygen (anaerobically) and produces landfill gas. Landfill gas, primarily methane, can be captured to minimize greenhouse gas emissions and for energy projects. Hybrid (Aerobic-Anaerobic) - The hybrid bioreactor landfill accelerates waste degradation by employing a sequential aerobic-anaerobic treatment to rapidly degrade organics in the upper sections of the landfill and collect gas from lower sections. Operation as a hybrid results in the earlier onset of methanogenesis compared to aerobic landfills The Solid Waste Association of North America (SWANA) has defined a bioreactor landfill as "any permitted Subtitle D landfill or landfill cell where liquid or air is injected in a controlled fashion into the waste mass in order to accelerate or enhance biostabilization of the waste." The United States Environmental Protection Agency (EPA) is currently collecting information on the advantages and disadvantages of bioreactor landfills through case studies of existing landfills and additional data so that EPA can identify specific bioreactor standards or recommend operating parameters.

Features Unique to Bioreactor Landfills: The bioreactor accelerates the decomposition and stabilization of waste. At a minimum, leachate is injected into the bioreactor to stimulate the natural biodegradation process. Bioreactors often need other liquids such as stormwater, wastewater, and wastewater treatment plant sludges to supplement leachate to enhance the microbiological process by purposeful control of the moisture content and differs from a landfill that simple recirculates leachate for liquids management. Landfills that simply recirculate leachate may not necessarily operate as optimized bioreactors.

J Biomech, 2005 Mar, 38(3), 543 - 9
3-D computational modeling of media flow through scaffolds in a perfusion bioreactor; Porter B et al.; Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro . In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs . Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture . We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds . Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity . Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media . The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution . Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes . This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.

J Biotechnol, 2005 Mar 2, 116(1), 61 - 77 Epub 2004 Nov 30.
Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus; Casas Lopez JL et al.; Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production . The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers . Pellets of approximately 1200mum initial diameter were reduced to a final stable size of approximately 900mum when the agitation intensity was >/=600rpm (u(t)>/=2.03ms(-1)) . A stable pellet diameter of approximately 2500mum could be attained in less intensely agitated cultures . These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air . Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere . This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin . Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function . The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers . Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.

Appl Microbiol Biotechnol . 2005 Jan 13; {Epub ahead of print}
Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745; Kollmann A et al.; Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi . The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity . We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes . versicolor . We identified 13 oligomers formed from 2,5-xylidine . (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25x10(-5) M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture . Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.

Ann N Y Acad Sci, 2004 Nov, 1027, 85 - 98
Modeling of phosphate ion transfer to the surface of osteoblasts under normal gravity and simulated microgravity conditions; Mukundakrishnan K et al.; We have modeled the transport and accumulation of phosphate ions at the remodeling site of a trabecular bone consisting of osteoclasts and osteoblasts situated adjacent to each other in straining flows . Two such flows are considered; one corresponds to shear levels representative of trabecular bone conditions at normal gravity, the other corresponds to shear level that is representative of microgravity conditions . The latter is evaluated indirectly using a simulated microgravity environment prevailing in a rotating wall vessel bioreactor (RWV) designed by NASA . By solving the hydrodynamic equations governing the particle motion in a RWV using a direct numerical simulation (DNS) technique, the shear stress values on the surface of the microcarriers are found . In our present species transfer model, osteoclasts release phosphate ions (Pi) among other ions at bone resorption sites . Some of the ions so released are absorbed by the osteoblast, some accumulate at the osteoblast surface, and the remainder are advected away . The consumption of Pi by osteoblasts is assumed to follow Michaelis-Menten (MM) kinetics aided by a NaPi cotransporter system . MM kinetics views the NaPi cotransporter as a system for transporting extracellular Pi into the osteoblast . Our results show, for the conditions investigated here, the net accumulation of phosphate ions at the osteoblast surface under simulated microgravity conditions is higher by as much as a factor of three . Such increased accumulation may lead to enhanced apoptosis and may help explain the increased bone loss observed under microgravity conditions.

Artif Organs, 2005 Jan, 29(1), 58 - 66
A novel bioartificial liver with culture of porcine hepatocyte aggregates under simulated microgravity; Hochleitner B et al.; An extracorporeal bioartificial liver device could provide vital support to patients suffering from acute liver failure . We designed a novel, customized bioreactor for use as a bioartificial liver (patent pending) . The Innsbruck Bioartificial Liver (IBAL) contains aggregates of porcine hepatocytes grown under simulated microgravity . The culture vessel rotates around its longitudinal axis and is perfused by two independent circuits . The circuit responsible for exchange of plasma components with the patient consists of a dialysis tube winding spirally around the internal wall of the culture vessel . IBAL was evaluated in vitro . Viability tests showed sufficient viability of hepatocytes for up to 10 days . Cytologic examination of samples from the bioreactor showed liver cell aggregates . These were also examined by electron microscopy . A number of biochemical parameters were analyzed . In conclusion, cell culture is possible for at least 10 days in the IBAL system, organoid hepatocyte aggregates are formed and synthetic activity of the hepatocytes was demonstrated.

Biotechnol Bioeng . 2005 Jan 10; {Epub ahead of print}
Novel application of oxygen-transferring membranes to improve anaerobic wastewater treatment; Kappell AS et al.; Anaerobic biological wastewater treatment has numerous advantages over conventional aerobic processes; anaerobic biotechnologies, however, still have a reputation for low-quality effluents and operational instabilities . In this study, anaerobic bioreactors were augmented with an oxygen-transferring membrane to improve treatment performance . Two anaerobic bioreactors were fed a synthetic high-strength wastewater (chemical oxygen demand, or COD, of 11,000 mg l(-1)) and concurrently operated until biomass concentrations and effluent quality stabilized . Membrane aeration was then initiated in one of these bioreactors, leading to substantially improved COD removal efficiency (>95%) compared to the unaerated control bioreactor ( approximately 65%) . The membrane-augmented anaerobic bioreactor required substantially less base addition to maintain circumneutral pH and exhibited 75% lower volatile fatty acid concentrations compared to the unaerated control bioreactor . The membrane-aerated bioreactor, however, failed to improve nitrogenous removal efficiency and produced 80% less biogas than the control bioreactor . A third membrane-augmented anaerobic bioreactor was operated to investigate the impact of start-up procedure on nitrogenous pollutant removal . In this bioreactor, excellent COD (>90%) and nitrogenous (>95%) pollutant removal efficiencies were observed at an intermediate COD concentration (5,500 mg l(-1)) . Once the organic content of the influent wastewater was increased to full strength (COD = 11,000 mg l(-1)), however, nitrogenous pollutant removal stopped . This research demonstrates that partial aeration of anaerobic bioreactors using oxygen-transferring membranes is a novel approach to improve treatment performance . Additional research, however, is needed to optimize membrane surface area versus the organic loading rate to achieve the desired effluent quality . (c) 2005 Wiley Periodicals, Inc.

Yi Chuan, 2004 Nov, 26(6), 977 - 83
{The application of chloroplast genome transformation in higher plant.}; Wang YF et al.; The chloroplast genome transformation of higher plant has become the research hotspot of plant genetic engineering with several advantages over nuclear genetic engineering, and it has become into a powerful approach for both basic and applied research . This paper presents a brief summary for the principle and methods of chloroplast genome transformation in higher plant . The particular emphasis was placed on its application in the basic and applied research . These applications include the studies of rearrangement of Rubisco and the structure, transcription, translation and RNA editing of genes in chloroplast; the producing of antibodies, vaccines, poly-beta-hydroxybutyrate and bio-elastic protein using chloroplast as bioreactor; the generating of transgenic plants with resistance to insect, disease, herbicide and drought; and the decreasing the gene flow of transgenic plants.

Yi Chuan, 2004 Sep, 26(5), 567 - 73
{Mammary Gland Dual-Expression Construct Containing Prolactin and Foreign DNAs Can Promote the Expression of Foreign Proteins in Mammary Cells.}; Liu FT et al.; To investigate the feasibility in the promotion of the expression of foreign proteins, such as human lactinferrin (hLF) and thrombopoietin (TPO), in the transgenic animals-mammary gland bioreactor by bovine prolactin (bPRL), two types of mammary gland dual-expression vectors containing bPRL and foreign DNAs were constructed . In one type of vector, both foreign and bPRL DNAs were linked by internal ribosome entry site (IRES) which shared one goat 6.7 kb of beta-casein promoter; while in the other type of vector, the transcriptions of the foreign gene aswell as bPRL were respectively directed by 6.7 kb goat beta-casein promoter and 2.0 kb of goat beta-casein promoter . After transfection of the vectors with lipofectin method, the expression of foreign proteins were measured by RT-PCR as well as ELISA assay . The results showed that bPRL could obviously promote the expression of foreign proteins (hLF and TPO) in cultured cells . The hLF expression level was increased from 12.6 mug/L to 18.4 mug/L and 37.2 mug/L in the COS-7 cells (African green monkey kidney cell) (P<0.05), and from 13.7 mug/L to 20.7 mug/L and 19.9 mug/L in the HC-11 cells (epithelial cell of mouse mammary gland) (P<0.05) by two vectors, respectively . TPO expression level was increased from 572 ng/L to 1340 ng/L in the COS-7 cell (P<0.05), and from 783 ng/L to 1040 ng/L in the HC-11 cell (P<0.05) by the vector which have two promoters . This work indicate that bovine prolactin can effectively increase the expression of foreign genes in mammal cells.

Yi Chuan, 2004 Jul, 26(4), 537 - 43
{Achievements and Applications in Making Chicken Chimeras Using BCs.}; Yan HF et al.; The technology of producing chicken chimeras using blastodermal cells is very important not only in the field of transgenic chicken bioreactor but also in searching for efficient ways to conserve avian genetic resource . The basic processes for producing chicken chimeras consist of: (1) Setting up the color model; (2) Separating and dissociating of donor embryos; (3) Compromising of the recipient embryos; (4) Windowing and recovering the recipient eggs; (5) Cells injecting; (6) Method of hatching . The progress, obstacles and prospects of producing chicken chimeras via BCs were discussed in this paper.

Yi Chuan, 2003 Jan, 25(1), 107 - 12
{Plants as bioreactor for the production of pharmaceutical proteins.}; Xiao NZ et al.; Transgenic plant as bioreactor has been used to produce recombinant proteins for medicinal purposes,including mammalian antibodies,blood substitutes and vaccines.As the demand for biopharmaceuticals is expected to increase,transgenic plants have the potential to provide virtually unlimited quantities of proteins for use as tools in both human health care and the bioscience.This paper reviews the recent developments in this field and the prospect of commercial applications.

J Biotechnol, 2005 Feb 23, 115(4), 379 - 386 Epub 2004 Nov 21.
Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica; Fickers P et al.; The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene . Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering . In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene . We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures . Batch culture led to a lipase production of 26450Uml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources . Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246Uml(-1) after 80h of cultivation . In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.

J Biotechnol, 2005 Feb 23, 115(4), 333 - 43 Epub 2004 Nov 24.
Comparison of different fungal enzymes for bleaching high-quality paper pulps; Sigoillot C et al.; Wild and recombinant hydrolases and oxidoreductases with a potential interest for environmentally sound bleaching of high-quality paper pulp (from flax) were incorporated into a totally chlorine free (TCF) sequence that also included a peroxide stage . The ability of feruloyl esterase (from Aspergillus niger) and Mn(2+)-oxidizing peroxidases (from Phanerochaete chrysosporium and Pleurotus eryngii) to decrease the final lignin content of flax pulp was shown . Laccase from Pycnoporus cinnabarinus (without mediator) also caused a slight improvement of pulp brightness that was increased in the presence of aryl-alcohol oxidase . However, the best results were obtained when the laccase treatment was performed in the presence of a mediator, 1-hydroxybenzotriazol (HBT), enabling strong delignification of pulps . The enzymatic removal of lignin resulted in high-final brightness values that are difficult to attain by chemical bleaching of this type of pulp . A partial inactivation of laccase by HBT was observed but this negative effect was strongly reduced in the presence of pulp . The good results obtained with the same laccase expressed in A . niger at bioreactor scale, revealed the feasibility of using recombinant laccase for bleaching high-quality non-wood pulps in the presence of a mediator.

J Biotechnol, 2005 Feb 9, 115(3), 307 - 15
Efficient production of a soluble fusion protein containing human beta-defensin-2 in E . coli cell-free system; Chen H et al.; Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not cause microbial resistance . In order to produce hBD2 efficiently, an Escherichia coli cell-free biosynthesis system has been developed as an alternative method . A specific plasmid pIVEX2.4c-trxA-shBD2 was constructed for the cell-free expression of fusion protein (hBD2 linked with His-Tag and Trx-Tag) . This allowed enhancement of protein stability and facilitated downstream purification process . Significant amount of target fusion protein was synthesized in the batch-mode bioreactor by optimizing the reaction conditions . About five-fold improvement of productivity (ca . 2.0mg/ml soluble fusion protein) could be achieved by using a continuous exchange cell-free (CECF) system compared to batch system . One-step affinity chromatographic process was developed to recover high purity fusion protein (95.2%) with overall recovery ratio of about 50% . The fusion protein was cleaved by cyanogens bromide (CNBr), and the mature hBD2 had demonstrated strong inhibition on the growth of E . coli D31 at low concentration.

J Biotechnol, 2005 Feb 9, 115(3), 291 - 301 Epub 2004 Nov 05.
The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells; Heilmann K et al.; The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS) . Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA) . Cell number and antibody concentration were quantified as criteria for viability and productivity . Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy . The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production . In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production . In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption . It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.

Protein Pept Lett, 2005 Jan, 12(1), 85 - 8
Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma; Guan YX et al.; The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth . In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.

Res Microbiol, 2005 Jan-Feb, 156(1), 82 - 7
Optimisation of growth conditions for continuous culture of the hyperthermophilic archaeon Thermococcus hydrothermalis and development of sulphur-free defined and minimal media; Postec A et al.; The hyperthermophilic archaeon Thermococcus hydrothermalis was cultivated in continuous culture in a gas-lift bioreactor in the absence of elemental sulphur on both proteinaceous and maltose-containing media . Optimal conditions (pH, temperature and gas flow rate), determined on complex media that yielded maximal growth rate and maximal steady state cell density, were obtained at 80 degrees C, pH 6 and gas sparging at 0.2 v v(-1) min(-1) . Higher steady state cell densities were obtained on a medium containing maltose and yeast extract . In order to design a defined and minimal media, the nutritional requirements of T . hydrothermalis were then investigated using continuous culture in the absence of elemental sulphur in the gas-lift bioreactor . First, the complex nutriments were replaced and a defined medium containing maltose, 19 amino acids and the two nitrogenous bases adenine and thymine, was determined . Secondly, selective feedings and withdrawal of amino acids showed requirements for 14 amino acids.

Biotechnol Appl Biochem . 2005 Jan 6; {Epub ahead of print}
Application of radial-flow bioreactor on production of beta1, 3-N-acetylglucosaminyltransferase-2 fused with GFP uv using stably transformed insect cell lines; Kwon MS et al.; A radial-flow bioreactor (RFB) with a reactor volume of 5 ml was applied to produce human beta1, 3-N-acetylglucosaminyltransferase (beta3GnT) using two stably transformed insect cell lines . When air was supplied to the RFB, cell growth was stopped at 4-d culture and beta3GnT was not detected . However, with a supply of pure oxygen, the cell concentration, assumed from glucose consumption, increased 1.3 x 10 7 cells/ml . Insect cells attached to polyvinyl alcohol (PVA) matrixes packed in the RFB and grew confluently . The 5.6 mU/ml of beta3GnT was produced under the conditions of a pure oxygen supply and the addition of glucose and glutamine . This RFB was first applied on the beta3GnT production using stably transformed insect cells . The amount of beta3GnT production in only a 5 ml scale RFB was comparable to that of a 100 ml shaking flask culture.

Int J Artif Organs, 2004 Nov, 27(11), 962 - 70
Hydrodynamic stimulation and long term cultivation of nucleus pulposus cells: a new bioreactor system to induce extracellular matrix synthesis by nucleus pulposus cells dependent on intermittent hydrostatic pressure; Gokorsch S et al.; A novel bioreactor system was constructed to induce extracellular matrix (ECM) synthesis by intervertebral disc (ID) cells due to intermittent hydrostatic pressure . The developed system is completely sterilizable and reusable . It is viable for cultivation, immobilization, and stimulation of various other cell types and tissues especially for cartilage . The custom made lid allows long-run cultivation through semi-continuous operation . Manual interferences and therefore the risk of contamination are reduced . Sampling, medium changing and addition of supplements are easily performed from the connected conditioning vessel, which could be placed in an incubator . For the present investigations nucleus pulposus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs which were subjected to intermittent hydrostatic pressure . Afterwards the construct was biochemically examined . The proven constituents of ECM were found to be released in dependence of the magnitude and profile of the applied pressure.

Biotechnol Bioeng . 2005 Jan 5; {Epub ahead of print}
Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor; Klein J et al.; In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented . A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol . The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process . Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity . Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm(3) corresponding to 42.8% v/v of solid fraction . These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor . (c) 2004 Wiley Periodicals, Inc.

Zhonghua Yi Xue Za Zhi, 2004 Nov 2, 84(21), 1832 - 5
{In vitro immunocompatibility of a novel bioartificial liver reactor material: propylene-acidamide grafted polypropylene membrane.}; Peng CH et al.; OBJECTIVE: To evaluate the immunocompatibility of a novel bioartificial liver bioreactor material: propylene-acidamide grafted polypropylene membrane (PP-g-AAm) in vitro on peripheral blood mononuclear cells (PBMCs) . METHODS: Fifty milliliters of peripheral blood were collected from 30 healthy young people . PBMCs were extracted and inoculated on the 24-well culture plate preset with sterilized PP-g-AAm membrane and ungrafted polypropylene (PP) membrane . Automatic biochemical analyzer was used to detect the lactic dehydrogenase (LDH) value of the PMBCs after 2 and 6 hours . The PMBCs on these 2 kinds of membrane were cultured for 6 hours and then added with lipopolysaccharide and cultured continually . Six, twelve, and thirty-six hours later the supernatant was collected . ELISA was used to detect the values of tumor necrosis factor (TNF)alpha, interleukin (IL)-1beta, and IL-6 . Flow cytometry was used to detect the expression of CD69 antigen . Scanning electron microscopy was used to observe the adhesion of PMBCs on the materials . RESULTS: After 2 hours' epimembranous inoculation the LDH value was 43.50 U/L +/- 12.71 U/L in the PP group, significantly higher than that in the PP-g-AAm group (29.13 U/L +/- 8.74 U/L, P = 0.008) and the newly extracted PMBC group (0 h group, 19.89 U/L +/- 4.67 U/L, P = 0.000) . However, the difference between the 0 h group and PP-g-AAm group (P = 0.080) was insignificant . After 6 hours' epimembranous inoculation the LDH value was 50.25 U/L +/- 13.38 U/L in the PP group, and 32.50 U/L +/- 9.21 U/L in the PP-g-AAm group (P = 0.001), both significantly higher than that in the 0 h group (P = 0.000, and P = 0.019) . There was no significant difference between the values 2 hours and 6 hours after inoculation for the two groups . No expression of TNFalpha, IL-1beta, and IL-6 was found in the supernatant of the 2 groups without LPS stimulation . Expressions of TNFalpha, IL-1beta, and IL-6 could be found at a low level 12 hours after LPS stimulation for both groups and peaked 24 hours after LPS stimulation . The expressions of TNFalpha, IL-1beta, and IL-6 were lower in the PP-g-AAm group than in the PP group (P = 0.004, P = 0.003, and P = 0.022) . The TNFalpha, IL-1beta, and IL-6 levels all decreased after 36 hours . The CD69 antigen expression rates were 17.20% +/- 3.45%and 12.02% +/- 2.44% respectively in the PP group and PP-g-AAm group, both significantly higher than that of the blank control group (3.38% +/- 1.30%, both P = 0.000) . SEM showed that the PMBCs adhered on the PP-Aam membrane were significantly less then those adhered on the PP membrane . And the PMBCs adhered on the PP-g-AAm membrane were smaller and with less microvilli . CONCLUSION: PP-g-AAm membrane has weaker activation capability to PMBCs and has better immunocompatibility in comparison with the PP membrane.

IEEE Trans Nanobioscience, 2004 Dec, 3(4), 243 - 50
Complex permittivity measurement as a new noninvasive tool for monitoring in vitro tissue engineering and cell signature through the detection of cell proliferation, differentiation, and pretissue formation; Bagnaninchi PO et al.; In in vitro tissue engineering, microporous scaffolds are commonly used to promote cell proliferation and differentiation in three-dimensional structures . Classic measurement methods are particularly time consuming, difficult to handle, and destructive . In this study, a new nondestructive method based on complex permittivity measurement (CPM) is proposed to monitor and track the osteoblast and macrophage differentiation through their morphological variation upon cell attachment and proliferation inside the microporous scaffolds . CPM is performed using a vector network analyzer and a dielectric probe under sterile conditions in a laminar-flow hood . A suitable effective medium approximation (EMA) is applied to fit the data in order to extract the parameters of the different constituents . Our data show that the EMA depolarization factor can be monitored to assess the variation of cell morphology characterizing cell attachment . Discrimination between two batches of scaffolds seeded, respectively, with 2 million and 1 million osteoblast cells is possible; the ratio of their CPM-derived cell volume fractions is in agreement with the ratio of their cell seeding numbers . In addition, cell proliferation inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium and inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium supplemented to induce the formation of extracellular matrix is monitored via CPM over several days . CPM-determined cell volume fraction is compared to DNA assay cell counts . Extracellular matrix formation and cell presence was confirmed by scanning electron microscopy . A set of three signature parameters (epsilon'mem, epsilon'cyt, kappa'cyt) characteristic of cell line is extracted from CPM . Distinct signatures are recorded for osteoblasts and macrophages, thus confirming the ability of CPM to discriminate between different cell types . This study demonstrates the potential of CPM as a diagnostic tool to monitor quickly and noninvasively cell growth and differentiation inside microporous scaffolds . Our findings suggest that the use of CPM could be extended to many biomedical applications, such as drug detection and automation of tissue and bacterial cultures in bioreactors.

Appl Microbiol Biotechnol . 2005 Jan 4; {Epub ahead of print}
Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation; Wang W et al.; A single addition of 200 muM methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume) . Repeated elicitation with two additions of 200 muM MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels . The content of ginsenosides Rg(1), Re, Rb(1) and Rd in the ALR was increased from, respectively, 0.18+/-0.01, 0.21+/-0.01, 0.21+/-0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32+/-0.02, 0.36+/-0.02, 0.72+/-0.06 and 0.08+/-0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43+/-0.02, 0.46+/-0.03, 1.09+/-0.07 and 0.14+/-0.02 mg per100 mg DW with two additions of MJA . Interestingly, the activity of the Rb(1) biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb(1) content . In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted . The final cell density and total ginsenoside content in the ALR reached 27.3+/-1.5 g/l and 2.02+/-0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg(1), Re, Rb(1) and Rd was 111.8+/-4.7, 117.2+/-4.6, 290.2+/-5.1 and 32.7+/-8.1 mg/l, respectively . The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.

Yi Chuan, 2004 Jan, 26(1), 75 - 83
{Analysis of Codon Usage in Potato and Its Application in the Modification of t-PA Gene.}; Bai X et al.; Bioperl-1.0 was used under Hongqi LINUX system to programm the codon analysis software.According to the analysis of 98 codon DNA sequences with this software,the codon usage in potato was calculated and 4 codons have been inferred to the optimal codons.The codons of tissue plasminogen activator (t-PA) gene sequence have been reconstructed according to the results.The t-PA gene sequence containing the optimal codons of potato will be used for t-PA production by potato bioreactor.

Biotechnol Bioeng . 2004 Dec 29; {Epub ahead of print}
Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C; Yoon SK et al.; To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control . Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period . Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively . Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C . In addition, they increased with increasing culture pH . Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C . At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH . The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C . The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH . Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C . Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C . These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells . (c) 2004 Wiley Periodicals, Inc.

Biotechnol Bioeng . 2004 Dec 29; {Epub ahead of print}
Low-cost noninvasive optical CO(2) sensing system for fermentation and cell culture; Ge X et al.; High-throughput bioprocessing is a very promising technique for bioprocess development and optimization because of its high efficiency . The key to its development has been the availability of simple and inexpensive sensors to monitor the bioprocesses conducted in its small-scale bioreactors . Here we report on a low-cost noninvasive CO(2) sensing system suitable for any transparent vessel . The system was composed of a CO(2) sensing patch, a coaster, an interface, and a computer . The sensing film was prepared using the ion-pair technique . The coaster was a small self-made device with necessary optics and electronics for ratiometric measurements with a component cost of less than $100 . Results show that the system was stable and reliable despite its simplicity and low cost . The sensitivity of the CO(2) sensing system was not affected by pH, media type, or temperature . It was shown to be stable for at least 10 days, long enough for most bioprocesses . (c) 2004 Wiley Periodicals, Inc.

Huan Jing Ke Xue, 2004 Sep, 25(5), 102 - 5
{Printing and dyeing wastewater treatment using combined process of anaerobic bioreactor and MBR}; Zheng X et al.; This paper describes a labor-scale experiment for printing and dyeing wastewater treatment of woolen mill using a combined process of an anaerobic reactor and a membrane bioreactor (MBR) . The experimental results showed that when the concentration of COD, BOD5 and color in the influent were 128-321 mg/L, 36-95 mg/L and 40-70 dilution times (DT), the average concentrations of COD, BOD5, color and turbidity in the effluent were 36.9 mg/L, 3.7 mg/L, 21 DT and 0.24 NTU, respectively, and the corresponding removal rates were 80.3%, 95%, 59% and 99.3%, respectively . A new integrated membrane bioreactor by gravity drain of liquid level in the bioreactor was developed in this study . It not only lessens suction pump for effluent and controlling unit comparing to the traditional integrated membrane bioreactor, but also has the characters of high and continuous flux, concise configuration and simple operation and maintenance . According to the experimental results, the air flow rate through the membrane module is a significant factor to affect the flux rate and cake layer deposited on the membrane . With application of optimal air flow rate, it is effective to reduce membrane fouling and maintain high flux rate.

Biotechnol Bioeng . 2004 Dec 23; {Epub ahead of print}
Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag; Collen A et al.; The effect of induction of protein production was studied in bioreactor cultures of T . reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag . The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation . The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production . Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations . Induction of the cellulase genes followed a similar temporal pattern in both strains . The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed . Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter . The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein . The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions . However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain . (c) 2004 Wiley Periodicals, Inc.

J Agric Food Chem, 2004 Dec 29, 52(26), 7842 - 5
A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate; Je JY et al.; Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin . This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system . Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column . Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala . In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.

Biotechnol Bioeng . 2004 Dec 17; {Epub ahead of print}
Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: Metabolic response and production of recombinant protein; Sandoval-Basurto EA et al.; A significant problem of large-scale cultures, but scarcely studied for recombinant E . coli, is the presence of gradients in dissolved oxygen tension (DOT) . In this study, the effect of DOT gradients on the metabolic response of E . coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined . DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6% . Cells were continuously circulated between both vessels to simulate circulation times (t(c)) of 20, 50, 90, and 180 sec . A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20% . The performance of E . coli cultured under oscillating DOT was significantly affected, even at a t(c) of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions . Specific growth rate decreased linearly with t(c) to a maximum reduction of 30% at the highest t(c) tested . The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin . In these cases, the losses were 9%, 27%, and 20%, respectively, at t(c) of 20 sec and 65%, 94%, and 87%, respectively, at t(c) of 180 sec . Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses . The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity . The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E . coli exposed to DOT gradients . (c) 2004 Wiley Periodicals, Inc.

J Ind Microbiol Biotechnol . 2004 Dec 17; {Epub ahead of print}
Cometabolic reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor; Ginkel CG et al.; The discharge of bromate, a suspected carcinogen, will be restricted in the near future . To assess the possibility of biotechnological treatment of bromate-containing wastewaters, the removal of bromate by chlorate-reducing microorganisms was studied . The removal of bromate and chlorate was studied in laboratory gas-lift bioreactors supplied with hydrogen gas as electron donor in the absence of molecular oxygen . In these reactors, bromate was reduced cometabolically by chlorate-respiring microorganisms . To allow the cometabolic reduction of bromate, a chlorate:bromate molar ratio of at least 3:1 was required . The cometabolic conversion permitted almost complete reduction of bromate into bromide at hydraulic retention times of at least 6 h . Optimal bromate reduction activity was observed at approximately 35 degrees C . The pH optimum was between 7 and 8 . Bromate reduction in excess of 80% and a maximum bromate reduction rate of 2.3 g l(-1) day(-1) in a pilot-scale gas-lift bioreactor demonstrates that the process is sustainable.

Biotechnol Lett, 2004 Nov, 26(21), 1619 - 22
Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate; Kim YS et al.; Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150mu and cultured for 40days . Up to 100mu MJ inhibited the root growth but increase ginsenoside accumulation . In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 mu MJ peaked after 10days at 48mgg(-1) dry wt and then dropped sharply . Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.

Altern Lab Anim, 2004 Mar, 32(1), 25 - 35
Evaluation of a flat membrane hepatocyte bioreactor for pharmacotoxicological applications: evidence that inhibition of spontaneously produced nitric oxide improves cell functionality; Canova N et al.; A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes . In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days . Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production . Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB . The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way . The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity . In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.

J Neurosci Res, 2005 Jan 1-15, 79(1-2), 26 - 32
Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor; Sa Santos S et al.; We report the first approach for growth and maintenance of primary astrocytes on a fully controlled environment . For this purpose, cells were immobilized in Cytodex microcarriers and grown in a stirred tank bioreactor . The distribution of astrocytes at the microcarrier surface was visualized using confocal microscopy and glial fibrillary acidic protein (GFAP) labeling, a specific glial probe . Crucial bioreaction parameters such as agitation rate, microcarrier type, and concentration, as well as cell inoculum concentration were assessed . Cytodex 3 proved the best microcarrier for astrocyte growth, with the highest cell densities obtained for 6 g/l of Cytodex 3 using an inoculum of approx . 0.15 x 10(6) cells/ml in vessels operated at 60 rpm, using a refeed operational mode consisting of complete medium replacement every 5 days . Using such optimized conditions, cells were maintained in steady-state for approximately 24 days, allowing online monitoring and control of environmental variables such as temperature, pH, and O(2) . To test further the advantages of this fully controlled system, astrocytes were also subjected to hypoxic stress for 5 hr; the cell number was not affected by hypoxia but the glycolytic flux was enhanced during the stress imposed . The culture system described is a novel tool to study brain cell metabolism, allowing sampling over time and the monitoring of cellular behavior through stressful conditions and during recovery . (c) 2004 Wiley-Liss, Inc.

Hum Antibodies, 2004, 13(3), 69 - 79
Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for Membrane Anion Exchange Chromatography one-step purification; Jacobin MJ et al.; Purification of human IgM monoclonal antibodies (MAbs) has proved to be difficult . Since IgM Mabs tend to bind strongly to a variety of resin support surfaces, the number of chromatographic steps used in the purification of these biomolecules should be minimized . Here we describe procedures developed for the optimal production and purification of the human monoclonal IgM B7, which specifically binds to the myosin heavy chain of human ventricular myocardium . This property makes this antibody potentially useful for the diagnosis of myocardial necrosis . Several chromatographic techniques were evaluated (size exclusion, ion exchange, affinity chromatography) . The best results were obtained with anion exchange membrane chromatography using Sartobind Q15 (98% purity, 30% recovery) . IgM production was improved by the hollow fiber technology which permitted the use of serum-reduced medium and an increase in antibody concentration to an average production of 300-400 microg/ml, compared to 20 microg/ml in flask culture . Several flow-rates were also evaluated, the optimal being 20 ml/minute for 30% of recovery . Importantly, the purified IgM molecule was able to bind to human myosin in ELISA and Western-blotting, thus allowing the IgM to be kept intact for further radiolabeling.

Methods Mol Biol, 2005, 295, 55 - 70
Growing hybridomas; Entrican G et al.; Hybridomas can be grown in a number of ways to produce stocks of monoclonal antibodies . Smaller volumes may be produced in static flask culture, but if larger amounts are required, a bioreactor may be used.

Protein Expr Purif, 2005 Jan, 39(1), 61 - 70
Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen; Bahia D et al.; This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments . Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments . This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory . We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets . We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.

Toxicol Sci . 2004 Dec 8; {Epub ahead of print}
In Vitro Zonation and Toxicity in a Perfused Hepatocyte Co-Culture; Allen JW et al.; In vitro models that more closely represent the intricate architecture and functional diversity of the liver would be beneficial to toxicology . We have established a bioreactor culture system that recapitulates features of liver zonation in vitro, allowing investigation of compartmentalized drug metabolism and toxicity . In vitro zonation is induced by exposing hepatocyte cultures to oxygen and nutrient gradients in a parallel plate bioreactor . Gradients were modeled and experimentally validated over co-cultures of hepatocytes and fibroblasts that exhibit a stable hepatocyte phenotype in vitro . Co-cultures exposed to physiologic oxygen gradients exhibited zonal induction of CYP2B and CYP3A protein that mimics that found in vivo . Furthermore, exposure to a prototypic zonal hepatotoxin, APAP, resulted in maximal toxicity at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo . In conclusion, we have established a perfused hepatocyte co-culture system for molecular and applied investigation into zonation-dependent phenomena involving drug metabolism and toxicity.

Tissue Eng, 2004 Sep-Oct, 10(9-10), 1436 - 45
Tribology approach to the engineering and study of articular cartilage; Wimmer MA et al.; This study has been based on the assumption that articular motion is an important aspect of mechanotransduction in synovial joints . For this reason a new bioreactor concept, able to reproduce joint kinematics more closely, has been designed . The prototype consists of a rotating scaffold and/or cartilage pin, which is pressed onto an orthogonally rotating ball . By oscillating pin and ball in phase difference, elliptical displacement trajectories are generated that are similar to the motion paths occurring in vivo . Simultaneously, dynamic compression may be applied with a linear actuator, while two-step-motors generate the rotation of pin and ball . The whole apparatus is placed in an incubator . The control station is located outside . Preliminary investigations at the gene expression level demonstrated promising results . Compared with free-swelling control and/or simply compression-loaded samples, chondrocyte-seeded scaffolds as well as nasal cartilage explants exposed to interface motion both showed elevated levels of cartilage oligomeric matrix protein mRNA . The final design of the bioreactor will include four individual stations in line, which will facilitate the investigation of motion-initiated effects at the contacting surfaces in more detail.

Biomaterials, 2005 May, 26(15), 2509 - 16
An organic-inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor; Kataoka K et al.; Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes . A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL . The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them . In the present study, we examined the possible utility of a new porous organic-inorganic-hybrid scaffold in an RFB . The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol-gel method using sieved sucrose particles as a porogen . In the porous TEOS-PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold . When cultivated in PDMS-TEOS, HepG2 cells secreted a approximately three-fold greater amount of albumin than that secreted in a monolayer culture . For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions . The present results indicate that PDMS-TEOS may be a promising scaffold for developing such functional culture methods.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 138 - 147
Development of a small-scale bioreactor: Application to in vivo NMR measurement; Gmati D et al.; A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells . The bioreactor was made of a 10-mm NMR tube . NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter . Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model . Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min . Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed . Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion . In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta) . Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH . (c) 2004 Wiley Periodicals, Inc.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 157 - 63
Low-temperature pausing of cultivated mammalian cells; Hunt L et al.; There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches . We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels . High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks . After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C . Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures . Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures . The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells . (c) 2004 Wiley Periodicals, Inc.

J Hepatol, 2004 Dec, 41(6), 950 - 6
Development of a scaled up liver device incorporating cryo-preserved pig liver micro-organs; Gershonowitz A et al.; BACKGROUND/AIMS: Currently there is no effective therapy for most patients with fulminant or end stage liver disease . METHODS: Pig liver micro-organs (LMOs), which preserve liver micro-architecture and ensure a maximal 150-200mum distance from a source of nutrients and gases have been prepared and a method to cryo-preserve them has been developed . A new scaled-up extra-corporeal liver device termed aLIVE-H in which LMOs are exposed to liver-like hemodynamic conditions has also been developed . The purpose of this work is to test the safety and function of cryo-preserved LMOs and how the hemodynamic properties of the scaled up aLIVE device affect their function . RESULTS: Pig LMOs in aLIVE-H, transcribe albumin and Factor V at similar levels, irrespective of their position within the bioreactor, indicating that the hemodynamic features of the aLIVE-H device allow for homogeneous plasma distribution and proper function at different locations . Cryo-preserved LMOs transcribe albumin and Factor V at levels comparable to those transcribed by a normal pig liver . Connecting the aLIVE-H bioreactor to normal pigs did not affect key blood components and biochemical parameters . CONCLUSIONS: An extra-corporeal liver device aLIVE-H which imitates the hemodynamic and functional properties of the normal liver and incorporates cryo-preserved LMOs has been developed and characterized . aLIVE-H was found to perform key synthetic liver functions.

Chemosphere, 2005 Jan, 58(3), 299 - 310
Carbon/electron source dependence of polychlorinated biphenyl dechlorination pathways for anaerobic granules; Nollet H et al.; The effect of acclimating anaerobic granules from commercial bioreactors with different carbon/electron sources on their ability to reductively dechlorinate a tri-(2,3,4-CB) and heptachlorobiphenyl (2,2',3,3',4,5,6-CB) was studied . The anaerobic granules were first grown in upflow anaerobic sludge blanket (UASB) reactors fed with two different mixtures of carbon/electron sources, i.e., propionate/butyrate/methanol and formate/methanol . Differences in dechlorination patterns for 2,2',3,3',4,5,6-CB were observed in batch experiments inoculated with granules from these two sets of UASB reactors . Variation of the carbon/electron source, during the dechlorination process, had no effect on the dechlorination pathway, but the extents and rates of dechlorination were highest for ethanol and formate and lowest for pyruvate fed batches . Pre-acclimation of different anaerobic sludges to polychlorinated biphenyls (PCBs) shortened the lag period, but did not influence the PCB dechlorination pathway . This is the first time that similar acclimation conditions for several anaerobic microbial communities prior to inoculation were reported to yield similar substrate specificities for the reductive dechlorination of specific PCB congeners . This research demonstrates a successful strategy for the development of biocatalysts to serve as the inoculum of partially decontaminated sites in order to provide microorganisms with specificities complementary to those of naturally occurring dechlorinators.

Water Sci Technol, 2004, 50(9), 17 - 23
Use of microwave pretreatment for enhanced anaerobiosis of secondary sludge; Park B et al.; This work elucidates the effects of pretreatment of secondary sludge by microwave irradiation on anaerobic digestion . The soluble chemical oxygen demand (COD) concentration increased up to 22% as microwave irradiation time increased, which indicated the sludge particles disintegrated . Three identical automated bioreactors with working volume of 5 l were used as anaerobic digesters at mesophilic temperature (35 degrees C) . The reactors were separately fed with sludge with microwave pretreated- and control- sludge at different hydraulic retention times (HRT) . The volatile solid (VS) reduction in the control operation was approximately 23.2 +/- 1.3%, while it was 25.7 +/- 0.8% for the reactors with the pretreated sludge . The average biogas production rate with the pretreated sludge at 8, 10, 12, and 15 days HRTs was 240 +/- 11, 183 +/- 9, 147 +/- 8, and 117 +/- 7 ml/l/d respectively, while those with the control sludge were 134 +/- 12 and 94 +/- 7 ml/l/d at 10 and 15 days HRTs . Maximum rates of COD removal and methane production with the pretreated sludge were 64% and 79% higher than those of the control system, respectively.

Appl Microbiol Biotechnol . 2004 Dec 2; {Epub ahead of print}
Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors; Thanh NT et al.; The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated . Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50-400 muM); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration . The highest ginsenoside yield was obtained at 200 muM MJ . In the second experiment, 200 muM MJ was added on day 15 during the cultivation . The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment . Rb group gisnsenosides accumulated more than Rg group ginsenosides . Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly . Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides . These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.

Biomaterials, 2005 May, 26(14), 2001 - 12
Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system; Hu JC et al.; Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor . For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts . However, the PGA constructs contained more collagen for both culture conditions when compared to agarose . For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks . The mechanical properties of the PGA constructs also did not improve with culture time . Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period . As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.

Biomaterials, 2005 May, 26(14), 1857 - 75
The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review; Kannan RY et al.; The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors . Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential . The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues . Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link . In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1888 - 92
Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA; Clark KJ et al.; Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors . Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors . These enzymes are known to be detrimental to the quality of recombinant glycoproteins . Bioreactor temperature has been used to control cell growth and recombinant protein production rates . However, the effect of culture temperature on the production of proteases and sialidases has not been investigated . In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C . Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa) . The 50 kDa protease activity decreased similarly with time for both culture conditions . The 69 kDa protease activity increased with time for both culture conditions . The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase . Intracellular sialidase activity was present in both cultures . The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium . The initial peak in intracellular sialidase activity was followed by a first-order decay . The intracellular sialidase activities for the two culture conditions were not significantly different . The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. , , . Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1802 - 9
A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage; Chen HC et al.; A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage . The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium . The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation . Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition . The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures . In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading . With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1788 - 96
High-level scu-PA production by butyrate-treated serum-free culture of recombinant CHO cell line; Kim JS et al.; The MGpUK-5 cell line, transformed with a single-chain urokinase-type plasminogen activator (scu-PA) minigene, generated mRNA transcripts and scu-PA titers corresponding to 65% or 86% of the amount generated before serum-free adaptation, despite significant loss of scu-PA gene copies during adaptation to serum-free culture . To further augment scu-PA production, a culture strategy employing sodium butyrate was explored . In 60-mL spinner flask cultures, sodium butyrate in the concentration range 1-10 mM allowed scu-PA production 2- to 3-fold higher than that in the negative control culture . Its productivity-enhancing activity was dependent on cell density in a range of 1-5 x 10(6) cells/mL, generating 72,200 +/- 8,100 IU/mL (480 +/- 50 mg/L) in 60-mL spinner flask cultures . To confirm this result, cells were grown to 4.4 x 10(6) cells/mL and treated with 5 mM sodium butyrate in a 2.5-L perfusion culture . The scu-PA titer increased more than 2-fold, and specific production rate of scu-PA increased 3-fold by this treatment . Overall, this perfusion culture gave rise to 1.7 x 10(8) IU scu-PA (1.1 g), comparable to total scu-PA production in a batch butyrate-treated culture performed at a 25-L bioreactor scale (1.3-3.5 g) . Our results suggest that sodium butyrate treatment on high-density culture enables scu-PA production in gram quantities.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1725 - 32
Polymer development for enhanced delivery of phenol in a solid-liquid two-phase partitioning bioreactor; Prpich GP et al.; Two-Phase Partitioning Bioreactors (TPPBs) have traditionally been used to partition toxic concentrations of xenobiotics from a cell-containing aqueous phase by means of an immiscible organic solvent and to deliver these substrates back to the cells on the basis of metabolic demand and the maintenance of thermodynamic equilibrium between the phases . A limitation of TPPBs, which use organic liquid solvents, is the possibility that the solvent can be bioavailable, and this has therefore limited organic liquid TPPBs to the use of pure strains of microbes . Solid polymer beads have recently been introduced as a replacement for liquid organic solvents, offering similar absorption properties but with the capability to be used with mixed microbial populations . The present work was aimed at identifying a polymer with a greater capacity for and more rapid uptake and release of phenol for use as the second phase in a mixed culture TPPB . Polarity and hydrogen bonding capabilities between polymer and phenol were considered in the screening and selection process of candidate polymers . Hytrel (a copolymer of poly(butylene terephthalate) and butylene ether glycol terephthalate) polymer beads, offered improved capacity (19 mg phenol/g polymer at a fixed initial phenol concentration of 2000 mg/L) and a greater diffusivity (1.54 x 10(-7) cm2/s) when compared to the capacity and diffusivity of previously used EVA (ethylene vinyl acetate) beads (12.4 mg phenol/g polymer and 3.73 x 10(-9) cm2/s, respectively) . Hytrel polymer beads were then used in a TPPB for the investigation of various substrate feeding strategies (fed-batch, bead replacement, and concentrated spikes of phenol), with rapid and complete phenol degradation shown in all cases.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1718 - 24
A novel parallel shaken bioreactor system for continuous operation; Akgun A et al.; A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale . The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid . As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force . Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas . Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously . By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time . Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1710 - 7
Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture; Nehring D et al.; The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production . If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates . In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration . The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter . Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained . When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL . At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium . As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy . Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.

Aviakosm Ekolog Med, 2000, 34(5), 51 - 9
{Transmission and exchange of genetic information during bacterial conjugation in ground-based simulations of the factors of orbital flight}; Zerov IuP et al.; Control laboratory experiments on bacterial conjugation under simulated spaceflight conditions were performed with the use of new equipment (bioreactor RECOMB-2 and container BIOMAGNISTAT) within the RSA-NASA science program . External parameters were selected and the plan of simulation of a space experiment was verified to ensure high efficiency of the conjugative transfer of chromosomal and plasmid DNA and storage of hybrids on the ground . Genetic analysis of conjugative hybrids E . coli supported the hypothesized possibility of transfer of a whole bacterial chromosome during conjugation that will lead to relative stabilization of the diploid state . Earlier this hypothesis was used to interpret results of experiments performed on MIR in 1992-1993 . Hence, the ground laboratory investigations proved the conclusion about high probability of transfer of large fragments or even a whole chromosome during space flight . Screening of the geomagnetic field by BIOMAGNISTAT increases the probability of conjugative contacts between cells and is likely to slightly inhibit the processes of recombination.

ASAIO J, 2002 Jan-Feb, 48(1), 12 - 6
Application of stereolithography for scaffold fabrication for tissue engineered heart valves; Sodian R et al.; A crucial factor in tissue engineering of heart valves is the functional and physiologic scaffold design . In our current experiment, we describe a new fabrication technique for heart valve scaffolds, derived from x-ray computed tomography data linked to the rapid prototyping technique of stereolithography . To recreate the complex anatomic structure of a human pulmonary and aortic homograft, we have used stereolithographic models derived from x-ray computed tomography and specific software (CP, Aachen, Germany) . These stereolithographic models were used to generate biocompatible and biodegradable heart valve scaffolds by a thermal processing technique . The scaffold forming polymer was a thermoplastic elastomer, a poly-4-hydroxybutyrate (P4HB) and a polyhydroxyoctanoate (PHOH) (Tepha, Inc., Cambridge, MA) . We fabricated one human aortic root scaffold and one pulmonary heart valve scaffold . Analysis of the heart valve included functional testing in a pulsatile bioreactor under subphysiological and supraphysiological flow and pressure conditions . Using stereolithography, we were able to fabricate plastic models with accurate anatomy of a human valvular homograft . Moreover, we fabricated heart valve scaffolds with a physiologic valve design, which included the sinus of Valsalva, and that resembled our reconstructed aortic root and pulmonary valve . One advantage of P4HB and PHOH was the ability to mold a complete trileaflet heart valve scaffold from a stereolithographic model without the need for suturing . The heart valves were tested in a pulsatile bioreactor, and it was noted that the leaflets opened and closed synchronously under subphysiological and supraphysiological flow conditions . Our preliminary results suggest that the reproduction of complex anatomic structures by rapid prototyping techniques may be useful to fabricate custom made polymeric scaffolds for the tissue engineering of heart valves.

Space Med Med Eng (Beijing), 2001 Apr, 14(2), 149 - 53
{Prospect of the Advanced Life Support Program Breadboard Project at Kennedy Space Center in USA}; Guo SS et al.; The Breadboard Project at Kennedy Space Center in NASA of USA was focused on the development of the bioregenerative life support components, crop plants for water, air, and food production and bioreactors for recycling of wastes . The keystone of the Breadboard Project was the Biomass Production Chamber (BPC), which was supported by 15 environmentally controlled chambers and several laboratory facilities holding a total area of 2150 m2 . In supporting the Advanced Life Support Program (ALS Program), the Project utilizes these facilities for large-scale testing of components and development of required technologies for human-rated test-beds at Johnson Space Center in NASA, in order to enable a Lunar and a Mars mission finally.

Cell Motil Cytoskeleton, 2001 Nov, 50(3), 161 - 72
Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments; Eichenmuller B et al.; The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus . To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components . To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells . After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture . EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography . In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP . EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer . In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain . Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM . Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin .

Biotechnol Bioeng, 2002 Mar 20, 77(6), 704 - 16
Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants; Sauerwald TM et al.; Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors . Such cell death may occur from necrosis but is more commonly associated with apoptosis . During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell . XIAP is the most potent caspase inhibitor encoded in the mammalian genome . The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells . CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation . In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin . Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs . Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment . Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases . Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments . With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns . While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus . In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol . Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines .

Biotechnol Bioeng, 2002 Mar 20, 77(6), 685 - 92
Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors; Ozuer A et al.; Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues . The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield . An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity . Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle . However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus . The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications . In this work, the dependence of the vector yield on the genetic background of the virus is examined . In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system . After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost .

Environ Toxicol Chem, 2002 Jan, 21(1), 1 - 8
Alpha,beta-unsaturated sulfophenylcarboxylates as degradation intermediates of linear alkylbenzenesulfonates: evidence for omega-oxygenation followed by beta-oxidations by liquid chromatography-mass spectrometry; Eichhorn P et al.; Liquid chromatography with an electrospray interface to a mass spectrometer (LC-ES-MS) and LC-ES coupled to a tandem MS (LC-ES-MS/MS) were used to detect and identify intermediates excreted transiently during the aerobic degradation of linear alkylbenzenesulfonates (LAS) in fixed-bed bioreactors (FBBR) . The inoculum for the FBBR was the microflora of the River Rhine, Germany . Two major phenomena were observed on the addition of 100 mg/L LAS to the system, sorption and then biodegradation . Disappearance due to sorption was followed in an inhibited FBBR . Biodegradation of LAS started on day 7 and was accompanied by the transient excretion of intermediates, which were later largely degraded . We detected not only the sulfophenylcarboxylates (SPCs) observed previously but also the alpha,beta-unsaturated SPCs (SPC-2H), which have not been reported before . Experiments with the (4-sulfophenyl)dodecanes (C12-LAS), which had minor contaminants of C11-LAS, showed C12-, C10-, C8-, C6-, and C4-SPCs when LAS was degraded as well as traces of C9-, C7-, and C5-SPCs . Signals from the SPC-2H species were usually some 10% of those from the corresponding SPCs . Samples from these experiments were also examined by gas chromatography-mass spectrometry (GC-MS), but no desulfonated intermediates were detected . We interpret the data to mean that the only attack on LAS was by (omega-oxygenation; there was no visible initial desulfonation . The products of omega-oxygenation were oxidized to the corresponding SPC and subject to beta-oxidation, as evidenced not only by the pattern of C-2 units in the excreted SPCs but also in the corresponding series of SPC-2H, representing the intermediates in beta-oxidation.

Crit Rev Biotechnol, 2001, 21(4), 233 - 94
Hydrodynamic and mass transfer characteristics of three-phase gaslift bioreactor systems; Petersen EE et al.; This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors . The factors affecting the mixing and volumetric mass transfer coefficient (k(L)a), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed . Measurement methods, modeling and empirical correlations are reviewed and compared . To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature . This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions . The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k(L)a.

Int J Artif Organs, 2001 Nov, 24(11), 807 - 13
The effect of serum from liver cancer patients on the growth and function of primary and immortalised hepatocytes; Grant MH et al.; A limiting factor in the efficacy of bioartificial liver (BAL) for the treatment of liver failure is the toxicity of the patients' serum to the hepatocytes in the device . This study investigates the interaction of liver cancer patient serum with primary and immortalised rat hepatocytes . Liver cancer serum increased the growth rate of immortalised hepatocytes, without affecting reduced glutathione levels . The activities of DT-diaphorase and pi glutathione-S-transferase (GST), enzymes associated with de-differentiation, were also increased . Exposure of primary hepatocytes to liver cancer serum resulted in a decrease in cytochrome P450 (CYP) content, and in P450 dependent metabolism of testosterone . Formation of 2-alpha- and 6-beta- hydroxy testosterone was decreased . These reactions are predominantly associated with CYP 2C11 and 3A1 respectively in normal rat liver . The activity of total GST was also decreased, although that of the pi isoenzyme of GST was not affected . Our results suggest that exposure of hepatocytes in a bioreactor to liver cancer patient serum will result in overgrowth of cells, if proliferating cells are being used, and in de-differentiation . The serum may have to be pretreated with adsorbants to remove toxins prior to BAL treatment.

Int J Artif Organs, 2001 Nov, 24(11), 793 - 8
Experimental evaluation of a cell module for hybrid liver support; Gerlach JC et al.; Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy . Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery . Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells . The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density . Treatment started after complete recovery from anesthesia and was performed continuously . A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group . Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

Ann N Y Acad Sci, 2001 Nov, 944, 334 - 43
Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver; MacDonald JM et al.; A novel "multicoaxial hollow fiber bioreactor" has been developed consisting of four concentric tubes, the two innermost tubes are called hollow fibers . Bioartificial livers are created by culturing liver progenitors in the space between the two innermost hollow fibers and with culture media contained in the two compartments (intracapillary and extracapillary) sandwiching the cell compartment . The outermost compartment is used for gas exchange . A hydrodynamic model has recently been established to predict the optimum hydraulic permeability and bioreactor operational parameters to create the physicochemical environment found in the liver acinus . However, perfusion with serum-free hormonally-defined media and inoculation of cells introduces membrane fouling into the equation, and this parameter must be incorporated into the model . Using commercially available semipermeable hollow fibers (1 mm {0.65 microm pores} and 3 mm {0.1 microm pores} outer diameters {o.d}), the primary cause of resistance is the middle hollow fiber . Preliminary studies using bioreactors inoculated with isolated rat hepatocytes and perfused with serum-containing culture media demonstrated that the middle hollow fiber is the primary site of fouling, and this fouling ultimately causes cell mortality by blocking the transfer of nutrients . Experiments were performed to determine the best commercially available middle hollow fiber for construction of bioreactors and two 3-mm outer-diameter middle hollow fibers were compared: polypropylene and polysulfone, with 0.2 microm and 0.1 microm pore sizes, respectively . Dead-ended and cross flow configurations were compared for their effectiveness at reducing membrane fouling in the middle hollow fiber by determining the change in resistance with time . The results demonstrate that the 0.2-microm pore size polypropylene hollow fiber is the best choice for construction of the multicoaxial hollow-fiber bioreactor, and that cross flow results in two orders of magnitude lower resistance than dead-ended flow after 36 h.

Ann N Y Acad Sci, 2001 Nov, 944, 308 - 19
Development of a hybrid liver support system; Sauer IM et al.; Hybrid liver systems are being developed as temporary extracorporeal liver support therapy . The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy . In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors . These systems are capable of supporting essential liver functions . Animal experiments verify the possibility of upscaling bioreactors for clinical treatment . However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings . The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure . Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research . For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed . The construction is based on capillaries for hepatocyte aggregate immobilization . Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network . Cells are perfused via independent capillary membrane compartments . Decentralized oxygen supply and carbon dioxide removal with low gradients is possible . The parallel use of identical units enables easy upscaling . Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising.

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 561 - 5
{Studies on the cell suspension culture of Saussarea medusa in a stirred tank bioreactor}; Huang Y et al.; The cell suspension culture of Saussarea medusa in a 2L aerated and agitated bioreactor with a four-pitch-blade impeller was investigated . The effects of agitation speed, aeration and inoculum size on cell growth and flavonoids production were studied and it was found that cells had optimum growth and flavonoids production when cultivated at 75 r/min, 700-1000 L/min and an inoculum of 4.0-5.0 g/L . A high cell biomass of 13.8 g/L and flavonoids production of 416 mg/L were achieved after 12 days of cultivation . Time course study revealed that flavonoids biosynthesis was growth-associated . The studies on aggregates size distribution in the bioreactor showed that the aggregates break-up caused by hydrodynamic stress might adversely affect cell growth and lead to significant reduction of cell biomass and flavonoids production.

Water Sci Technol, 2001, 44(10), 197 - 202
Sludge reduction potential of metazoa in membrane bioreactors; Luxmy BS et al.; In order to check the sludge reduction capacity of metazoa in a membrane bioreactor (MBR), pilot-scale studies were conducted . Three MBRs had been set in a wastewater treatment plant at Tokyo, Japan and they were receiving real wastewater . Initially pH inside the three MBRs was controlled as pH 7, 6 and 5 respectively . Then metazoa population was monitored along with MLSS change . It was found that the presence or absence of the metazoa population did not have any significant effect on the increasing pattern of MLSS . In the MBR with pH 6 highest accumulation of sludge was observed though a high and steady level of metazoa (1,000-2,000 per ml) was present there . But in this MBR a lot of metazoa attached in the membrane was also observed and here the increase in transmembrane pressure was less than in the other two . So, metazoa population especially the attached one in the membrane plays an effective role in fouling control of the membrane . Presence of attached media may provide a suitable niche for metazoa in the process . So, attached media known as DB lace was also inserted in MBRs for testing its capacity along with inoculum of oligochaete worms . Accumulation of sludge was not satisfactory in the attached string and it seems that inoculated worm could not adjust to the environment as they were not sludge originated . So, in the next experimental stage, attached media was inserted in the form of a bundle and this time no inoculation of worm was used . A steady metazoa population was observed in the system but the accumulation of sludge in the attached media was the same as before.

Ann Biomed Eng, 2001 Nov, 29(11), 963 - 73
Dynamic in vitro quantification of bioprosthetic heart valve leaflet motion using structured light projection; Iyengar AKS et al.; Quantification of heart valve leaflet deformation during the cardiac cycle is essential in understanding normal and pathological valvular function, as well as in the design of replacement heart valves . Due to the technical complexities involved, little work to date has been performed on dynamic valve leaflet motion . We have developed a novel experimental method utilizing a noncontacting structured laser-light projection technique to investigate dynamic leaflet motion . Using a simulated circulatory loop, a matrix of 150-200 laser light points were projected over the entire leaflet surface . To obtain unobstructed views of the leaflet surface, a stereo system of high-resolution boroscopes was used to track the light points at discrete temporal points during the cardiac cycle . The leaflet surface at each temporal point was reconstructed in three dimensions, and fit using our biquintic hermite finite element approach (Smith et al., Ann . Biomed . Eng . 26:598-611, 2001) . To demonstrate our approach, we utilized a bovine pericardial bioprosthetic heart valve, which revealed regions of complex flexural deformation and substantially different shapes during the opening and closing phases . In conclusion, the current method has high spatial and temporal resolution and can reconstruct the entire surface of the cusp simultaneously . Because it is completely noncontacting, this approach is applicable to studies of fatigue and bioreactor technology for tissue engineered heart valves.

Ann Biomed Eng, 2001 Nov, 29(11), 947 - 55
Analysis of oxygen transport to hepatocytes in a flat-plate microchannel bioreactor; Roy P et al.; Oxygen transfer to cultured hepatocytes in microchannel parallel-plate bioreactors with and without an internal membrane oxygenator was investigated with a mathematical model and the results were corroborated with fluorescence imaging experiments . The consumption of oxygen by hepatocytes was assumed to follow Michaelis-Menten kinetics . Our simulations indicate that under conditions of low Peclet (Pe) number (<80) and fixed Damlkohler number (= 0.14, corresponding to rat hepatocytes) the cells are hypoxic in the bioreactor without an internal membrane oxygenator . Under the same conditions, the bioreactor with an internal membrane oxygenator can avoid cell hypoxia by appropriate selection of membrane Sherwood number and/or the gas phase oxygen partial pressure, thus providing greater control of cell oxygenation . At high Pe, both bioreactors are well oxygenated . Experimentally determined oxygen concentrations within the bioreactors were in good qualitative agreement with model predictions . At low Pe, cell surface oxygen depletion was predicted from analytically derived criteria . Hepatocytes with oxygen dependent functional heterogeneity may exhibit optimal function in the bioreactor with the internal membrane oxygenator.

Nucleic Acids Res . 2002 Jan 15;30(2):E9.
High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells; Durocher Y et al.; A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension . Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity . Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI . The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein-Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression . The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4 . We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette . Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively . The presence of serum had a positive effect on gene transfer and expression . Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h . Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor . Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.

Bioseparation, 2001, 10(1-3), 57 - 63
Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents; Lutkemeyer D et al.; This presentation compares three different expanded bed matrices . STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein . The capture of a mouse IgG1 and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively . The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat . In all cases the capture of the target proteins was comparable to conventional chromatographic systems . The purification success was mainly dependent on the selectivity of the ligand used . The affinity process resulted in a highly purified product . The ion exchanger and chelating material mainly concentrated the product . In all three cases 100 l of cell broth were successfully processed in one run . The robustness of the ion exchanger process was poor, because of strong cell matrix interaction . However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.

Biotechnol Bioeng, 2002 Feb 15, 77(4), 455 - 61
Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations; Shiao TL et al.; Overexpression of Arabidopsis thaliana genes for the fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, improved the tolerance of A . thaliana hairy roots to low oxygen conditions . Whereas the specific growth rate of untransformed hairy roots in shake flasks and in a multiple-tube recirculation bioreactor declined significantly with decreasing oxygen tension down to 25% air saturation, growth of the transformant root lines was maintained at rates similar to those achieved with full aeration . This work demonstrates that altering the expression of selected genes involved in anaerobic metabolism can alleviate the problems of oxygen deficiency in hairy root cultures caused by poor mixing and mass transfer conditions .

Biotechnol Bioeng, 2002 Feb 15, 77(4), 381 - 93
Validation of a model for process development and scale-up of packed-bed solid-state bioreactors; Weber FJ et al.; We have validated our previously described model for scale-up of packed-bed solid-state fermenters (Weber et al., 1999) with experiments in an adiabatic 15-dm(3) packed-bed reactor, using the fungi Coniothyrium minitans and Aspergillus oryzae . Effects of temperature on respiration, growth, and sporulation of the biocontrol fungus C . minitans on hemp impregnated with a liquid medium were determined in independent experiments, and the first two effects were translated into a kinetic model, which was incorporated in the material and energy balances of the packed-bed model . Predicted temperatures corresponded well with experimental results . As predicted, large amounts of water were lost due to evaporative cooling . With hemp as support no shrinkage was observed, and temperatures could be adequately controlled, both with C . minitans and A . oryzae . In experiments with grains, strong shrinkage of the grains was expected and observed . Nevertheless, cultivation of C . minitans on oats succeeded because this fungus did not form a tight hyphal network between the grains . However, cultivation of A . oryzae failed because shrinkage combined with the strong hyphal network formed by this fungus resulted in channeling, local overheating of the bed, and very inhomogeneous growth of the fungus . For cultivation of C . minitans on oats and for cultivation of A . oryzae on wheat and hemp, no kinetic models were available . Nevertheless, the enthalpy and water balances gave accurate temperature predictions when online measurements of oxygen consumption were used as input . The current model can be improved by incorporation of (1) gas-solids water and heat transfer kinetics to account for deviations from equilibrium observed with fast-growing fungi such as A . oryzae, and (2) the dynamic response of the fungus to changes in temperature, which were neglected in the isothermal kinetic experiments .

J Endocrinol, 2002 Jan, 172(1), 199 - 210
Production and purification of recombinant human inhibin and activin; Pangas SA et al.; Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion . Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers . Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein . To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein . These cells produce approximately 200 microg/ml per day total recombinant human inhibin . Conditioned cell media can be purified through column chromatography resulting in