Bioreactors

Tissue Eng, 2003 Jun, 9(3), 549 - 54
Design of a flow perfusion bioreactor system for bone tissue-engineering applications; Bancroft GN et al.; Several different bioreactors have been investigated for tissue-engineering applications . Among these bioreactors are the spinner flask and the rotating wall vessel reactor . In addition, a new type of culture system has been developed and investigated, the flow perfusion culture bioreactor . Flow perfusion culture offers several advantages, notably the ability to mitigate both external and internal diffusional limitations as well as to apply mechanical stress to the cultured cells . For such investigation, a flow perfusion culture system was designed and built . This design is the outgrowth of important design requirements and incorporates features crucial to successful experimentation with such a system.

Curr Opin Biotechnol, 2003 Jun, 14(3), 270 - 6
Diversity and dynamics of microbial communities in engineered environments and their implications for process stability; Briones A et al.; The availability of molecular biological tools for studying microbial communities in bioreactors and other engineered systems has resulted in remarkable insights linking diversity and dynamics to process stability . As engineered systems are often more manageable than large-scale ecosystems, and because parallels between engineered environments and other ecosystems exist, the former can be used to elucidate some unresolved ecological issues . For example, the process stability of methanogenic bioreactors containing well-defined trophic groups appears to depend on the diversity of the functional groups within each trophic level as well as on how these functional groups complement each other . In addition to using engineered systems to study general ecological questions, microbial ecologists and environmental engineers need to investigate conditions, processes, and interactions in engineered environments in order to make the ecological engineering of bioreactor design and operation more practicable.

Am J Reprod Immunol, 2003 Aug, 50(2), 113 - 23
Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer; Edwards JL et al.; In 1997, Wilmut et al . announced the birth of Dolly, the first ever clone of an adult animal . To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer . The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo . The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte . Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals . Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes . Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors . A major limitation of cloning procedures is the extreme inefficiency for producing live offspring . Dolly was the only live offspring produced after 277 attempts . Similar inefficiencies for cloning adult animals of other species have been described by others . Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life . Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

Mol Reprod Dev, 2003 Aug, 65(4), 389 - 95
Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos; Park TS et al.; This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain . EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene . During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population . Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation . Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene . The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism . As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively . The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs . In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production .

Am J Physiol Cell Physiol, 2003 Nov, 285(5), C1071 - 81 Epub 2003 Jul 02.
Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters; Bertrand A et al.; Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle . This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins . However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence . It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression . Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter . Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle . Return to a normal transcriptional state was observed 7-10 days after electroporation . Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle . We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced . However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context.

Appl Environ Microbiol, 2003 Jul, 69(7), 4076 - 86
Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats; Sarvari Horvath I et al.; Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments . Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells . The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth . A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism . There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate . Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid . Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+ . At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred . Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred . Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

Zhonghua Gan Zang Bing Za Zhi, 2003 Jun, 11(6), 358 - 60
{Preparation of hollow fiber bioreactor for culturing pig hepatocytes}; Wang YJ et al.; OBJECTIVE: To study the method of preparing the hollow fiber bioreactor for culturing pig hepatocytes . METHODS: Hepatocytes were isolated from experimental suckling minipigs by two-step perfusion with collagenase, and seeded onto hollow fiber bioreactor, then cultured with an artificial capillary cell culture system . The albumin-excretion, lidocaine-transforming rate, lactate dehydrogenase (LDH) release and the cell viability in bioreactors were examined . RESULTS: The porcine albumin could be detected by SDS/PAGE on the 2nd, 4th, 6th day . The rates of lidocaine-transforming ranged from 89.6% to 96.1% . The release of LDH into the culture medium increased from (23.7+/-4.6) U/L to (127.8+/-17.4) U/L (F=39.582, P<0.01) during the experiments, and the viability of pig hepatocytes in hollow fiber bioreactor reduced from 95.8%+/-0.3% to 83.8%+/-4.7% (t=5.135, P<0.01) . CONCLUSION: The hollow fiber bioreactor for culturing pig hepatocytes can be prepared by artificial capillary cell culture system, which provides a certain liver-specific function in 1 week.

Plant Cell Rep, 2003 Jul, 21(11), 1129 - 33 Epub 2003 Apr 29.
Effect of aggregate size in cell cultures of Saussurea medusa on cell growth and jaceosidin production; Zhao D et al.; Cell suspension cultures of Saussurea medusa were grown in shake flasks and a 5-l stirred tank bioreactor . Biomass and jaceosidin distribution in cell aggregates of different sizes were investigated during the cultivation period . The results showed that on day 10, jaceosidin accumulation showed an increase with increasing size of the cell aggregate to 4 mm in diameter, with the highest jaceosidin accumulation being 12.2 mg/g . An inverse tendency was observed with cell aggregates larger than 4 mm in diameter, with the lowest accumulation being 3.1 mg/g . However, all of the cell aggregates, despite their size, synthesized almost the same amount of jaceosidin at day 12 . Oxygen diffusion limitation and cell-cell contact may explain this behavior . In comparison with cells cultivated in shake flasks, decreased biomass and decreased jaceosidin concentration were observed when the cells were cultivated in a stirred tank bioreactor . The sublytic effects caused by the hydrodynamic stress in combination with insufficient nutrients in the bioreactor may cause cell damage.

Plant Cell Rep, 2003 Jul, 21(11), 1088 - 94 Epub 2003 Apr 16.
Selectable marker-free transgenic barley producing a high level of cellulase (1,4-beta-glucanase) in developing grains; Xue GP et al.; The use of barley grains as bioreactors for high-level production of cellulase (1,4-beta-glucanase) was investigated . A hybrid cellulase gene, cel-hyb1, driven by the rice GluB-1 promoter was expressed specifically in developing endosperm . Codon usage optimisation of cel-hyb1 increased its expression in barley grains 527-fold and led to cellulase production of up to 1.5% of total grain protein . CEL-HYB1 enzyme in barley grains was highly stable during post-harvest storage . Selectable marker gene ( hph) was subsequently eliminated from transgenic lines through segregation of hph from synthetic cel-hyb1 ( syn.cel-hyb1) in T1 progeny, using a binary plasmid containing hph and syn.cel-hyb1 in separate T-DNAs . These data suggest that barley grains can potentially be used for the commercial production of cellulase.

J Hazard Mater, 2003 Jun 27, 100(1-3), 79 - 94
Combined slurry and solid-phase bioremediation of diesel contaminated soils; Nano G et al.; This work investigates, at a laboratory and pilot-scale, the influence of various operating parameters on the combined slurry and solid-phase bioremediation technique for a diesel contaminated soil . For slurry-phase bioreactors (SPB), it has been found that, as far as famine conditions are attained at the end of the react cycle, a low hydraulic retention time and a low slurry recycle ratio allows for a better utilization of the reactor volume . A 7-day slurry-phase bioreactor treatment has been shown to provide enough contaminant removal allowing the soil drawn from the slurry-phase bioreactors to be fed effectively to the solid-phase bioreactors (SoPB) for completing the soil cleanup . However, an important improvement of the solid-phase bioreactor performance has been found using soil additives, namely sand and surfactants . While the first soil additive improves pile porosity and consequently oxygen diffusion, the latter increases contaminant bioavailability.

Water Res, 2003 Aug, 37(14), 3472 - 80
Bioremoval of cadmium from aqueous solution by black gram husk (Cicer arientinum); Saeed A et al.; Husk of black gram (Cicer arientinum), a waste of no commercial value, was investigated as a new biosorbent of cadmium from low concentration aqueous solutions . With 99.99% sorption efficiency from 10mg l(-1) cadmium solution, the biomass required at saturation was 0.8 g mg(-1) cadmium . Biosorption was rapid and equilibrium was achieved in 30 min . Among the various desorbing agents tested, 99.89% cadmium recovery was achieved with 0.1M HCl . Sorption efficiency of cadmium during six biosorption-desorption cycles in batch operations declined, which was traceable to 39.0% black gram husk (bgh) weight loss . This decline was only 9.71% when compensated for biomass loss, which is comparable to 10.45% decline during six cycles in fixed bed column bioreactor in which biomass loss was only 5.98% . On plotting breakthrough curves it was noted that bgh in the fixed bed column was capable of bringing down cadmium concentration from 10 to 0.1 mg l(-1) in 35.5 l volume . Biosorption of cadmium was not effected in the presence of other cations . Comprehensive characterization of parameters indicate bgh to be an excellent material for biosorption of cadmium to treat wastewaters containing low concentration of the metal . As an agrowaste, the advantage of application of this material as a metal biosorbent in a fixed bed column bioreactor system, in comparison with those based on immobilized algae or biomass of algal, fungal and bacterial origin, is considered.

Expert Opin Biol Ther, 2003 Jul, 3(4), 551 - 61
Progress and challenges for cell encapsulation in brain tumour therapy; Visted T et al.; Cell encapsulation provides a method to circumvent the host immune system by encapsulating cells or tissues in immunoisolating, semipermeable membranes before implantation . The technology has been widely studied with an aim of developing bio-organs transplantable into patients without the need of immunosuppression, and in cancer therapy, the principle of cell encapsulation may be further exploited . Encapsulated recombinant cells represent factories or bioreactors for the production of therapeutic proteins . By implanting the bioreactors in the vicinity of the tumour, long-term local de novo delivery of the therapeutic proteins may be achieved . Malignant brain tumours such as glioblastoma multiforme (GBM) remain highly lethal neoplasms, refractory to current therapies . Researchers and medical professionals are working on developing translational therapies to combat these aggressive tumours . Numerous clinical trials on gene therapy for glioma patients have been conducted over the last decade, but the results have largely been disappointing . Cell encapsulation represents an alternative method for local delivery of therapeutic proteins with antineoplastic properties to glioma patients . The concept has not yet reached clinical trials, but encouraging results have been achieved in rats bearing gliomas when implanting endostatin-secreting encapsulated cells into the rat brain . This review primarily focuses on the recent progress that has been made with cell encapsulation technology . In addition, the challenges this field faces before clinical application in brain tumour patients is discussed.

J Nat Prod, 2003 Jun, 66(6), 743 - 51
Isolation of halogenated monoterpenes from bioreactor-cultured microplantlets of the macrophytic red algae Ochtodes secundiramea and Portieria hornemannii; Barahona LF et al.; Field collections of the red macroalgae Ochtodes secundiramea and Portieria hornemannii exhibit site-to-site variations in halogenated monoterpene (HMT) content . In contrast, microplantlets of O . secundiramea and P . hornemannii established through cell and tissue culture techniques had remarkably similar HMT profiles when cultivated in a photobioreactor under identical, controlled conditions . Both algae shared Apakaochtodene B (6) as the only cyclic HMT, 10E-bromomyrcene (3) and 10E-bromo-3-chloro-alpha-myrcene (4) as the dominant acyclic HMTs, and myrcene (1) as their common precursor . Furthermore, HMT yields were comparable between organisms (0.9-1.3 micromol/g dry mass of 6; 3.4-4.4 micromol/g of 3) . P . hornemannii microplantlets also contained 7-chloromyrcene (9) as the dominant compound (37-73 micromol/g), suggesting additional chlorination capacity . Proposed pathways for HMT biosynthesis shared by P . hornemannii and O . secundiramea microplantlets possessed two common manifolds: (a) bromonium ion (Br(+))-catalyzed cyclization of 1, followed by chlorination to yield 6; (b) Markovnikov addition of Br(+) at Delta(6,10) of 1 to yield 3 with a marked selectivity (>50:1) for the E isomer, followed by chlorination of 3 to 4 . This study demonstrated that bioreactor tissue culture is a new venue for bioprospecting and production of natural compounds from marine macroalgae under a controlled environment.

Biotechnol Bioeng, 2003 Sep 5, 83(5), 583 - 94
Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor; Wu JY et al.; This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers . The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results . Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations . Thus, new correlations describing the above-mentioned relationships are suggested . The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher . For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon) . A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186 . For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5 . The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers .

Appl Microbiol Biotechnol, 2003 Oct, 62(5-6), 503 - 6 Epub 2003 Jun 26.
Composites of bacterial cellulose and paper made with a rotating disk bioreactor; Mormino R et al.; Suspended solids in the nutrient medium for Acetobacter xylinium in a rotating disk bioreactor become incorporated into the gelatinous mat of bacterial cellulose as it forms . Embedding fibers of ordinary cellulose creates composites with enhanced strength and the toughness of bacterial cellulose . Purified cellulose and elongated fibers from paper are incorporated differently than are spherical particles such as silica gel . About 90% of the final cellulose can come from scrap paper, and dried composite sheets were much stronger than plain bacterial cellulose per unit area.

J Craniofac Surg, 2003 May, 14(3), 340 - 7
Perfusion bioreactor for vascular tissue engineering with capacities for longitudinal stretch; Mironov V et al.; Arterial growth during embryonic vascular development is associated with longitudinal strain . The longitudinal strain is an important element of the embryonic vascular mechanical environment (EVME) . Thus, a perfusion bioreactor for vascular tissue engineered constructs must include the functional capacity for longitudinal strain . To accomplish this goal, a perfusion bioreactor with the capacity for longitudinal strain was developed . The bioreactor includes two media perfusion systems: one for the inside perfusion and one for the outside perfusion of the cardiovascular engineered tubular construct (CETC) . The watertight perfusion chamber allows periodic changing of longitudinal strain of the construct during mechanical conditioning . The range of the longitudinal strain is 0% to 200% . The biomechanical properties of the CETC are controlled by a pressure transducer and a digital TV camera . The pressure transducer and TV camera are connected to a computer . This allows the recording of a relationship between the radius of the VTEG and pressure in both static and dynamic regimens . This bioreactor can perform biomechanical conditioning with longitudinal strain.

Artif Organs, 2003 Jul, 27(7), 613 - 22
Bioartificial liver inoculated with porcine hepatocyte spheroids for treatment of canine acute liver failure model; Chen Z et al.; The aim of this study was to evaluate a novel bioartificial system in a canine model of acute liver failure . An acute liver failure model in canines was induced by an end-side portocaval shunt combined with common bile duct ligation and transection . The bioartificial liver system, which utilized blood perfusion through a hollow fiber bioreactor from BIOLIV A3A inoculated with 1.0 - 3.1 x 1010 porcine hepatocyte spheroids, was developed for the treatment of acute liver failure . Sixteen acute liver failure model canines were divided between a group treated with bioartificial liver (n=8) and a control group (n=8) for 5 h . Blood alanine aminotransferase (ALT), alkaline phosphatase (AKP), total bilirubin (TBi), direct bilirubin (DBi), prothrombin time (PT), ammonia levels, and the ratio of branched chain to aromatic amino acids (Fischer's ratio) were determined . ALT, AKP, TBi, DBi, and ammonia levels were significantly elevated, PT was significantly prolonged, and Fischer's ratio decreased significantly in the canine model of the two groups on day 14 after operation compared to baseline . There were no significant differences between the two groups in laboratory data before treatment . In canines treated with the bioartificial liver system, ALT, AKP, TBi, DBi, and ammonia levels decreased significantly, PT was significantly shortened, Fischer's ratio was significantly elevated after treatment, and the survival rate by day 7 after treatment was 100% . In canines in the control group, on the other hand, there were no significant differences in ALT, AKP, TBi, DBi, PT, and ammonia levels between pretreatment and posttreatment, though these indices decreased to a slight degree after treatment . The survival rate by day 7 after treatment was 62.5% in the control group . Fischer's ratio decreased after treatment . ALT, AKP, TBi, DBi, PT, and ammonia levels in the bioartificial liver system group were lower, and Fischer's ratio and survival rate were higher than those in the control group after treatment . These results indicate that the novel bioartificial liver system we developed has a significant impact on the course of canine acute liver failure model and has potential advantages for clinical use in patients with acute liver failure.

Ann Chim, 2003 Apr, 93(4), 381 - 8
Membrane bioreactor processes: a must for the wastewater treatment plants of the Lagoon of Venice; Cecchi F et al.; A pilot scale experimentation of a membrane bioreactor has been progressing since 1999 at Fusina-Venice wastewater treatment plant . Results showed that the limits introduced by Law Decree 30 July 1999 (Decree "Ronchi-Costa") can be fulfilled (except for Arsenic) and treated water is suitable for reuse purposes . According to the results reported in this paper, sizing and involved costs for the membranes application at Fusina wastewater treatment plant are presented . This can be the proper engineering solution for the Integrated Project Fusina.

Poult Sci, 2003 Jun, 82(6), 927 - 30
Validating the hen as a bioreactor for the production of exogenous proteins in egg white; Harvey AJ et al.; Increased demand for the production of human biopharmaceuticals in transgenic organisms has led to an intensive effort to develop the hen as a bioreactor producing exogenous proteins in egg white via transgenesis . To date, however, robust methods for transgenic modification of the avian genome have been lacking . We have used a replication-defective retroviral vector derived from avian leukosis virus (ALV) to generate transgenic chickens expressing bacterial beta-lactamase secreted into serum and egg whites through several generations . Expression was driven by the ubiquitous cytomegalovirus (CMV) promoter . Here we describe results from a transgenic lineage (Harvey et al., 2002a,b) in which (1) the transgene was stably transmitted from a G1 founder male (5657) through several generations without silencing, (2) the protein was biologically active, and (3) the level of expression in egg whites was doubled in a G3 homozygote.

J Biotechnol, 2003 Jun 26, 103(2), 191 - 6
High-density tissue-like cultivation of JAR choriocarcinoma cells for the in vitro production of human xylosyltransferase; Kuhn J et al.; Human xylosyltransferase is the chain-initiating enzyme involved in the biosynthesis of glycosaminoglycans . Large amounts of xylosyltransferase are required to study the biochemical properties of the native enzyme . To achieve this goal a scale-up of animal cell culture systems was inevitable due to the small amounts of the enzyme present in tissues, e.g . only 0.5 microg XT can be obtained from a chick embryo . JAR choriocarcinoma cells cultured with 10% fetal calf serum were found to secrete xylosyltransferase with relatively high activities (1.10 mU l(-1)) . To reduce contaminating proteins JAR cells were adapted to serum-free conditions . Xylosyltransferase activities up to 0.22 mU l(-1) were determined in the harvested cell culture supernatant . Scaling-up of JAR cell culture in the hybrid hollow fiber bioreactor Tecnomouse resulted in the production of 15.8 mU or 270 microg XT in 0.5 l of XT-enriched cell culture supernatant using 57 l of serum-free cell culture medium . The XT activity per ml harvest solution was 200-280-fold higher in this cell culture supernatant than in cell culture flasks . In addition, the specific XT activity of the bioreactor product was 6 microU mg(-1) of total protein, which is 2-fold higher than that obtained under static culture conditions . This study clearly demonstrates the successful high-density, tissue-like cultivation of JAR choriocarcinoma cells in a hollow fiber bioreactor resulting in an effective production of native human xylosyltransferase.

ScientificWorldJournal, 2002 May 08, 2, 1243 - 6
Polar organic pollutants in groundwater: experimental approaches to biodegradation during subsoil passage; Knepper TP; A selection of polar organic compounds was investigated for their biodegradation on a laboratory scale fixed-bed bioreactor and the decline of the parent compounds besides the formation of metabolites was monitored . Of particular interest was the investigation into the degradation of pesticides, especially isoproturon (IPU), surfactants and industrial by-products of chemical synthesis . The results from the laboratory degradation experiments are compared to findings in groundwater.

ScientificWorldJournal, 2001 May 01, 1, 194 - 5
Bioreactors for decreasing the growth of brain tumors; Carroll RS et al.; Malignant gliomas are the most common primary brain tumors . They are highly aggressive tumors characterized by a recurrence rate of virtually 100% . Despite significant advances in neuroimaging and neurosurgical techniques, the median survival time of patients with glioblastoma multiforme remains 12 to 18 months . Malignant gliomas are characterized by rapidly dividing cells, which invade into the normal brain, and a high degree of vascularity . Recent experimental evidence indicates that tumor-related angiogenesis contributes significantly to the malignant phenotype.

Med Biol Eng Comput, 2003 May, 41(3), 271 - 82
Large-scale expansion of mammalian neural stem cells: a review; Kallos MS et al.; A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neural stem cells (NSCs) as therapeutic agents . The expected demand for treatment from the millions of afflicted individuals, coupled with the expected demand from biotechnology companies creating therapies, has fuelled the need to develop large-scale culture methods for these cells . The rapid pace of discovery in this area has been assisted through the use of animal model systems, enabling many experiments to be performed quickly and effectively . This review focuses on recent developments in expanding human and murine NSCs on a large scale, including the development of new serum-free media and bioreactor protocols . In particular, engineering studies that characterise important scale-up parameters are examined, including studies examining the effects of long-term culture of NSCs in suspension bioreactors . In addition, recent advances in the human NSC system are reviewed, including techniques for the evaluation of NSC characteristics.

Osteoarthritis Cartilage, 2003 Jun, 11(6), 403 - 11
A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro; Grogan SP et al.; OBJECTIVE: To characterise in vitro engineered cartilaginous constructs made employing a novel static, scaffold-free and closed chamber system . DESIGN: Chondrocytes derived from slaughter age pigs (3-6 months) were seeded at high density (20 x 10(6)) into cylindrical chambers (1.0 x 0.5cm) and were maintained between an upper and a lower membrane (100 kDa) for 21 days and subsequently cultured in open culture for 7 additional days . RESULTS: Viable constructs produced were approximately 10 mmx2mm in size and were stable enough to be handled by surgical pincers . Histology and electron microscopy evaluations revealed a neo-cartilage structure of high cell density with a comprehensive extracellular matrix . Predominately collagen type II and negligible amounts of collagen types I and X were detected using RT-PCR and SDS-PAGE analyses . CONCLUSIONS: In this study, we provide evidence of a scaffold-free system that can produce immature hyaline-like cartilaginous constructs suitable for in vivo implantation, or that may be useful for in vitro studies of events related to the process of chondrogenesis.

Huan Jing Ke Xue, 2003 Mar, 24(2), 125 - 9
{Treatment of wastewater from a toilet for reclamation with a airlift external recirculated membrane bioreactor}; Xu H et al.; A new type membrane bioreactor(MBR) coupled with a separated immersed membrane unit was studied for treatment of wastewater from a toilet and for reclamation of the treated water . The results showed that the effluent of the MBR was stable with COD < 47 mg/L, BOD5 < 8.5 mg/L, NH3-N < 20 mg/L, when the influent concentration was COD 440-970 mg/L, BOD5 307-612 mg/L, NH3-N 59-111 mg/L . The treated water could meet the standard of reused water for toilet washing issued by the Ministry of Construction P.R . China . The sludge loading, volume loading, HRT and their impaction to COD of effluent were studied . Appropriate sludge loading, volume loading were respectively 0.1 kg/(kg.d), 1 kg/(m3.d) and HRT 7-8 hour . The effect of mixed liquid suspendend solid(MLSS), temperature and permeating time of the MBR to the membrane fluxes were discussed in this paper.

Huan Jing Ke Xue, 2003 Mar, 24(2), 66 - 72
{Numerical simulation of moisture movement in landfill bioreactors under the condition of leachate recirculation}; Wang H et al.; Landfill bioreactor is a new international development field in the municipal solid waste management . It can reduce the difficulty of leachate treatment and accelerate the landfill stability process . The general moisture distribution rules in landfills were analyzed in this paper and a three dimensional transient mathematical model was established to simulate the saturated-unsaturated moisture movement under the condition of leachate recirculation to landfills . The model was solved by Galerkin finite element method and tested with Theis well formula . The functions of different leahcate recirculation designs in landfill bioreactors were implemented in the model . Thus, the moisture movement in the landfill could be simulated under the condition of leachate recirculation, which provides theoretical basis for the effective design and operation of landfill bioreactor.

Biotechnol Bioeng, 2003 Aug 20, 83(4), 444 - 53
Optimal covalent immobilization of glucose oxidase-containing liposomes for highly stable biocatalyst in bioreactor; Wang S et al.; The glucose oxidase-containing liposomes (GOL) were prepared by entrapping glucose oxidase (GO) in the liposomes composed of phosphatidylcholine (PC), dimyristoyl L-alpha-phosphatidylethanolamine (DMPE), and cholesterol (Chol) and then covalently immobilized in the glutaraldehyde-activated chitosan gel beads . The immobilized GOL gel beads (IGOL) were characterized to obtain a highly stable biocatalyst applicable to bioreactor . At first, the glutaraldehyde concentration used in the gel beads activation as well as the immobilizing temperature and time were optimized to enhance the immobilization yield of the GOL to the highest extent . The liposome membrane composition and liposome size were then optimized to obtain the greatest possible immobilization yield of the GOL, the highest possible activity efficiency of the IGOL, and the lowest possible leakage of the entrapped GO during the GOL immobilization . As a result, the optimal immobilization conditions were found to be as follows: the liposome composition, PC/DMPE/Chol = 65/5/30 (molar percentage); the liposome size, 100 nm; the glutaraldehyde concentration, 2% (w/v); the immobilizing temperature, 4 degrees C; and the immobilizing time, 10 h . Furthermore, the optimal IGOL prepared were characterized by its rapidly increasing effective GO activity to the externally added substrate (glucose) with increasing temperature from 20 to 40 degrees C, and also by its high stability at 40 degrees C against not only the thermal denaturation in a long-term (7 days) incubation but also the bubbling stress in a bubble column . Finally, compared to the conventionally immobilized glucose oxidase (IGO), the higher operational stability of the optimal IGOL was verified by using it either repeatedly (4 times) or for a long time (7 days) to catalyze the glucose oxidation in a small-scale airlift bioreactor .

J Immunol Methods, 2003 Jun 1, 277(1-2), 171 - 83
Mammary gland-specific secretion of biologically active immunosuppressive agent cytotoxic-T-lymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4Ig) in milk by transgenesis; Lui VC et al.; A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival . Tolerance induction is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side effects . The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient's immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical transplantation . To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals . Prior to the generation of transgenic farm animals, we tested our strategy in mice . Using the promoter of the sheep beta-lactoglobulin gene, we expressed our CTLA4Ig chimeric gene in the mammary gland of transgenic mice . The yield of CTLA4Ig was fivefold higher in transgenic milk than that from transfected cells . Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation . We showed that the production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that were fed with the transgenic milk . The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is feasible; further studies involving farm animals are warranted.

Appl Biochem Biotechnol, 2003 Apr-Jun, 109(1-3), 3 - 13
Combining mixing regimes for optimized anaerobic wastewater treatment; Kleerebezem R; Operational practice of high-rate anaerobic bioreactors such as upflow anaerobic sludge bed (UASB) reactors is generally based on maximization of the biomass concentration and, in the case of more than one reactor compartment, operation in parallel . In this article, a modeling approach is used to postulate that the treatment performance of anaerobic bioreactors can be improved by simple operational measures . To achieve minimized effluent soluble substrate concentrations, operation of two reactors in series combined with active exchange of biomass between both reactors is suggested . In this way, substrate concentrations lower than the minimum achievable concentration in a completely mixed reactor can be achieved . It is furthermore suggested that maximized biomass concentrations (and solid retention times {SRTs}) do not necessarily lead to minimized effluent concentrations of organic material . At elevated SRTs, the soluble microbial products resulting from biomass turnover are shown to represent the main fraction of soluble organic material in the effluent of the reactor, limiting treatment efficiency.

Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 193 - 9
Optimization of submerged-culture conditions for mycelial growth and exo-biopolymer production by Auricularia polytricha (wood ears fungus) using the methods of uniform design and regression analysis; Xu CP et al.; This paper is concerned with the optimization of submerged culture conditions for mycelial growth and exo-biopolymer production by Auricularia polytricha by one-factor-at-a-time and uniform design (UD) methods . First, the one-factor-at-a-time method was adopted to investigate the effects of environmental factors (i.e., initial pH and temperature) and variables of medium components (i.e., carbon, nitrogen and mineral sources) on mycelial growth and exo-biopolymer production . Sucrose, yeast extract, and K2HPO4 were identified to be the most suitable carbon, nitrogen, and mineral sources, respectively . The optimal temperature and initial pH for mycelial growth and exo-biopolymer production were found to be 25 degrees C and 5.0, respectively . Subsequently, the concentrations of sucrose, yeast extract, and K2HPO4 were optimized using the UD method . The optimal concentrations for the enhanced production were determined as 6% (w/v) sucrose, 2.5% (w/v) yeast extract, and 0.3% (w/v) K2HPO4 for mycelial yield, and 6% (w/v) sucrose, 1.28% (w/v) yeast extract, and 0.3% (w/v) K2HPO4 for exo-biopolymer production, respectively . Subsequent experiments confirmed the validity of the models . This optimization strategy in shake-flask culture led to a mycelial yield of 6.14 g/l, and exo-biopolymer production of 2.12 g/l, respectively, which were considerably higher than those obtained in the preliminary studies . By using the optimized medium, the maximum concentrations of mycelial biomass and exo-biopolymer in a 5 litre stirred-tank bioreactor indicated 35.3 g/l and 3.1 g/l, respectively.

J Radiat Res (Tokyo), 2002 Dec, 43 Suppl, S213 - 8
Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation; Green LM et al.; Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation . Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure . Tissue samples were fixed and fluorescently labeled for actin and microtubules . Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry . ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant . Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected . With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures . Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue . Thyroxin release paralleled cell survival in the bioreactors and control cultures . Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

Water Sci Technol, 2003, 47(7-8), 157 - 63
Use of active barriers to reduce eutrophication problems in urban lakes; Hart B et al.; Excessive concentration of phosphorus is one of the main causes of algal blooms and eutrophic conditions in lakes . In many urban lakes, it appears that a large proportion of the phosphorus in the water column comes from the sediments, particularly when these are anaerobic . Sub-aqueous capping is a relatively new method that has become an attractive option for isolating contaminated sediments from the environment, thus preventing or delaying the release of contaminants into surface waters . Active barrier materials (i.e . capping layers that consist of one or more reactive components) are gaining increasing attention for their greater efficiency in inactivating contaminants held in sediment layers . This paper reports laboratory bioreactor experiments to test the effectiveness of three forms of calcium carbonate (CaCO3) in reducing the release of phosphorus from anaerobic sediment from Lake Carramar, a small urban lake in Melbourne . Two of the CaCO3 active barrier materials tested proved to be quite effective, the most effective materials being the fine particle size, precipitated forms of CaCO3 . Over the 20-day experimental period, a 2% layer of the German material SoCal reduced the amount of phosphorus released by almost 100 times over that occurring with no barrier . The Australian product ESCal, while not as effective as the SoCal, still reduced the phosphorus released by around 15 times that with no barrier . A finely ground Lilydale limestone was essentially ineffective in reducing phosphorus release from the sediments . A preliminary cost-benefit analysis suggests that SoCal is unlikely to be attractive for use in Australia, given the estimated application cost of around 3,800 dollars per tonne . However, although the ESCal is slightly less effective in retaining phosphorus, its potential application cost estimated at 2,000 dollars per tonne, makes it an attractive option . On the basis of these most promising preliminary results, we intend to further test the use of the ESCal . Further investigations will include: longer term laboratory studies using ESCal, optimisation of the barrier layer and methods for applying this material, mesocosms and full lake studies, and risk assessment studies to ensure there are no adverse ecological effects from its use.

Oncol Rep, 2003 Jul-Aug, 10(4), 783 - 9
Evaluating prostate cancer cell culturing methods: a comparison of cell morphologies and metabolic activity; Winkenwerder JJ et al.; LNCaP prostate cancer cells were grown under four unique cultivation conditions . Two types of bioreactor systems were used to observe the influence of low-shear culture conditions allowing for three-dimensional growth: a) a perfusion rotating wall vessel (RWV) bioreactor; and b) a high aspect ratio vessel (HARV) RWV bioreactor, with periodic medium exchanges (fed-batch) . In addition, two growth methods utilized tissue culture flasks (TCFs): a) unaltered or conventional TCFs; and b) poly(2-hydroxyethyl methacrylate) {poly(HEMA)} coated TCFs, to inhibit cell attachment . Comparisons were drawn based on qualitative observation of cell morphology and quantitative metabolic data . Similar cellular metabolism was demonstrated for cells grown under each condition . The degree of aggregation, however, varies considerably . Spherical shaped aggregates with diameters of 1 to 3 mm were produced when cells were grown within the perfusion-RWV bioreactor . All other growth conditions produced irregular shaped aggregates of various sizes . Quantitative results demonstrated the expected glucose utilization concomitant with lactate accumulation . Immunohistochemical evaluations were unremarkable for all four cultivation conditions . Results demonstrate that use of the perfusion-RWV bioreactor is advantageous in obtaining spherical aggregates, while grown in a controlled environment.

Biotechnol Prog, 2003 May-Jun, 19(3), 1064 - 70
The snowball effect in fed-batch bioreactions; Chang DM; The bioreactor will play an important role in future biological manufacturing . For economic profit, important profiles of the feed rate in fed-batch cultures have been discussed . Unfortunately, the optimal feed rate is less robust . In these studies there exists the snowball effect in a substrate-inhibited bioprocess, in which substrate is accumulated due to uncertain parameters in the model or feed-rate error . The snowball effect also exists in multi-substrate-limited processes . In further studies, the interaction between the substrates has been higher in essential substrates than in growth-enhancing substrates . In a typical fed-batch bioreactor, the amount of the product can be reduced to 1% or less when the snowball effect arises . A new control structure, i.e., an off-line optimized feedforward controller added to a gain-scheduling PI(2)D feedback controller, is proposed to eliminate the troublesome snowball effect . The proposed control strategy recovers the yield up to 95% . Moreover, the robustness of the proposed control structure is demonstrated by simulation.

Biotechnol Prog, 2003 May-Jun, 19(3), 1045 - 8
Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture; Durrschmid MP et al.; Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle . In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells . Both systems are fully scaleable and can be used for industrial high cell density bioprocesses . As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB) . CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands) . Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields . For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.

Biotechnol Prog, 2003 May-Jun, 19(3), 1026 - 8
Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor; Chattopadhyay S et al.; The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide . Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively . The batch kinetics was used to identify the mathematical model . The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor . An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.

Biotechnol Prog, 2003 May-Jun, 19(3), 833 - 43
Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium; Hesse F et al.; The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated . For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line . First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system . During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system . The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated . Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation . This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%) . Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor . For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales . The application of protein supplements did not lead to a significant improvement in the cultivation conditions . The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters . The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate . The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.

Biotechnol Prog, 2003 May-Jun, 19(3), 828 - 32
Hydrogen production with immobilized sewage sludge in three-phase fluidized-bed bioreactors; Wu SY et al.; Municipal sewage sludge was immobilized with a modified alginate gel entrapment method, and the immobilized cells were used to produce hydrogen gas in a three-phase fluidized bed . The hydrogen-producing fluidized beds were operated at different liquid velocity (U(0)) and hydraulic retention time (HRT) . The results show that in response to operating liquid velocities, the fluidized-bed system had three flow regimes, namely, plug flow, slug flow, and free bubbling . Pressure fluctuation analysis was used to analyze the hydrodynamic properties in this three-phase fluidized bed when it was under a steady-state production of biogas . With a steady-state biogas production rate (U(g)) of 0.196 mL/s/L, a transition state occurred at a liquid velocity (U(0)) of 0.85 cm/s . As U(0) < 0.85 cm/s, the system was basically a nonhomogeneous fluidized bed, whereas the bed became homogeneous when U(0) was higher than 0.85 cm/s . The fluidized bed can be stably carried out at high loading rates (HRT as low as 2 h) . Hydrogen fermentation results show that the maximal hydrogen production rate was 0.93 L/h/L and the best yield (Y(H)2(/sucrose)) was 2.67 mol H(2)/mol sucrose.

Plant Cell Rep, 2003 Jun, 21(9), 844 - 50 Epub 2003 Apr 11.
Cost-effective in vitro propagation methods for pineapple; Firoozabady E et al.; We have developed an efficient and cost-effective method for commercial micropropagation of Smooth Cayenne pineapple . In vitro shoots were used as starting materials, and either longitudinal sections of the shoots or leaf bases were used as the explants to regenerate shoots . When these explants were used, the axillary meristems, which usually remain quiescent during shoot multiplication, were able to form new shoots . Subsequent to the regeneration step, additional multiplication was achieved inside a 10-l Nalgene vessel with shoots immersed in liquid medium for 5-10 min/h (periodic immersion bioreactor, PIB) . The shoots were then induced to form roots and transferred to soil . Using the above micropropagation method and the PIB, we produced 6,000-8,000 shoots from two initial shoots in less than 6 months . The clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.

Plant Cell Rep, 2003 Feb, 21(6), 538 - 48 Epub 2003 Jan 09.
Developing a scale-up system for the micropropagation of cucumber (Cucumis sativus L.): the effect of growth retardants, liquid culture and vessel size; Konstas J et al.; We investigated the effect of the growth retardant flurprimidol, the phase of the culture medium (solid versus liquid) and the size of the liquid culture vessel (250-ml flask versus 2.5-l airlift bioreactor) on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants . Flurprimidol at concentrations of 0.1-2 mg l(-1) caused considerable growth retardation but increased, albeit slightly, the number of branches and buds and stimulated (solid medium) or reduced (liquid medium) the accumulation of NO(3)-and PO(4)(3-) . Flurprimidol had varying effects on the accumulation of soluble sugars and antioxidant compounds . Bioreactor-derived plants showed an increased fresh weight and size but a decreased content of macronutrients, solid sugars, ascorbic acid and free antioxidant phenolics . The majority (95%) of the plants were successfully acclimatized after being graft on squash . The perspective for an efficient, commercial-level use of bioreactors in combination with growth retardants of this commercially important vegetable species is discussed.

Best Pract Res Clin Endocrinol Metab, 2003 Jun, 17(2), 211 - 22
Skeletal muscle as an artificial endocrine tissue; Goldspink G; Muscle has the ability to take up and express engineered genes and, because it is a post-mitotic tissue, their half-life of expression is prolonged . Although muscle is not regarded as a secretory tissue, in many cases, the gene products enter the systemic circulation . The possibility exists, therefore, of using this approach to alter levels of endocrine and paracrine factors . As a therapeutic procedure, this method has an advantage over the administration of the peptide/protein, which has a relatively short half-life and requires repeated injections . Engineered genes in plasmid or viral vectors under the control of a muscle-specific regulatory sequence may be introduced by intramuscular injection or by the introduction of transfected myoblasts . The latter is also being used in bioreactors to produce medicinal proteins/peptides in vitro as these offer some advantages over bacterial expression systems . However, for gene therapy purposes, there are still safety issues to be addressed.

Ann N Y Acad Sci, 2003 Mar, 984, 492 - 501
Turnup turndown of membrane operation of membrane bioreactors; Howell JA et al.; Membrane bioreactors can be operated with intermittent permeation and continuous aeration . Aeration close to the surface of a submerged membrane helps to maintain a membrane surface that is free from fouling . The conditions under which this occurs depend on the interaction between flux and aeration rate . Increased flux is possible without severe fouling if the aeration rate is increased . Results of performing membrane operation under the dual intermittency of aeration rate and permeation rate, with permeation also interrupted on a regular cycle, are presented . The results show that membrane plants designed for optimal operation at moderate flux can survive effectively with higher flux operation for restricted periods . Designing to account for such effects could reduce overall plant costs.

Ann N Y Acad Sci, 2003 Mar, 984, 420 - 35
Why and how membrane bioreactors with unsteady filtration conditions can improve the efficiency of biological processes; Daubert I et al.; A membrane bioreactor (MBR), an association of a bioreactor with a crossflow filtration unit, enables continuous processes with total cell retention within the reactor to be realized . Provided that high dilution rates can be applied and that inhibition processes are avoided, very high biomass concentrations can be reached, thereby improving the volumetric productivities . These membrane bioreactors have been successfully applied to various microbial bioconversion, such as alcoholic fermentation, solvents, organic acid production, starters, and wastewater treatment . On the basis of the biological reaction characteristics and bibliographic results, the potentialities and bottlenecks of this methodology are discussed . Depending on the application, it is shown how the performance of the membrane bioreactor can be enhanced by acting either on the biological reaction achievement, by controlling the balance between cell growth and death, or on the dilution rate, by increasing the permeate flux through the filtration unit . This discussion is based on results obtained in specific biological treatments applied to polluted liquid and gas.

Ann N Y Acad Sci, 2003 Mar, 984, 411 - 9
Membrane bioreactors for treating waste streams; Howell JA et al.; Membrane bioreactors (MBRs) have a number of advantages for treating wastewater containing large quantities of BOD . This paper reviews the inherent advantages of an MBR, which include high potential biomass loadings, lower sludge yields, and retention of specialized organisms that may not settle well in clarifiers . A major problem in effluent treatment occurs when mixed inorganic and organic wastes occur with high concentrations of pollutants . Inorganics that might cause extremes of pH and/or salinity will inhibit microbial growth and only specialized organisms can survive under these conditions . Refractory organics are only biodegraded with difficulty by specialized organisms, which usually do not resist the extreme inorganic environments . The use of membrane bioreactors to help separate the micro-organisms from the inorganic compounds, yet permit the organics to permeate, has been developed in two different designs that are outlined in this paper . The use of membrane contactors in a multimembrane stripping system to treat acidic chlorinated wastes is proposed and discussed.

Biotechnol Bioeng, 2003 Aug 5, 83(3), 321 - 34
Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24; Muthing J et al.; R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma . The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the C(H)2 domain of each heavy chain, which is necessary for its in vivo effector function . The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO(2)/HCO(3) (-) (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium . Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode . Specific growth rates (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 0.40 d(-1)) . N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS) . The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation . In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained . The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411 . Different culture buffering gave rise to altered glycosylation pattern of the MAb R24 . Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity .

Waste Manag, 2003, 23(4), 339 - 44
Water leaching of titanium from ore flotation residue; Jaworska MM et al.; Copper ore tailings were tested for the stability of titanium submitted to water leaching in three different reactor systems (agitated vessel, bioreactor and percolated fixed-bed column) . For each of these systems, titanium extraction did not exceed 1% of the available metal . Biomass removed from ore residue adsorbed a small part of the titanium with sorption capacities below 20-30 mg g(-1), but most of this biomass was sequestered in the ore residue . Oxygen and carbon dioxide concentrations were monitored and changes in concentration correlated with bacteria development at the initial stage of the process and to fungal development in the latter stages.

Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 111 - 7
CO2 hydration by immobilized carbonic anhydrase; Bhattacharya S et al.; The enzyme carbonic anhydrase (isoform II) from bovine and human erythrocytes was immobilized using different covalent coupling methods on inert matrices . Immobilized carbonic anhydrase may enable concentration of CO2 for Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase)-catalysed fixation in bioreactors . In the present study the activity of carbonic anhydrase with respect to hydration of CO2 using soluble and immobilized enzymes was determined . The stability of the immobilization matrix, the properties of the immobilized enzymes subjected to a variation in operation variables and the activity profile with respect to storage are reported . Immobilization imparted greater thermal and storage stability and enhanced reusability.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 2002 Oct, 37(5), 329 - 32
{Tissue-engineered auricled cartilage: an experimental study}; Zhang R et al.; OBJECTIVE: To study the feasibility of engineering auricled neocartilage with chitosan/polylacticacid-polycrylactone (PLA-PCL) network scaffolds and to search the difference between dynamic and silent chondrocytal culture techniques . METHODS: Chondrocytes from auricled cartilage of 4 weeks old New Zealand White rabbit were seeded onto chitosan/PLA-PCL network . Ten cell-polymer scaffolds were divided into two groups: dynamic group (n1 = 5) with rotating bioreactors and silent group (n2 = 5) with ordinary dishes to culture . Using scan electroscope, grossly histological and immunohistological techniques, the morphological evaluation was done individually at 1st week in vitro, 4th and 8th week in vivo . RESULTS: Chondrocytes adhered and grew up well on the network, but more quantities of chondrocytes, Glycoaminoglygan (GAG) and type II collagen were found in dynamic group . There was an obvious difference between dynamic and silent group (P < 0.05) . CONCLUSION: Chitosan/PLA-PCL network scaffold is good for adhesions and growth of chondrocytes . Furthermore, dynamic cell culture method is better than silent method for formation of neocartilage.

Water Res, 2003 Jul, 37(12), 2937 - 43
Effects of substrate concentrations on the growth of heterotrophic bacteria and algae in secondary facultative ponds; Kayombo S et al.; This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon . The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors . From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model . The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation . The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l . The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l . The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l . The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass) . The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate . Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.

Neurosurgery, 2003 Jun, 52(6), 1411 - 22; discussion 1422-4
The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy; Dunn IF et al.; Malignant gliomas are among the most challenging of all cancers to treat successfully, being characterized not only by aggressive proliferation and expansion but also by inexorable tumor invasion into distant brain tissue . Although considerable progress has been made in the treatment of these tumors with combinations of surgery, radiotherapy, and chemotherapy, these efforts have not been curative . Neurosurgeons as oncologists have increasingly turned their attention to therapies on a molecular scale . Of particular interest to neurosurgeons is the ability to deliver therapy locally to the tumor site or to take advantage of existing immunological mediators, enhancing drug concentrations or therapeutic cell numbers while bypassing the blood-brain barrier to maximize efficacy and minimize systemic toxicity . Exciting local-therapy approaches have been proposed for these devastating tumors . In this review, we discuss the potential applications of bioreactors, neural stem cells, immunotherapies, biodegradable polymers, and convection-enhanced drug delivery in the treatment of malignant gliomas . These approaches are at different stages of readiness for application in clinical neurosurgery, and their eventual effects on the morbidity and mortality rates of gliomas among human patients are difficult to ascertain from successes in animal models . Nevertheless, we are entering an exciting era of "nanoneurosurgery," in which molecular therapies such as those discussed here may routinely complement existing surgical, radiological, and chemotherapeutic approaches to the treatment of neuro-oncological disease . The potential to deploy any of a number of eloquently devised molecular therapies may provide renewed hope for neurosurgeons treating malignant gliomas.

Int J Artif Organs, 2003 Apr, 26(4), 319 - 30
Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor; Marlovits S et al.; This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel . Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks . Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy . During serial monolayer cultures chondrocytes dedifferentiated to a "fibroblast-like" structure and produced predominantly collagen type I . When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time . Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected . Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils . This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.

Environ Microbiol, 2003 Jun, 5(6), 453 - 61
Bacterial community structure and function in a metal-working fluid; van der Gast CJ et al.; The diversity of bacterial populations colonizing spatially and temporally separated samples of the same metal-working fluid (MWF) formulation was investigated . Analyses were performed with a view to improve strategies for bioaugmentation of waste MWF in bioreactor systems and prevention of in-use MWF biodeterioration in engineering workshops . Significantly, complementary phenotypic, genotypic and in situ methods revealed that the bacterial communities in operationally exhausted MWFs had low diversity and were similar in species composition from different locations and uses . Of the 179 bacterial isolates studied, only 11 genera and 15 species were identified using fatty acid methyl ester (FAME) analysis, with culture independent analyses by 16S rDNA denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization being congruent with these FAME data . In order to gain some insight into functional role of detected populations, we correlated the MWF chemical composition and potential pollution load with bacterial abundance and community composition detected within samples.

Artif Cells Blood Substit Immobil Biotechnol, 2003 May, 31(2), 117 - 26
Artificial liver support in the third millennium; Chamuleau RA; Analogous to the artificial kidney there is a need for an effective and safe liver support system to bridge patients with hepatic failure to liver transplantation or own liver regeneration . An overview is given of the biological and non-biological systems used in clinical practice in the past and at present . The conclusion is drawn that only the biological systems might have the potential to prolong life significantly in patients with acute liver failure . The systems with this potential are summarised . Both in Europe and the USA good bioreactors are available . Most of them are based on porcine hepatocytes, which have immunological and zoonotic drawbacks . What is missing is the well differentiated human hepatocyte in sufficient amounts . Successful development of this cell will be the crown on bioartificial liver research in the third millenium.

Adv Biochem Eng Biotechnol, 2003, 81, 95 - 150
Molecular biology of stress genes in methanogens: potential for bioreactor technology; Conway de Macario E et al.; Many agents of physical, chemical, or biological nature, have the potential for causing cell stress . These agents are called stressors and their effects on cells are due to protein denaturation . Cells, microbes, for instance, perform their physiological functions and survive stress only if they have their proteins in the necessary concentrations and shapes . To be functional a protein shape must conform to a specific three-dimensional arrangement, named the native configuration . When a stressor (e.g., temperature elevation or heat shock, decrease in pH, hypersalinity, heavy metals) hits a microbe, it causes proteins to lose their native configuration, which is to say that stressors cause protein denaturation . The cell mounts an anti-stress response: house-keeping genes are down-regulated and stress genes are activated . Among the latter are the genes that produce the Hsp70(DnaK), Hsp60, and small heat protein (sHsp) families of stress proteins . Hsp70(DnaK) is part of the molecular chaperone machine together with Hsp40(DnaJ) and GrpE, and Hsp60 is a component of the chaperonin complex . Both the chaperone machine and the chaperonins play a crucial role in assisting microbial proteins to reach their native, functional configuration and to regain it when it is partially lost due to stress . Proteins that are denatured beyond repair are degraded by proteases so they do not accumulate and become a burden to the cell . All Archaea studied to date possess chaperonins but only some methanogens have the chaperone machine . A recent genome survey indicates that Archaea do not harbor well conserved equivalents of the co-chaperones trigger factor, Hip, Hop, BAG-1, and NAC, although the data suggest that Archaea have proteins related to Hop and to the NAC alpha subunit whose functions remain to be elucidated . Other anti-stress means involve osmolytes, ion traffic, and formation of multicellular structures . All cellular anti-stress mechanisms depend on genes whose products are directly involved in counteracting the effects of stressors, or are regulators . The latter proteins monitor and modulate gene activity . Biomethanation depends on the concerted action of at least three groups of microbes, the methanogens being one of them . Their anti-stress mechanisms are briefly discussed in this Chapter from the standpoint of their role in biomethanation with emphasis on their potential for optimizing bioreactor performance . Bioreactors usually contain stressors that come with the influent, or are produced during the digestion process . If the stressors reach levels above those that can be dealt with by the anti-stress mechanisms of the microbes in the bioreactor, the microbes will die or at least cease to function . The bioreactor will malfunction and crash . Manipulation of genes involved in the anti-stress response, particularly those pertinent to the synthesis and regulation of the Hsp70(DnaK) and Hsp60 molecular machines, is a promising avenue for improving the capacity of microbes to withstand stress, and thus to continue biomethanation even when the bioreactor is loaded with harsh waste . The engineering of methanogenic consortia with stress-resistant microbes, made on demand for efficient bioprocessing of stressor-containing effluents and wastes, is a tangible possibility for the near future . This promising biotechnological development will soon become a reality due to the advances in the study of the stress response and anti-stress mechanisms at the molecular and genetic levels.

Adv Biochem Eng Biotechnol, 2003, 81, 31 - 56
Metabolic interactions between methanogenic consortia and anaerobic respiring bacteria; Stams AJ et al.; Most types of anaerobic respiration are able to outcompete methanogenic consortia for common substrates if the respective electron acceptors are present in sufficient amounts . Furthermore, several products or intermediate compounds formed by anaerobic respiring bacteria are toxic to methanogenic consortia . Despite the potentially adverse effects, only few inorganic electron acceptors potentially utilizable for anaerobic respiration have been investigated with respect to negative interactions in anaerobic digesters . In this chapter we review competitive and inhibitory interactions between anaerobic respiring populations and methanogenic consortia in bioreactors . Due to the few studies in anaerobic digesters, many of our discussions are based upon studies of defined cultures or natural ecosystems.

Adv Biochem Eng Biotechnol, 2003, 80, 115 - 48
Membrane-assisted extractive bioconversions; Fernandes P et al.; This chapter summarizes the use of membrane reactors in extractive bioconversions as process integration systems leading to in situ product recovery . Several membrane reactor configurations are analyzed, taking into account the type of bioconversion, biocatalyst type and location (either in the aqueous phase or in the membrane), membrane chemistry and morphology, solvent (extractant) type and its biocompatibility . Modeling of liquid-liquid extractive membrane bioreactors operation is also analyzed considering kinetics and mass-transfer aspects . The chapter includes examples from the authors' laboratory as well as other published in the field . Both enzyme and whole cell-based bioconversions are considered . Relevant aspects related to the solvent (extractant) toxicity and how the membrane could protect the biocatalytic activity are analyzed . Trends in this field are also given.

Adv Biochem Eng Biotechnol, 2003, 80, 19 - 68
Integration of physiology and fluid dynamics; Schmalzriedt S et al.; The purpose of strategies for the integration of fluid dynamics and physiology is the development of more reliable simulation tools to accelerate the process of scale-up . The rigorous mathematical modeling of the richly interactive relationship between the dynamic response of biosystems and the physical environment changing in time and space must rest on the link between coupled momentum, energy and mass balances and structured modeling of the biophase . With the exponential increase in massive computer capabilities hard- and software tools became available for simulation strategies based on such holistic integration approaches . The review discusses fundamental aspects of application of computational fluid dynamics (CFD) to three-dimensional two-phase turbulence flow in stirred tank bioreactors . Examples of coupling momentum and material balance equations with simple unstructured kinetic models for the behavior of the biophase are used to illustrate the application of these strategies to the selection of suitable impeller configurations . The examples reviewed in this paper include distribution of carbon and energy source in fed batch cultures as well as dissolved oxygen fields during aerobic fermentations . A more precise forecasting of the impact of the multitude of interactions must, however, rest upon a rigorous understanding of the response of the cell factory to the complex dynamic stimulation due to space- and time-dependent concentration fields . The paper also introduces some ideas for fast and very fast experimental observations of intracellular pool concentrations based on stimulus response methods . These observations finally lead to a more complex integration approach based on the coupling of CFD and structured metabolic models.

J Invest Surg, 2003 Mar-Apr, 16(2), 83 - 92
Recovery of preservation-injured primary human hepatocytes and nonparenchymal cells to tissuelike structures in large-scale bioreactors for liver support: an initial transmission electron microscopy study; Gerlach JC et al.; This study investigated large-scale regeneration and tissue reorganization of adult human liver cells from preservation injured transplant organs . The use of basement membrane protein gels and growth factor enriched culture medium in standard culture flasks promotes liver tissue formation in isolated rat and pig hepatocytes, resulting in prolongation of phenotypic stability and metabolic competence of primary cells in vitro . A special bioreactor construction for high-density three-dimensional cell recovery was developed and isolation of cells from discarded human donor livers was enabled . In vitro regeneration of adult human liver cells isolated from preservation-injured organs took place over a period of 2 weeks in a purpose-built bioreactor . Basement membrane protein and growth factors were avoided . Reorganization of tissue structures was studied using transmission electron microscopy (TEM) . This showed regeneration and tissue reorganization of adult human cells from preservation-injured organs by coculture with nonparenchymal cells in the bioreactor . The majority of the aggregated hepatocytes in the bioreactors showed morphological similarities to those in vivo (although not re-formed to hepatocyte plates), exhibiting cell-cell junctions and reconstituted bile canaliculi-like spaces between neighboring hepatocytes . Perfusion channels appeared regularly between cell aggregates . The arrangement of nonparenchymal cells between the hepatocyte aggregates exhibited similarities to liver sinusoids . Endothelial cells often covered the aggregates and formed a borderline to the perfusion channels between the capillaries . Similar to the space of Disse, further nonparenchymal cells were located between the endothelial cells and the parenchymal aggregates . Deposits of biomatrix fibers occurred spontaneously . The regenerated cell mass was close to that of a single liver lobe . In conclusion, the further optimization of bioreactors that enable cell recovery from preservation injury may lead to the utilization of cells from discarded whole or split transplants for extracorporeal temporary liver support therapy or hepatocyte transplantation.

J Agric Food Chem, 2003 May 21, 51(11), 3455 - 9
The effect of SO2 on the production of ethanol, acetaldehyde, organic acids, and flavor volatiles during industrial cider fermentation; Herrero M et al.; SO(2) is widely used in cider fermentation but also in other alcoholic beverages such as wine . Although the authorized limit is 200 ppm total SO(2), the International Organizations recommend its total elimination or at least reduction due to health concerns . Addition of SO(2) to apple juice at levels frequently used in industrial cidermaking (100 mg/L) induced significantly higher acetaldehyde production by yeast than that obtained without SO(2) . Although the practical implications of acetaldehyde evolution under cidermaking conditions has been overcome by research and few data are available, this compound reached levels in two 2000 L bioreactors that may have prevented the occurrence of simultaneous alcoholic and malolactic fermentation . It was observed that malolactic fermentation had a positive effect promoting reduction of acetaldehyde levels in cider fermented with juice, SO(2)-treated or not . The addition of SO(2) clearly delayed malolactic fermentation comparing to the control, affecting not the onset of the malolactic fermentation but the rate of malic acid degradation . This compound, however, had a stimulatory effect on alcoholic fermentation.

Biotechnol Bioeng, 2003 Jul 5, 83(1), 93 - 103
The use of dinoflagellate bioluminescence to characterize cell stimulation in bioreactors; Chen AK et al.; Bioluminescent dinoflagellates are flow-sensitive marine organisms that produce light emission almost instantaneously upon stimulation by fluid shear in a shear stress dose-dependent manner . In the present study we tested the hypothesis that monitoring bioluminescence by suspended dinoflagellates can be used as a tool to characterize cellular response to hydrodynamic forces in agitated bioreactors . Specific studies were performed to determine: (1) impeller configurations with minimum cell activation, (2) correlations of cellular response and an integrated shear factor, and (3) the effect of rapid acceleration in agitation . Results indicated that (1) at a volumetric mass transfer coefficient of 3 x 10(-4) s(-1), marine impeller configurations were less stimulatory than Rushton configurations, (2) bioluminescence response and a modified volumetric integrated shear factor had an excellent correlation, and (3) rapid acceleration in agitation was highly stimulatory, suggesting a profound effect of temporal gradients in shear in increasing cell stimulation . By using bioluminescence stimulation as an indicator of agitation-induced cell stimulation and/or damage in microcarrier cultures, the present study allows for the verification of hypotheses and development of novel mechanisms of cell damage in bioreactors .

Biotechnol Bioeng, 2003 Jul 5, 83(1), 45 - 52
Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions; Xie L et al.; Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date . One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal . In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures . Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity . This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media . These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions . To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80 . At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement . Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions . After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale .

Microb Cell Fact . 2003 Apr 11;2(1):3.
Metabolic networks of microbial systems; Bhattacharya S et al.; In contrast to bioreactors the metabolites within the microbial cells are converted in an impure atmosphere, yet the productivity seems to be well regulated and not affected by changes in operation variables . These features are attributed to integral metabolic network within the microorganism . With the advent of neo-integrative proteomic approaches the understanding of integration of metabolic and protein-protein interaction networks have began . In this article we review the methods employed to determine the protein-protein interaction and their integration to define metabolite networks . We further present a review of current understanding of network properties, and benefit of studying the networks . The predictions using network structure, for example, in silico experiments help illustrate the importance of studying the network properties . The cells are regarded as complex system but their elements unlike complex systems interact selectively and nonlinearly to produce coherent rather than complex behaviors.

J Biomed Mater Res A, 2003 May 1, 65(2), 144 - 57
Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes; Fey-Lamprecht F et al.; A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney . Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP) . Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between . The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP) . Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days . Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology . As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration . Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli . In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney .

Appl Microbiol Biotechnol, 2003 Jul, 62(1), 1 - 20 Epub 2003 May 06.
Insect cell culture for industrial production of recombinant proteins; Ikonomou L et al.; Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production . Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus . Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture . Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media . The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated . Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing . Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells . Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed . Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.

Environ Sci Technol, 2003 Apr 15, 37(8), 1698 - 704
Bioreactors for removing methyl bromide following contained fumigations; Miller LG et al.; Use of methyl bromide (MeBr) as a quarantine, commodity, or structural fumigant is under scrutiny because its release to the atmosphere contributes to the depletion of stratospheric ozone . A closed-system bioreactor consisting of 0.5 L of a growing culture of a previously described bacterium, strain IMB-1, removed MeBr (> 110 micromol L(-1)) from recirculating air . Strain IMB-1 grew slowly to high cell densities in the bioreactor using MeBr as its sole carbon and energy source . Bacterial oxidation of MeBr produced CO2 and hydrobromic acid (HBr), which required continuous neutralization with NaOH for the system to operate effectively . Strain IMB-1 was capable of sustained oxidation of large amounts of MeBr (170 mmol in 46 d) . In an open-system bioreactor (10-L fermenter), strain IMB-1 oxidized a continuous supply of MeBr (220 /micromol L(-1) in air) . Growth was continuous, and 0.5 mol of MeBr was removed from the air supply in 14 d . The specific rate of MeBr oxidation was 7 x 10(-16) mol cell(-1) h(-1) . Bioreactors such as these can therefore be used to remove large quantities of contaminant MeBr, which opens the possibility of biodegradation as a practical means for its disposal.

Thorac Cardiovasc Surg, 2003 Apr, 51(2), 78 - 83
The relevance of large strains in functional tissue engineering of heart valves; Mol A et al.; BACKGROUND: Exposing the developing tissue to flow and pressure in a bioreactor has been shown to enhance tissue formation in tissue-engineered heart valves . Animal studies showed excellent functionality in these valves in the pulmonary position . However, they lack the mechanical strength for implantation in the high-pressure aortic position . Improving the in vitro conditioning protocol is an important step towards the use of these valves as aortic heart valve replacements . In this study, the relevance of large strains to improve the mechanical conditioning protocol was investigated . METHODS: Using a newly developed device, engineered heart valve tissue was exposed to increasing cyclic strain in vitro . Tissue formation and mechanical properties were analyzed and compared to unstrained controls . RESULTS: Straining resulted in more pronounced and organized tissue formation with superior mechanical properties over unstrained controls . Overall tissue properties improved with increasing strain levels . CONCLUSIONS: The results demonstrate the significance of large strains in promoting tissue formation . This study may provide a methodological basis for tissue engineering of heart valves appropriate for systemic pressure applications.

J Environ Monit, 2003 Apr, 5(2), 336 - 40
Determination of ethanolamine, ethylene glycol and triethylene glycol by ion chromatography for laboratory and field biodegradation studies; Mrklas O et al.; The determination of alkanolamines and glycols in groundwater and subsurface environments is essential for environmental assessment, remediation and monitoring for selected industrial sites . Monoethanolamine (MEA), ammonium, sodium, magnesium and calcium detection was performed using cation exchange chromatography (IC) with suppressed conductivity detection . Acetate, chloride, nitrite, nitrate, phosphate, sulfate and oxalate were monitored employing anion exchange chromatography with suppressed conductivity . Detection of ethylene glycol (MEG) and triethylene glycol (TEG) and ethanol was carried out using ion exclusion chromatography with pulsed amperometric detection . Effective determination of MEA, MEG and TEG in complex groundwater matrices without compound transformation offered improved monitoring capabilities . This study presents robust analytical tools for MEA, MEG and TEG determination in biodegradation studies . Using ion chromatography offered significant advantages for the analyses of groundwater samples and laboratory bioreactor monitoring.

Anal Bioanal Chem, 2003 Jun, 376(3), 342 - 8 Epub 2003 May 01.
Optical sensor systems for bioprocess monitoring; Ulber R et al.; Bioreactors are closed systems in which microorganisms can be cultivated under defined, controllable conditions that can be optimized with regard to viability, reproducibility, and product-oriented productivity . To drive the biochemical reaction network of the biological system through the desired reaction optimally, the complex interactions of the overall system must be understood and controlled . Optical sensors which encompass all analytical methods based on interactions of light with matter are efficient tools to obtain this information . Optical sensors generally offer the advantages of noninvasive, nondestructive, continuous, and simultaneous multianalyte monitoring . However, at this time, no general optical detection system has been developed . Since modern bioprocesses are extremely complex and differ from process to process (e.g., fungal antibiotic production versus mammalian cell cultivation), appropriate analytical systems must be set up from different basic modules, designed to meet the special demands of each particular process . In this minireview, some new applications in bioprocess monitoring of the following optical sensing principles will be discussed: UV spectroscopy, IR spectroscopy, Raman spectroscopy, fluorescence spectroscopy, pulsed terahertz spectroscopy (PTS), optical biosensors, in situ microscope, surface plasmon resonance (SPR), and reflectometric interference spectroscopy (RIF).

Trends Biotechnol, 2003 May, 21(5), 233 - 40
Hematopoietic stem cells: from the bone to the bioreactor; Cabrita GJ et al.; The ex vivo expansion of human hematopoietic stem cells is a rapidly developing area with a broad range of biomedical applications . The mechanisms of renewal, differentiation and plasticity of stem cells are currently under intense investigation . However, the complexity of hematopoiesis, the heterogeneity of the culture population and the complex interplay between the culture parameters that significantly influence the proliferation and differentiation of hematopoietic cells have impaired the translation of small scale results to the highly demanded large-scale applications . The better understanding of these mechanisms is providing the basis for more rational approaches to the ex vivo expansion of hematopoietic stem cells . Efforts are now being made to establish a rational design of bioreactor systems, allowing the modeling and control of large-scale production of stem cells and the study of their proliferation and differentiation, under conditions as similar as possible to those in vivo.

Ann Biomed Eng, 2003 Apr, 31(4), 391 - 402
Phenotype modulation in vascular tissue engineering using biochemical and mechanical stimulation; Stegemann JP et al.; Biochemical stimulation was applied in combination with cyclic mechanical strain to engineered vascular constructs made of isolated smooth muscle cells in a three-dimensional (3D) collagen type 1 matrix . Platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) were added exogenously to the medium used to culture the constructs . Mechanical stimulation was applied using a bioreactor system that imparted a 10% circumferential strain at a frequency of 1 Hz . The parameters studied were gel compaction, cell proliferation, and expression of the contractile protein smooth muscle alpha-actin (SMA) . Mechanical stimulation caused a characteristic increase in gel compaction and cell proliferation, relative to statically cultured controls . Stimulation with PDGF increased cell proliferation and decreased SMA expression in 3D gels, but inhibited the effects of mechanical stimulation and produced a more open matrix structure . TGF-beta strongly inhibited cell proliferation and increased SMA expression, especially in the presence of mechanical strain, and resulted in a dense matrix . These results show that cell phenotype can be modulated in engineered blood vessels by applying selected combinations of biochemical and mechanical stimuli, and suggest that such control over cell function can be used to tailor the properties of engineered tissues.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 471 - 80
Effects of pressure pulsation on oxygen transfer rate measured by sulfite method; Huang WC et al.; Pressure pulsation (PP) was investigated for its effects on oxygen transfer rate (OTR) measured by sulfite oxidation . By manipulating airflow rate, 0.41- 1.2 vvm, and a control valve in a 4-L bioreactor, the frequency of PP was varied at different gas pressures3-15 psig . A mathematical model of OTR was built and compared to experimental data . OTR was also examined at constant gas pressure, 4.5-15.0 psig . The results indicate a good agreement between measurement and model prediction . OTR above 9 psig during PP showed significant enhancement at 25 degrees C . This proves that PP not only affects the elevation of DO level, but also increases the interfacial area and mass transfer coefficient.

Appl Biochem Biotechnol, 2003 Spring, 105 -108, 383 - 92
Measurement of rheological properties of corn stover suspensions; Pimenova NV et al.; Corn stover is currently being evaluated as a feedstock for ethanol production . The corn stover suspensions fed to reactors typically range between 10 and 40% solids . To simulate and design bioreactors for processing highly loaded corn stover suspensions, the rheologic properties of the suspension must be measured . In systems with suspended solids, rheologic measurements are difficult to perform owing to settling in the measurement devices . In this study, viscosities of corn stover suspensions were measured using a helical ribbon impeller viscometer . A calibration procedure is required for the impeller method in order to obtain the shear rate constant, k, which is dependent on the geometry of the measurement system . The corn stover suspensions are described using a power law flow model.

Hybrid Hybridomics, 2003 Feb, 22(1), 11 - 6
Recognition of multiple Mycoplasma bovis antigens by monoclonal antibodies; Denes B et al.; To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034 . Three days after the last immunization the spleen of the immunized mouse was removed aseptically . The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol . The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium . Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA) . The results showed that 63 antibody-producing hybridomas had been obtained . For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting . According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa . The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method . The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland) . Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin . Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day . The isotype of the antibodies was determined by ELISA . The antibodies produced by the 12 cell lines tested were assigned to the IgG(1) subclass according to the heavy chain . Ten cell lines produced kappa and two produced lambda light-chain antibody . Possible cross-reactions of the produced monoclonal anti-M . bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure . All of the 12 antibodies tested gave a reaction with the antigen of M . bovis strain designated 26034 . MAbs 3G12 (67, 69, and 72 kDa) and 5B8 (22, 25, and 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species . The other antibodies reacted with the M . bovigenitalium F7, M . anseris 8389, M . oculi, and M . gallisepticum s6 Holland antigens . Owing to its high specificity and affinity, the antibody produced by the cell line 3G12 is primarily considered suitable for use in immunodiagnostic tests of M . bovis infections.

Planta Med, 2003 Apr, 69(4), 344 - 9
Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor; Palazon J et al.; We tested the effect of three variables: the bioreactor system (Wave or Spray reactor), medium exchange and culture period, on the capacity of a selected hairy root line of Panax ginseng to produce ginsenosides . Among the reactors, the Wave bioreactor appeared to be the most efficient in promoting hairy root line growth . Periodic exchanges of the medium and a longer culture period increased the growth rate of cultured hairy root line and, consequently, its capacity to produce ginsenosides . Under established optimum conditions (medium exchange every 14 days over a culture period of 56 days using the Wave bioreactor), the initial root fresh weight was enhanced more than 28-fold, giving a root biomass of 284.9 g L(-1) and a ginsenoside content of 145.6 mg L(-1) . It is noteworthy that this ginsenoside production exceeded by almost 3-fold that obtained during the shake flask culture of our hairy root line, although it often happens that the scale-up from shake flask to a bioreactor culture results in reduced productivities . To our knowledge this is the first time that a Wave bioreactor has been used for hairy root culture.

Novartis Found Symp, 2003, 249, 34 - 46; discussion 46-51, 170-4, 239-41
The fundamentals of tissue engineering: scaffolds and bioreactors; Vunjak-Novakovic G; Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation . One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate, and (iii) bioreactors that provide the conditions necessary for the cells to regenerate functional cartilaginous tissues . Here we explore the paradigm of tissue-engineered cartilage repair that is based on the generation of immature but functional constructs in vitro, and the remodelling and maturation of these constructs in vivo.

ISA Trans, 2003 Apr, 42(2), 289 - 303
Simulation results for on-line optimization of a batch bioreactor using nonlinear filtering and optimal control; Dondo R et al.; The computation of optimal control profiles for batch bioreactors is based on the use of simple and empirical dynamic models . Since these models present some level of uncertainty, the difference between the model dynamics and the reactor dynamics can have significant effects in the reliability of the calculated profile . To develop near optimal control trajectories considering this drawback, we propose to calculate successive control profiles on a moving time horizon using a mathematical model in which the kinetic parameters are estimated by an observer . The desired objective is to generate a near optimal control trajectory adapted to the "running" fermentation . This idea results in a nonlinear estimator plus an optimizer arrangement that so far has not been applied to batch fermentors . Numerical simulations are performed on xanthan-gum batch fermentations and reasonably good results are obtained.

Huan Jing Ke Xue, 2003 Jan, 24(1), 91 - 7
{Filtration capability of the bio-dynamic membrane}; Fan B et al.; The filter modules made of common mesh material were submerged in to a bioreactor to form a dynamic-membrane bioreactor . This paper studied the filtration capability of the dynamic bio-membrane forming on 100-micron Dacron mesh material . The outstanding solid-liquid separation capability of the dynamic bio-membrane was mainly contributed to the gel-layer attaching on the mesh material . The dynamic bio-membrane could checked 30%-60% TOC of various molecule weights ranging < 3000 to > 10(5), whereas its separation accuracy was not high enough to intercept all the organics of molecules weights larger than 10(5) . The tap water filtration resistance of the dynamic bio-membrane sheet with gel-layer attaching on was about 1.9 x 10(9) m-1, which almost equaled to that of the untapped sheet . The resistance caused by cake-layer contributed about 70% to the total filtration resistance of the dynamic bio-membrane on working . The untapped dynamic bio-membrane sheet showed an initial resistance to tap water permeation, but the one with gel-layer attaching on had little initial resistance . The reason might be that the gel-layer eliminated the hydrophobicity of the mesh material.

In Vitro Cell Dev Biol Anim, 2002 Oct, 38(9), 493 - 504
Three-dimensional growth of endothelial cells in the microgravity-based rotating wall vessel bioreactor; Sanford GL et al.; We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d . Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d . These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology . The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope . Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material . These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB) . The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures . The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME {N(G)-nitro-L-arginine-methylester} . Cells in the SFB responded similarly . The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression . We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.

Environ Technol, 2003 Mar, 24(3), 369 - 76
Treatment of metal-contaminated water and vertical distribution of metal precipitates in an upflow anaerobic bioreactor; Quan ZX et al.; A lab-scale upflow anaerobic bioreactor filled with granular sludge and cow manure was operated for 140 days to determine the mechanism of metal removal and the vertical distribution of metal precipitates . Heavy metal ions were removed in the order of Cu2+, Cd2+, Zn2+, Fe2+ and Mn2+ with respect to the height in the reactor . The solid phase analysis showed that the heavy metals were mostly precipitated in the form of metal sulfides by sulfate reduction The contents of metal precipitates in the reactor were as follows: (i) Cd and Zn were highest in the bottom, (ii) Fe was highest at the low-middle layer, and (iii) Mn was increased with the height in the reactor . The vertical distribution of metal sulfides in the reactor was directly related to the solubility product (Ksp) . Results obtained in this study suggest a feasibility of the application to separate precipitation metal-containing wastewater.

Biotechnol Bioeng, 2003 Jun 30, 82(7), 766 - 77
Experimental investigations of multiple steady states in aerobic continuous cultivations of Saccharomyces cerevisiae; Lei F et al.; The steady-state behavior of a glucose-limited, aerobic, continuous cultivation of Saccharomyces cerevisiae CEN.PK113-7D was investigated around the critical dilution rate . Oxido-reductive steady states were obtained at dilution rates up to 0.09 h(-1) lower than the critical dilution rate by operating the bioreactor as a productostat, where the dilution rate was controlled on the basis of an ethanol measurement . Thus, the experimental investigations revealed that multiple steady states exist in a region of dilution rates below the critical dilution rate . The existence of multiple steady states was attributed to two distinct physiological effects occurring when growth changed from oxidative to oxido-reductive: (i) a decrease in the efficiency of ATP production and utilization (at ethanol concentrations below 3 g/L) and (ii) repression of the oxidative metabolism (at higher ethanol concentrations) . The first effect was best observed at low ethanol concentrations, where multiple steady states were observed even when no repression of the oxidative metabolism was evident, i.e., the oxidative capacity was constant . However, at higher ethanol concentrations repression of the oxidative metabolism was observed (the oxidative capacity decreased), and this resulted in a broader range of dilution rates where multiple steady states could be found .

Cytometry A, 2003 May, 53(1), 22 - 7
Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay; Brott DA et al.; BACKGROUND: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion . However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out . METHODS: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry . RESULTS: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations . The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells . The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13 . However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells . CONCLUSIONS: The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations . The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM . These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures .

J Ind Microbiol Biotechnol, 2003 May, 30(5), 302 - 7 Epub 2003 Apr 17.
Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides; Schiraldi C et al.; There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry . In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures . The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791) . Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E . coli cells subjected to prior thermal permeabilisation . Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75 degrees C) . Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest . Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio . The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75 degrees C . The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml(-1) h(-1) (g biocatalyst)(-1) with ~40% conversion when using a 30% (w/v) solution.

Appl Microbiol Biotechnol, 2003 May, 61(3), 261 - 7 Epub 2003 Feb 20.
Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors; Munoz R et al.; An algal-bacterial consortium formed by Chlorella sorokiniana and a phenanthrene-degrading Pseudomonas migulae strain was able to biodegrade 200-500 mg/l of phenanthrene dissolved in silicone oil or tetradecane under photosynthetic conditions and without any external supply of oxygen . Phenanthrene was only removed when provided in organic solvent, which confirms the potential of two-phase systems for toxicity reduction . Phenanthrene was degraded at highest rates when provided in silicone oil rather than in tetradecane since this solvent probably sequestered the PAH, reducing its mass transfer to the aqueous phase . The influence of phenanthrene concentration, amount of inoculum and light intensity on pollutant removal was also investigated and, under the best conditions, phenanthrene was degraded at 24.2 g m(-3).h(-1) . In addition to being cost-effective and mitigating the release of greenhouse gases into the atmosphere, photosynthetic oxygenation was especially beneficial to the use of two-phase partitioning bioreactors since it prevented solvent emulsification and/or volatilization and evidence was found that the microalgae release biosurfactants that could further enhance phenanthrene degradation.

Transplantation, 2003 Apr 15, 75(7), 916 - 22
Successful multilineage engraftment of human cord blood cells in pigs after in utero transplantation; Fujiki Y et al.; BACKGROUND: Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo . The aim of this study was to accomplish xenotransplantation of human HSPCs into pig . METHODS: Total mononuclear or CD34-positive HSPCs obtained from human cord blood were xenotransplanted percutaneously under an ultrasonographic guidance into preimmune pig fetuses . Peripheral blood and bone marrow (BM) cells of recipient pigs were collected and analyzed for the presence of human cells by a polymerase chain reaction to detect human specific Alu sequence on DNA extracted from those cells . Fluorescence-activated cell sorting (FACS) analysis was also performed to detect human hematopoietic cells . RESULTS: Transplantation of human cord blood cells into pig fetuses aged less than 52 days postcoitus resulted in a good engraftment rate . In one case, engraftment was detected up to 315 days posttransplantation by polymerase chain reaction . Human hematopoietic cells were detectable also by FACS in peripheral blood and BM . Furthermore, human CD34+ HSPCs were also observed in the BM of recipients . Those CD34+ cells in BM were sorted by FACS and subjected to further analyses . First, in vitro colony formation assay resulted in formations of multilineage colonies . Second, when they were transplanted into an immunodeficient mouse they were engrafted in the mouse . CONCLUSIONS: These data indicate an engraftment of human HSPCs in pig BM . In utero transplantation of human HSPCs into a preimmune pig fetus is useful to establish a pig reproducing human hematopoiesis.

Water Res, 2003 Apr, 37(8), 1932 - 8
Effect of tube size on performance of a fixed-film tubular bioreactor for conversion of hydrogen sulfide to elemental sulfur; Syed MA et al.; A fixed-film continuous-flow photobioreactor was used for the removal of hydrogen sulfide from synthetic industrial wastewater and conversion of it to elemental sulfur using sixteen 150 mm length Tygon tubes as the active part of the reactor . Three different tube sizes (internal diameters (ID) 6.4, 3.2, and 1.6mm) were used to investigate the effect of tube size on reactor performance . The reactor with 1.6mm tube attained a higher sulfide loading (1451 mg/Lh) than previously reported in the literature . High bacterial concentration, light to volume ratio and intimate contact between attached bacteria and influent sulfide resulted in excellent performance of the 1.6mm tube reactor.

Chemosphere, 2003 Jun, 51(9), 835 - 44
Porous organoclay composite for the sorption of polycyclic aromatic hydrocarbons and pentachlorophenol from groundwater; Ake CL et al.; Complex mixtures of hazardous chemicals such as polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and groundwater can have severe and long-lasting effects on health . The evidence that these contaminants can cause adverse health effects in animals and humans is rapidly expanding . The frequent and wide-spread occurrence of PAHs in groundwater makes appropriate intervention strategies for their remediation highly desirable . The core objective of this research was to assess the ability of a clay-based composite to sorb and remove toxic contaminants from groundwater at a wood-preserving chemical waste site . Treatment efficiencies were evaluated using either effluent from an oil-water separator (OWS) or a bioreactor (B2) . The effluent water from these units was passed through fixed bed columns containing either an organoclay composite or granular activated carbon . The sorbent columns were placed in-line using existing sampling ports at the effluent of the OWS or B2 . Individual one-liter samples of treated and untreated effluent were collected in Kimax bottles over the course of 78 h (total of 50 samples) . Subsequently each sample was extracted by solid phase extraction methodology, and pentachlorophenol (PCP) and PAH concentrations were quantitated via GC/MS . Columns containing porous organoclay composite, i.e . sand-immobilized cetylpyridinium-exchanged low-pH montmorillonite clay (CP/LPHM), were shown to reduce the contaminant load from the OWS effluent stream by 97% . The concentrations of benzo{a}pyrene (BaP) and PCP were considerably reduced (i.e . >99%) . An effluent stream from the bioreactor was also filtered through columns packed with composite or an equivalent amount of GAC . Although the composite reduced the majority of contaminants (including BaP and PCP), it was less effective in diminishing the levels of lower ring versus higher ring PAHs . Conversely, GAC was more effective in removing the lower ring PAHs, except for naphthalene and PCP . The effectiveness of sorption of PCP from the OWS effluent by the composite was confirmed using a PCP-sensitive adult hydra bioassay previously described in our laboratory . The findings of this initial study have delineated differences between CP/LPHM and GAC for groundwater remediation, and suggest that GAC (instead of sand) as the solid support for organoclay may be more effective for the treatment of contaminated groundwater under field conditions than GAC or CP/LPHM alone . Further work is ongoing to confirm this conclusion.

Biomaterials, 2003 Jun, 24(14), 2523 - 32
A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials; Engelmayr GC Jr et al.; Dynamic flexure is a major mode of deformation in the native heart valve cusp, and may effect the mechanical and biological development of tissue engineered heart valves (TEHV) . To explore this hypothesis, a novel bioreactor was developed to study the effect of dynamic flexural stimulation on TEHV biomaterials . It was implemented in a study to compare the effect of uni-directional cyclic flexure on the effective stiffness of two candidate TEHV scaffolds: a non-woven mesh of polyglycolic acid (PGA) fibers, and a non-woven mesh of PGA and poly L-lactic acid (PLLA) fibers, both coated with poly 4-hydroxybutyrate (P4HB) . The bioreactor has the capacity to dynamically flex 12 rectangular samples (25 x 7.5 x 2mm) under sterile conditions in a cell culture incubator . Sterility was maintained in the bioreactor for at least 5 weeks of incubation . Flexure tests to measure the effective stiffness in the "with-flexure" (WF) and opposing "against-flexure" (AF) directions indicated that dynamically flexed PGA/PLLA/P4HB scaffolds were approximately 72% (3 weeks) and 76% (5 weeks) less stiff than static controls (p<0.01), and that they developed directional anisotropy by 3 weeks of incubation (stiffer AF, p<0.01) . In contrast, both dynamically flexed and static PGA/P4HB scaffolds exhibited a trend of decreased stiffness with incubation, with no development of directional anisotropy . Dynamically flexed PGA/P4HB scaffolds were significantly less stiff than static controls at 3 weeks (p<0.05) . Scanning electron microscopy revealed signs of heterogeneous P4HB coating and fiber disruption, suggesting possible explanations for the observed mechanical properties . These results indicate that dynamic flexure can produce quantitative and qualitative changes in the mechanical properties of TEHV scaffolds, and suggest that these differences need to be accounted for when comparing the effects of mechanical stimulation on the development of cell-seeded TEHV constructs.

Crit Rev Biotechnol, 2003, 23(1), 61 - 93
Enzyme reactor design under thermal inactivation; Illanes A et al.; Temperature is a very relevant variable for any bioprocess . Temperature optimization of bioreactor operation is a key aspect for process economics . This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability . Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation . Enzyme thermal inactivation is a very complex process from a mechanistic point of view . However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor . More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions . This review focuses primarily on reactor design and operation under modulated thermal inactivation . It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved . More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

Yi Chuan Xue Bao, 2002 Dec, 29(12), 1057 - 62
{Murine beta-casein gene sequences direct a human tissue plasminogen activator variant expressed in the milk of transgenic mice}; Lin FY et al.; To investigate the ability of our cloned murine beta-casein locus to direct the exogenous gene expression in the milk of transgenic mice, the human t-PA variant mammary gland expression vector under the control of murine beta-casein gene regulatory elements was constructed, in which the human t-PA variant signal-pro peptide sequence was replaced with murine beta-casein signal peptide sequence and the human t-PA variant mature peptide cDNA was inserted into the second exon of beta-casein gene . The fusion gene was microinjected in the fertilized mice eggs . A total of 285 embryos were microinjected and transferred into 13 surrogate mother mice . Twelve positive transgenic mice were identified through PCR and Southern blot analysis among 42 new born mice . Human t-PA variant was expressed in the milk of 7 transgenic mice, the highest expression level attained to 3.6593 micrograms/ml . The results demonstrated that the murine beta-casein gene regulatory elements can direct the human t-PA variant gene successfully express in the milk of transgenic mice . It lays great foundation for the research on the beta-casein knock-in mice mammary gland bioreactor model construction.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 291 - 6 Epub 2003 Apr 12.
Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent; MacLeod CT et al.; Mycobacterium PYR-1 was used in a two-phase partitioning bioreactor (TPPB) to degrade low and high molecular weight polycyclic aromatic hydrocarbons . TPPBs are characterized by a cell-containing aqueous phase, and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells based on their metabolic demand and the thermodynamic equilibrium of the system . A bioavailable solvent, that is, a solvent usable as a carbon source, was used as the organic layer . Although bioavailable solvents are traditionally deemed unsuitable for use in TPPBs, bis(ethylhexyl) sebacate had superior chemical properties to other solvents examined and was cost-effective . In this system, 1 g of phenanthrene and 1 g of pyrene were completely degraded within 4 days, at rates of 168 mg l(-1) day(-1) and 138 mg l(-1 )day(-1), respectively, based on a 3-l aqueous volume . This is the highest pyrene degradation rate reported in the literature to date . Significant degradation of naphthalene and anthracene was also obtained . This work demonstrates that bioavailable solvents can be successfully used in TPPB systems, and may change the protocols commonly used to select solvents for TPPBs in the future.

Eur J Gynaecol Oncol, 2003, 24(1), 7 - 11
Monoclonal antibodies in the treatment of ovarian cancer; Markowska J; Monoclonal antibody application in ovarian cancer started eight years after the discovery of Kohler and Milstein, with the studies of Bast et al . which led to the introduction of CA 125 antigen estimation to everyday clinical routine . At present, monoclonal antibodies serve as a therapeutic tool for variable targets with the potential to be applied in multiple clinical situations . In the coming decade, the drug market is likely to be flooded with monoclonal antibodies . Most of the available monoclonal antibodies will be produced in bioreactors, using transgenic plants or transgenic animals . Apart from mouse antibodies, chimeric, humanised or human antibodies, oncology will certainly employ chimeric structures with antibody fragments built into the structures of receptors or growth factors . Irrespective of their origin, they will constitute an integral part of medical practice.

Biodegradation, 2002, 13(5), 317 - 28
The oxidation, fate and effects of iron during on-site bioremediation of groundwater contaminated by a mixture of polychlorophenols; Langwaldt JH et al.; Kinetics of simultaneous iron and polychlorophenol (CP) oxidation by groundwater enriched cultures were studied in laboratory and during actual remediation in order to reveal the fate and effects of iron on aerobic on-site bioremediation of boreal groundwater . 2,4,6-tri- (TCP), 2,3,4,6-tetra- (TeCP) and pentachlorophenol (PCP) were degraded in fluidized-bed bioreactor (FBR) by over 99%, over 99%, and over 96%, respectively . The oxygen consumption rate for CP-biodegradation was 1.31 micromol DO L(-1) min(-1) and 0.29 micromol DO L(-1) min(-1) for iron oxidation, i.e . approximately 12% of the oxygen was consumed by iron oxidation during normal FBR operation . Mineralization of CPs was confirmed by DOC removal and chloride release of 158% and 78%, respectively . Excess DOC removal was due to partial degradation of the natural organic matter (NOM) (1.1 mg L(-1) or 24% DOC removal) in the groundwater . Removal of NOM consumed 0.91 micromol DO L(-1) min(-1) . Iron oxidation in the FBR was over 94% of which chemical Fe(II) oxidation accounted for up to 10% . Fe(III) partially accumulated (58 to 69%) in the system . The TCP- and CP-biodegradation consumed DO at two times higher rates than the Fe(II)-oxidation in both, laboratory and full-scale, respectively . The batch assays at various TCP and Fe(II) ratios and DO concentrations showed simultaneous oxygen consumption by TCP and Fe-oxidizers and that increased Fe concentrations do not outcompete the bioremediation of CP's for available oxygen.

Lett Appl Microbiol, 2003, 36(5), 321 - 6
Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii; Kim SW et al.; AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii . METHODS AND RESULTS: The P . sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor . The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1) . The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions . CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production . Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production . SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P . sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.

Arthritis Rheum, 2003 Apr, 48(4), 1047 - 56
Matrix fixed-charge density as determined by magnetic resonance microscopy of bioreactor-derived hyaline cartilage correlates with biochemical and biomechanical properties; Chen CT et al.; OBJECTIVE: To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow- fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage . METHODS: Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC . The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method . The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests . RESULTS: S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time . There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region . Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli . The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end . Linear correlations were established among FCD, proteoglycan content, and biomechanical properties . CONCLUSION: HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment . In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.

J Colloid Interface Sci, 2003 Apr 15, 260(2), 332 - 8
Zeta potential measurement for air bubbles in protein solutions; Phianmongkhol A et al.; Protein adsorption at gas-liquid interfaces is important in a number of processes including foam formation in bioreactors, foam fractionation for protein recovery, and production of protein based food and drinks . The physical properties of the gas-liquid interface will influence foam stability; important properties will include both surface rheological and electrokinetic properties . While surface rheological properties of gas-protein solution interfaces have been reported, there are no published values for electrokinetic properties at such interfaces . In this paper, zeta potential values of gas bubbles in solutions of three proteins, measured using a microelectrophoresis technique, are reported . The three proteins chosen were BSA, beta-casein, and lysozyme; these proteins have all been used previously in protein foaming studies . The effect of protein concentration and ionic strength is considered . For BSA and beta-casein, zeta potential was found to increase with increasing protein concentration and ionic strength . For air bubbles in lysozyme solutions, measured zeta potential was zero . zeta potential values for air bubbles in some binary protein mixtures are also presented.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 121 - 6
Bioelectrocatalysis-based application of quinoproteins and quinoprotein-containing bacterial cells in biosensors and biofuel cells; Ikeda T et al.; Electrochemical studies on the applied aspects of quinoproteins are briefly reviewed . Catalytic reactions of quinoprotein enzymes can be connected to electrochemical reactions directly or by the mediation of molecules functioning as electron acceptors of the enzymes . Such an enzyme-electrochemical reaction is called bioelectrocatalysis . It provides a novel method of kinetic analysis of enzyme catalysis and even whole bacterial cell catalysis . The principle of bioelectrocatalysis is first described, then, the bioelectrocatalysis-based application of quinoproteins in biosensors is mentioned . Characteristics and performance of this type of biosensor is explained by citing our own work . Possible application in bioreactors and biofuel cells is also mentioned.

World J Gastroenterol, 2003 Apr, 9(4), 829 - 32
The development of a new bioartificial liver and its application in 12 acute liver failure patients; Ding YT et al.; AIM: Bioartificial liver is a hope of supporting liver functions in acute liver failure patients . Using polysulfon fibers, a new bioartificial liver was developed . The aim of this study was to show whether this bioartificial liver could support liver functions or not . METHODS: Hepatocytes were procured from swine using Seglen's methods . The bioartificial liver was constructed by polysulfon bioreactor and more than 10(10) hepatocytes . It was applied 14 times in 12 patients, who were divided into 7 cases of simultaneous HBAL and 5 cases of non-simultaneous HBAL . Each BAL treatment lasted 6 hours . The general condition of the patients and the biochemical indexes were studied . RESULTS: After treatment with bioartificial liver, blood ammonia, prothrombin time and total bilirubin showed significant decrease . 2 days later, blood ammonia still showed improvment . within one month period, 1 case (1/7) in simultaneous group died while in non-simultaneous group 2 cases (2/5) died . The difference was significant . Mortality rate was 25 % . CONCLUSION: The constructed bioartificial liver can support liver functions in acute liver failure . The simultaneous HBAL is better than non-simultaneous HBAL.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 676 - 9
Batch kinetics of Pseudomonas sp . growth on benzene . Modeling of product and substrate inhibitions; Monero A et al.; Batch tests of benzene degradation were performed in liquid phase at 30 degrees C, pH 6.8 +/- 0.2, and 200 rpm in two 3-L stirred tank bioreactors, using the benzene-degrading bacterium Pseudomonas sp . NCIMB 9688 . A relatively high starting biomass level (220-270 mg(X)/L) and starting benzene concentration ranging from 20 to 200 mg(S)/L were selected as conditions to investigate possible inhibition phenomena . Volumetric as well as specific rates of biomass formation and substrate consumption were calculated from experimental data of both growth and benzene degradation and used to propose and check a new overall kinetic model for cell growth simultaneously accounting for both product and substrate inhibitions . The results of the present study evidenced the occurrence of a competitive-type product inhibition due to 2-hydroxymuconic semialdehyde (K(iP)' = 0.902 mg(S)/L), which was stronger than the uncompetitive-type inhibition exerted by substrate (K(iS) = 7.69 mg(S)/L).

Biotechnol Prog, 2003 Mar-Apr, 19(2), 639 - 46
On-line optimization of recombinant product in a fed-batch bioreactor; Mahadevan R et al.; In this paper, an efficient scheme for on-line optimization of a recombinant product in a fed-batch bioreactor is presented . This scheme is based on the parametrization of the system states and the elimination of a subset of the dynamic equations in the mathematical model of the fed-batch bioreactor . The fed-batch bioreactor considered here involves the production of chloramphenicol acetyltransferase (CAT) in a genetically modified E . coli . The optimal inducer and the glucose feed rates are obtained using the proposed optimization approach . This approach is compared with the traditional optimization approach, where all the states and the manipulated variables are parametrized . The approach presented in this paper results in a 5-fold improvement in the computational time for the recombinant product optimization . The optimization technique is employed in an on-line optimization scheme, when parametric drift and a disturbance in the manipulated variable is present . Feedback from the process is introduced through resetting the initial conditions of the model and through an observer for estimating the time varying parameter . The simulation results indicated improvement in the amount of product formed, when the optimal profile is regenerated during the course of the batch.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 510 - 21
Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development; Saini S et al.; A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading . This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment . Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth . Construct seeding efficiency with chondrocytes is greater than 95% within 24 h . Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage . Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage . In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation . In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition . The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation . Notably, construct matrix accumulation can be manipulated by hydrodynamic loading . This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development . In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 501 - 9
Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production; Lee YY et al.; Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors . Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted . Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation . Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth . A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium . This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation . The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures . In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved . An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture . Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture . The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production . The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 418 - 27
Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor; Huang YM et al.; Macrophytic marine red algae are a diverse source of bioactive natural compounds . "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified . The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture . Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5) . Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed . Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase . The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1) . Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate . Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate . However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 400 - 4
A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor; Kim HJ et al.; To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized . The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system . The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems . The immobilized enzyme was more stable than the free enzyme at the same pH and temperature . Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability . A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode . The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.

Environ Technol, 2003 Feb, 24(2), 249 - 56
Impact of soluble organic compounds on permeate flux in an aerobic membrane bioreactor; Cicek N et al.; The relative impact of mixed liquor suspended solids and soluble organic compounds on the permeate flux in an aerobic membrane bioreactor (MBR) were investigated during long-term operation . A statistical correlation analysis performed on data obtained over an approximately 700 day operational period revealed that permeate flux was strongly correlated to soluble organic compounds such as soluble sugars and proteins and was not correlated to total mixed liquor COD . Organic compounds with sizes less than 0.10 microm exhibited the strongest correlation to permeate flux . Specific filtration tests conducted on the MBR showed that the effect of soluble COD was most pronounced in the range of 110-210 mg 1(-1) . A critical level of soluble COD was established at 500 mg 1(-1) after which point no correlation was present . The effluent quality remained high throughout the study at below 5 mg 1(-1) total COD, indicating that the membrane was able to retain most organic compounds regardless of mixed liquor soluble COD content . It was concluded that MBR permeate fluxes are enhanced when operating at conditions where bio-degradation is improved and soluble organic compounds are reduced.

J Huazhong Univ Sci Technolog Med Sci, 2002, 22(4), 288 - 91
Qualitative study of three cell culture methods; Wang A et al.; Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed . The results showed that the level of LDH was decreased over time in culture . However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC) . The levels of AST and ALT did not change significantly over the investigated time . The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC . The decline of CYP 1A was faster in MC than in SC . This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC . In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC . Taken together, it was indicated each investigated model had its advantages and disadvantages . It was also underlined that various in vitro models may address different questions.

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 679 - 86 Epub 2002 Dec 19.
Effect of nutrient limitation on product formation during continuous fermentation of xylose with Thermoanaerobacter ethanolicus JW200 Fe(7); Hild HM et al.; Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract . This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions . Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1) . Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1) . Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1) . The latter was also reflected in a shift from ethanol to acetate production at lower growth rates . An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 671 - 8 Epub 2002 Dec 19.
A novel circulating loop bioreactor with cells immobilized in loofa ( Luffa cylindrica) sponge for the bioconversion of raw cassava starch to ethanol; Roble ND et al.; A circulating loop bioreactor (CLB) with cells immobilized in loofa sponge was constructed for simultaneous aerobic and anaerobic processes . The CLB consists of an aerated riser and a non-aerated downcomer column connected at the top and bottom by cylindrical pipes . Ethanol production from raw cassava starch was investigated in the CLB . Aspergillus awamori IAM 2389 and Saccharomyces cerevisiae IR2 immobilized on loofa sponge were placed, respectively, in the aerated riser column and non-aerated downcomer column . Both alpha-amylase and glucoamylase activities increased as the aeration rate was increased . Ethanol yield and productivity increased with an increase in the aeration rate up to 0.5 vvm, but decreased at higher aeration rates . The CLB was operated at an aeration rate of 0.5 vvm for more than 600 h, resulting in an average ethanol productivity and yield from raw cassava starch of 0.5 g-ethanol l(-1) x h(-1) and 0.45 g ethanol/g starch, respectively . In order to increase ethanol productivity, it was necessary to increase the dissolved oxygen (DO) concentration in the riser column and decrease the DO concentration in the downcomer column . However, increasing the aeration rate resulted in increases in the DO concentration in both the riser and the downcomer columns . At high aeration rate, there was no significant difference in the DO concentration in the riser and downcomer columns . The aeration rate was therefore uncoupled from the liquid circulation by attaching a time-controlled valve in the upper connecting pipe . By optimizing the time and frequency of valve opening, and operation at high aeration rate, it was possible to maintain a very high DO concentration in the riser column and a low DO concentration in the downcomer column . Under these conditions, ethanol productivity increased by more than 100%, to 1.17 g l(-1) x h(-1).

Int J Artif Organs, 2003 Feb, 26(2), 139 - 48
Modulation of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) gene expression in isolated porcine hepatocytes perfused within a radial-flow bioreactor after low-temperature storing; Mischiati C et al.; Due to the scarcity of available human livers, porcine hepatocytes are currently being evaluated as a xenogeneic cell source for extracorporeal bioartificial liver (BAL) . Hypothermic storage of isolated porcine hepatocytes could support stocking of cell-loaded bioreactors for BAL use and may provide bioreactors ready to be used at the patient's bedside . For the development of this technology, it is of utmost importance to ensure cell viability and differentiated functions after low-temperature storage and following warm reperfusion . We compared cell viability, functional activity and apoptosis in isolated porcine hepatocytes which were perfused within a radial-flow bioreactor (RFB), stored at 4 degrees C and then reperfused at 37 degrees C . RFBs were loaded with 8 x 10(9), > or = 90% viable hepatocytes at 37 degrees C for 3 h . RFBs were then flushed with 4 degrees C University of Wisconsin solution (UW) and subsequently stored for 24 h or 48 h . RFBs were then reperfused for 8 h with recirculating medium plus serum at 37 degrees C . Cytochrome P450 (CYP) activity was studied before and after cold storage by means of monoethylglycinexylide (MEGX) detection in the effluent medium, after repeated lidocaine injections . After reperfusion experiments, hepatocytes were harvested for total RNA isolation . Reverse transcriptase-polymerase chain reaction (RT-PCR) was used in order to amplify specific mRNAs for Bcl-2 and Bax genes, by using appropriate primers; beta-actin primers were used as control . Total RNA was extracted by northern blotting analysis and for Bcl-2, Bax and beta-actin RNA messenger detection, RT-PCR amplification was used . Freshly isolated hepatocytes perfused into the RFB showed a progressive increase of MEGX while a loss in Bax expression was paralleled by an increase in Bcl-2 expression, in comparison to starting hepatocytes . After 4 degrees C storage and warm reperfusion, MEGX production was preserved in 24 h- and 48 h-stored bioreactors as well as a sharp increase of Bcl-2 and a decrease of Bax mRNAs . Our study suggests that refrigeration of hepatocyte-bioreactors is a suitable strategy to maintain both viability and function of isolated hepatocytes, for up to 48 h a time-length that is compatible with long-distance delivery of ready-to-use bioreactors.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Mar 25, 786(1-2), 143 - 51
Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector; Favre LI et al.; Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric-dimeric IgA (IgA(p/d)) antibody associated with secretory component (SC) . SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia . This property makes SIgA suitable as potential mucosal vaccine delivery vector . It remains that production and purification of SIgA is a complex task since IgA(p/d) and SC are naturally synthesized by two different cell types . Furthermore, only IgA(p/d) are capable to associate with SC . Thus, we sought to separate IgA(p/d) and monomeric IgA (IgA(m)) antibodies secreted by hybridoma cells in CELLine bioreactors . To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system . In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors . To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgA(p/d), and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads . Biochemical characterization based on SDS-PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure . Such preparations are currently used to immunize mice to induce mucosal and systemic responses.

Hum Gene Ther, 2003 Feb 10, 14(3), 243 - 54
Optimization of the generation and propagation of gutless adenoviral vectors; Sakhuja K et al.; Adenoviral vectors devoid of all viral coding regions are referred to by many names, including gutless vectors . Gutless vectors display reduced toxicity and immunogenicity, increased duration of transgene expression, and increased coding capacity compared to early generation vectors, which contain the majority of the viral backbone genes . However, the production of gutless vectors at a scale and purity suitable for clinical use has limited the utility of this technology . In this work we describe the optimization of the production of gutless vectors . We constructed an improved helper virus and generated an alternative gutless vector producer cell line, PERC6-Cre . We demonstrated increased gutless vector yields, minimal helper virus contamination, and no replication-competent adenovirus contamination using the optimized system . Furthermore, the PERC6-Cre cells were adapted to serum-free suspension culture and high-titer gutless vector preparations were produced using bioreactor technology, suggesting the feasibility of gutless vector scale-up for clinical use . Finally, we observed that helper virus lacking a packaging signal could be packaged at a low frequency, revealing an inherent limitation to the differential packaging strategy for gutless vector propagation.

J Gravit Physiol, 2001 Jul, 8(1), P13 - 6
Space bioreactors: their use, their future; Walther I; NASA: The use of bioreactors during space flight is discussed . The major elements of a bioreactor are a culture chamber, sensors, a control unit with feedback, as gas exchange system, a pump, fresh culture medium, and a waste reservoir . Types of bioreactors developed for use in space include the Woodlawn Wanderer 9 apparatus, the Space tissue loss system, rotating wall vessel, dynamic cell culture system and the SBR I . Future development for space bioreactors include improvements for cultivation of mammalian cells and tissue engineering and the transfer of bioreactor technology for earth-bound instruments .

Tissue Eng, 2003 Feb, 9(1), 51 - 62
Functional assessment of tissue-engineered meniscal cartilage by magnetic resonance imaging and spectroscopy; Neves AA et al.; A perfusion bioreactor system was used to grow bioartificial meniscal cartilage tissue in vitro . Magnetic resonance imaging and magnetic resonance spectroscopy methods were used to characterize the flow and perfusion profiles and the growth, distribution, and bioenergetics of the fibrochondrocytes in the resulting constructs . These measurements were correlated with each other and with subsequent histologic analysis . The study has demonstrated that these noninvasive magnetic resonance methods will be useful for designing bioreactor operation strategies and cell scaffolds that lead to the production of tissue-engineered meniscal cartilage constructs with properties resembling those of the native tissue.

Tissue Eng, 2003 Feb, 9(1), 9 - 26
Articular cartilage bioreactors and bioprocesses; Darling EM et al.; This review summarizes the major approaches for developing articular cartilage, using bioreactors and mechanical stimuli . Cartilage cells live in an environment heavily influenced by mechanical forces . The development of cartilaginous tissue is dependent on the environment that surrounds it, both in vivo and in vitro . Chondrocytes must be cultured in a way that gives them the proper concentration of nutrients and oxygen while removing wastes . A mechanical force must also be applied during the culturing process to produce a phenotypically correct tissue . Four main types of forces are currently used in cartilage-culturing processes: hydrostatic pressure, direct compression, "high"-shear fluid environments, and "low"-shear fluid environments . All these forces have been integrated into culturing devices that serve as bioreactors for articular cartilage . The strengths and weaknesses of each device and stimulus are explored, as is the future of cartilage bioreactors.

Huan Jing Ke Xue, 2002 Nov, 23(6), 51 - 6
{A submerged dynamic membrane bioreactor for domestic wastewater treatment}; Fan B et al.; The dynamic membrane forming on the surface of 0.1 mm Dacron mesh was utilized instead of static membrane, e.g . hollow fiber membrane, to develop a submerged dynamic membrane bioreactor (DMBR) . The DMBR had high quality effluents when treating domestic wastewater at HRT = 3.5 h . The dynamic membrane could work steadily at a pressure no more than several centimeters water head drop . The highest SS concentration in the effluents was 4.05 mg/L, whereas the measured SS concentrations were zero in most circumstances . At condition of MLSS = 7000-8000 mg/L, the effluent head drop was only 5.8 mm when flux = 14.9 L/(m2.h), and it had little change during a continuous run for 27 days . The dynamic membrane averagely removed 28.74% of COD and 36.9% of TOC in the supernatant of the mixed liquor . The DMBR could remove NH3-N more than 96% when DO in the mixed liquor was no less than 2-3 mg/L.

Nat Mater, 2002 Sep, 1(1), 42 - 4
Living bacteria in silica gels; Nassif N et al.; The encapsulation of enzymes within silica gels has been extensively studied during the past decade for the design of biosensors and bioreactors . Yeast spores and bacteria have also been recently immobilized within silica gels where they retain their enzymatic activity, but the problem of the long-term viability of whole cells in an inorganic matrix has never been fully addressed . It is a real challenge for the development of sol-gel processes . Generic tests have been performed to check the viability of Escherichia coli bacteria in silica gels . Surprisingly, more bacteria remain culturable in the gel than in an aqueous suspension . The metabolic activity of the bacteria towards glycolysis decreases slowly, but half of the bacteria are still viable after one month . When confined within a mineral environment, bacteria do not form colonies . The exchange of chemical signals between isolated bacteria rather than aggregates can then be studied, a point that could be very important for 'quorum sensing'.

Bioresour Technol, 2003 Jul, 88(3), 167 - 77
Production of mycelium biomass and ethanol from paper pulp sulfite liquor by Rhizopus oryzae; Taherzadeh MJ et al.; The cultivation conditions for Rhizopus oryzae grown in synthetic medium and paper pulp spent sulfite liquor (SSL) were investigated to achieve high biomass and ethanol yields using shake flasks and bioreactors . The fungus assimilated the hexoses glucose, mannose and galactose, and the pentoses xylose and arabinose as well as acetic acid which are present in SSL . The assimilation of hexoses was faster than pentoses during cultivation in a synthetic medium . However, all sugars were assimilated concomitantly during growth in SSL supplemented with ammonium, magnesium, calcium, phosphate, sulfate and trace amounts of some other metal ions (SSL-S) . The medium composition had an important influence on biomass yield . The highest biomass yields, viz . 0.18 and 0.43 g biomass/g sugar were obtained, when the cells were cultivated in shake flasks with a synthetic medium containing glucose as carbon and energy source and SSL-S, respectively . The corresponding yields in a bioreactor with more efficient aeration were 0.22 and 0.55 g/g . In addition to the biomass, ethanol, lactic acid, and glycerol were important extracellular metabolites of the cultivation with maximum yields of 0.37, 0.30 and 0.09 g/g, respectively . When the source of sugars in the medium was exhausted, the fungus consumed the metabolites produced, such that the liquid medium was depleted of potential oxidizable nutrients . In general, there was a direct competition between lactic acid and ethanol among the metabolites . Poor medium compositions and cultivation conditions resulted in higher yields of lactic acid, whereas the ethanol and biomass yields were higher in rich media . SSL-S supported good growth of mycelium and a high ethanol yield.

J Biotechnol, 2003 Mar 20, 101(3), 253 - 65
Catalytic properties of endoxylanase fusion proteins from Neocallimastix frontalis and effect of immobilization onto metal-chelate matrix; Mesta L et al.; The production of hybrid enzymes with novel properties and the research for new methods for enzyme immobilization in bioreactors are of major interest in biotechnology . We report here the second part of a study concerning the improvement of the properties of the endoxylanase XYN3A4 from the anaerobic fungi Neocallimastix frontalis . The effects of gene fusion and immobilization on metal-chelate matrix are also compared for the reference enzymes XYN3, XYN3A, XYN4 used for the construction of the fusion protein XYN3A4 . The influence of the metal ion in the immobilization process was first investigated and best immobilization yields were obtained with the Cu(II) ion whereas best coupling efficiencies were reached with the Ni(II) ion . It was also observed that XYN3, XYN3A and XYN34 had a lower rate of hydrolysis when immobilized on Ni(II)-IDA and more difficulties to accomodate small substrates than the soluble enzymes . Nevertheless, a major difference was noted during the hydrolysis of birchwood xylan and it appears that the reaction using the immobilized XYN3A4 chimeric enzyme leads to the accumulation of a specific product.

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 118 - 28 Epub 2003 Feb 08.
Scale-up of stirring as foam disruption (SAFD) to industrial scale; Hoeks FW et al.; Foam disruption by agitation-the stirring as foam disruption (SAFD) technique-was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1 . The dominating mechanism behind SAFD-foam entrainment-was also demonstrated at production scale . The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption . This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications . Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e . the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw . The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming . In this way a substantial increase in the volumetric productivity of the bioreactor was achieved . Design recommendations for the application of SAFD are given in this paper . Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised.

In Vitro Cell Dev Biol Anim, 2002 Sep, 38(8), 436 - 9
Three-dimensional binding of epidermal growth factor peptides in colonic tissues produced from rotating bioreactor; Kaeffer B et al.; Epidermal growth factor peptide binding was analyzed on primary cultures of colonic cells and along crypts by fluorescent laser-scanning confocal microscopy, using a three-dimensional image analysis software (Quant3D, Linux/Unix) . Structural, proliferative units from primary cultures grown in rotating bioreactor for 41 d were arranged according to a tubular symmetry or on a parallelepiped sheet . Mean width, height, and depth of 23 tissue-like masses (+/- standard error) were 125 microm (+/-16), 152 microm (+/-23), and 29 microm (+/-3), respectively . Mean density of nuclei in tissue-like masses, expressed as the number of nuclei per cubic millimeter (+/- standard error of the mean), was 1.8 x 10(5) (+/-0.7 x 10(5)) nuclei per cubic millimeter, which corresponded to a density that was five to six times lower than that estimated for the colonic crypt isolated by chelation . Spots of high epidermal growth factor (EGF) peptide binding that corresponded to microlesions in crypt monolayers or to active colonization of microcarriers by epithelial and stromal cells in tissue-like masses were observed . The relative intensities of EGF peptide binding that were obtained below cell position 8 on crypts were very homogeneous and were representative of the profile obtained with crypts isolated from adult rats adapted to a normal diet and used to develop primary cultures of colonocytes in our laboratory . A microscopic multidimensional analytic system to record the expression profiles of biomarkers along intestinal tissues should enhance the use of primary cultures of colonocytes for in vitro testing of new food products.

Chemosphere, 2003 Apr, 51(4), 295 - 303
Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor; Moldes D et al.; The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in a fixed-bed tubular bioreactor, filled with cubes of nylon sponge, operating in semi-solid-state conditions, was studied . Maximum individual manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1293 and 225 U/l were detected.The in vitro decolourisation of two structurally different dyes (Poly R-478, crystal violet) by the extracellular liquid obtained in the above-mentioned bioreactor was monitored in order to determine its degrading capability . The concentration of some compounds (sodium malonate, manganese sulphate) from the reaction mixture was optimised in order to maximise the decolourisation levels . A percentage of Poly R-478 decolourisation of 24% after 15 min of dye incubation was achieved.On the other hand, a methodology for a long treatment of these dyes based on the continuous addition of MnP enzyme and H(2)O(2) was developed . Moreover, this enzymatic treatment was compared with a photochemical decolourisation process . The former allowed to maintain the degradation rate almost constant for a long time, resulting in a decolourisation percentage of 70% and 30% for crystal violet and Poly R-478, respectively, after 2 h of treatment . As for the latter, it was not able to degrade Poly R-478, whereas crystal violet reached a degradation of 40% in 2 h.

Metab Brain Dis, 2002 Dec, 17(4), 485 - 91
Bioartificial liver support anno 2001; Chamuleau RA; Despite maximal intensive care, mortality of acute fulminant hepatic failure is high: 60%-75% in several studies . In addition patients with chronic liver insufficiency suffer from a bad quality of life: all patients suffer from fatigue; symptoms of hepatic encephalopathy, jaundice, and itching are often present . Analogous to artificial kidney treatment in patients with renal failure, an artificial liver assist device is needed not only to bridge patients with fulminant hepatic failure to liver transplantation or own liver regeneration, but also to improve the quality of life of patients with chronic liver insufficiency . Several modalities of artificial liver support are under investigation, like plasma exchange, haemodialysis, haemadsorption, albumin dialysis, liver cell transplantation, and the bioartificial liver . Artificial livers based on only supportive detoxification function do not show significant improvement of survival in controlled studies . Bioartificial liver support systems have also the potential to support hepatic synthetic functions . Bioreactors can be charged with freshly isolated or cryopreserved porcine hepatocytes, but also by human hepatoma cell lines . Several uncontrolled studies in humans show safety of such a treatment, even by using porcine cells . Transmission of porcine endogenous retrovirus to recipients has not been found . Furthermore, beneficial effects have been reported on symptoms of hepatic encephalopathy, on the height of intracranial pressure and on hemodynamic parameters . By using porcine cells immunological problems (e.g., serum sickness) can be expected during treatments longer than one week . However, "proof of the pudding" in the sense of improvement of survival is not yet available . The creation of a "liver dialysis unit" in the near future depends mainly on the development of well-differentiated immortalized human hepatocytes . Some progress in this field has already been obtained.

Metab Brain Dis, 2002 Dec, 17(4), 477 - 84
Concept for modular extracorporeal liver support for the treatment of acute hepatic failure; Sauer IM et al.; Acute liver failure has a poor prognosis . The introduction of liver transplantation as a therapeutic option reduced mortality to 20-40%.With the growing disparity between the number of organ donations and the number of patients waiting for liver transplantation, efforts have been made to optimize the allocation of organs and to design extracorporeal methods to support the failing liver . The modular extracorporeal liver support is a concept for the treatment of hepatic failure . The CellModule is a multicompartment bioreactor for extracorporeal liver support therapy . The construction provides efficient integrated oxygenator functions and decentralized mass transfer is effected by a woven array of capillary systems . The bioreactor promotes primary human liver cells to spontaneous neo-formation of liver sinusoidal structures in vitro . Small capillary subunits, in which interwoven membrane links represent the liver lobuli, are simultaneously perfused . The used cell mass of 400-600 g enabled the clinical application of a liver lobe equivalent hybrid organ . The DetoxModule enables albumin-dialysis for removal of albumin-bound toxins; a DialysisModule for continuous veno-venous hemofiltration can be added to the system, in the case of hepato-renal failure.

Hepatology, 2003 Mar, 37(3), 665 - 73
CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver; Iwahori T et al.; CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver . The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5 . A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells . The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture . Western blot analysis also demonstrated an increase in expression of the CYP3A protein . When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6 beta-hydroxy testosterone as a metabolite was formed . Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptor alpha(RXR alpha), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin . Similarly, the assay with a probe of HNF-4 alpha-binding motif indicated the formation of a large protein complex in the RFB culture . Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4 alpha, the large complexes binding to response elements of PXR or HNF-4 alpha in the RFB culture may contribute to up-regulation of CYP3A4 mRNA . In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4.

Water Res, 2003 Apr, 37(7), 1515 - 26
Regulatory role of n-propanol in propylene glycol biomethanization under overload; Seok J et al.; This work examines the transient response of an anaerobic fluidized bed bioreactor to an overload of propylene glycol (PG), the primary component in aircraft de-icing waste . Under favorable operating conditions, PG was converted to n-propanol (n-PrOH) and propionate (HPr), and subsequently n-PrOH was completely converted into HPr . HPr was then fully degraded to methane and carbon dioxide via acetate . Under an overload condition, n-PrOH conversion to propionate was completely blocked but propionate degradation continued, contrary to free-energy computations in which n-PrOH should rapidly degrade and HPr should accumulate . When the imposed overload condition was relieved, the accumulated n-PrOH was rapidly converted into propionate . n-PrOH, then, could act as a temporal sink for reducing equivalents (XH(2)) and could regulate the overall PG methanazation process . n-PrOH should be monitored along with typical VFAs such as HPr to avoid sudden VFA accumulation and thus to optimize process performance for PG methanization .

Biotechnol Bioeng, 2003 May 5, 82(3), 306 - 12
Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate-reducing bacteria; Utgikar VP et al.; The adverse effects of copper and zinc on an acetate-utilizing mixed cultures of sulfate-reducing bacteria (SRB) at concentrations below the toxic concentration (minimum metal concentration at which no sulfate reduction is observed) are reported in this paper . Mathematical models were developed to incorporate the toxic and inhibitory effects (defined as the reduction in bacterial population upon exposure to the metal and the decrease in the metabolic rate of sulfate reduction by the SRB, respectively) into the sulfate-reduction biokinetics . The characteristic toxicity and inhibition constants were obtained from the measurements of bacterial populations and dissolved metal concentrations in serum bottle studies conducted at 35 degrees C and pH 6.6 . Both copper and zinc had toxic and inhibitory effects on SRB . The toxicity constants for copper and zinc were 10.6 and 2.9 mM(-1), respectively, indicating that exposure to copper resulted in a higher mortality of SRB than did exposure to zinc . The values of the inhibition constants were found to be 17.9 +/- 2.5 and 25.2 +/- 1.0 mM(-1) for copper and zinc, respectively . This implies that dissolved zinc was slightly more inhibitory to SRB than copper . The models presented in the paper can be used to predict the response of a sulfate-reduction bioreactor to heavy metals during acid mine drainage treatment .

Biotechnol Bioeng, 2003 May 5, 82(3), 253 - 62
Formation of steady-state oxygen gradients in vitro: application to liver zonation; Allen JW et al.; We have developed a perfusion bioreactor system that allows the formation of steady state oxygen gradients in cell culture . In this study, gradients were formed in cultures of rat hepatocytes to study the role of oxygen in modulating cellular functions . A model of oxygen transport in our flat-plate reactor was developed to estimate oxygen distribution at the cell surface . Experimental measurements of outlet oxygen concentration from various flow conditions were used to validate model predictions . We showed that cell viability was maintained over a 24-h period when operating with a physiologic oxygen gradient at the cell surface from 76 to 5 mmHg O(2) at the outlet . Oxygen gradients have been implicated in the maintenance of regional compartmentalized metabolic and detoxification functions in the liver, termed zonation . In this system, physiologic oxygen gradients in reactor cultures contributed to a heterogeneous distribution of phosphoenolpyruvate carboxykinase (predominantly localized upstream) and cytochrome p450 2B (predominantly localized downstream) that correlates with the distribution of these enzymes in vivo . The oxygen gradient chamber provides a means of probing the oxygen effects in vitro over a continuous range of O(2) tensions . In addition, this system serves as an in vitro model of zonation that could be further extended to study the role of gradients in ischemia-reperfusion injury, toxicity, and bioartificial liver design .

Water Res, 2003 Mar, 37(6), 1296 - 307
Biokinetic modeling of in situ bioremediation of BTX compounds-impact of process variables and scaleup implications; Nakhla G; The impact of three process parameters, i.e . ground water velocity, oxygen-to-BTX mass ratio, and benzene, toluene and xylene (BTX) concentrations on first-order biodegradation kinetics in a pilot-scale in situ bioremediation system was assessed . Generally, first-order biodegradation coefficients decreased with ground water velocity, and increased with hydrogen peroxide dose and BTX concentration . First-order biodegradation rate coefficients for benzene, toluene, and o-xylene varied from 0.3 to 0.81, 0.24 to 0.72, and 0.21 to 0.63 d(-1), respectively . Biomass-specific first-order rate coefficients were insensitive to ground water velocity, and decreased with increasing BTX concentrations . At 10mg/l BTX concentration, the specific first-order coefficients increased with peroxide dose . However, at the 50mg/l BTX concentration and a peroxide dose of 1020 mg/l, a 30-70% reduction in specific first-order biodegradation coefficients was observed . BTX biodegradation kinetics in this pilot-scale system were approximately one-to-two orders of magnitude slower than in soil microcosms and mixed culture bioreactors, and about 200-300% higher than full-scale systems.

Water Res, 2003 Mar, 37(6), 1260 - 9
Toxicity of di-(2-ethylhexyl) phthalate on the anaerobic digestion of wastewater sludge; Alatriste-Mondragon F et al.; Previous studies on the microbial degradation of individual phthalic acid esters (PAEs) have demonstrated that the compounds with short ester hydrocarbon chains are easily biodegraded and mineralized, but PAEs with long ester chains are less susceptible to degradation and some of them are considered recalcitrant . Moreover, they inhibit methanogenesis . However, studies have not been made on the effect of feeding a combination of recalcitrant and biodegradable PAEs into anaerobic digesters treating wastewater sludge . The present study was conducted with wastewater sludge from the Los Angeles Bureau of Sanitation's Hyperion Treatment Plant . Di (2-ethylhexyl) phthalate (DEHP), the most common persistent PAE found in wastewater, and di-n-butyl phthalate (DBP), a common PAE with short ester chains, were sorbed into the sludge fed to a bench-scale digester for a period of 12 weeks . DEHP degradation was always poor, and accumulation of DEHP was correlated with inhibition of the microbial degradation of DBP and with process instability of the test digester . Inhibition of the DBP removal was completely reversed after DEHP addition was discontinued, but biogas production never recovered to the level observed in a control digester . Other process parameters of digester performance were not affected by DEHP accumulation . These results are similar to the toxic effects of long chain fatty acids on sludge digestion, suggesting that DEHP or its degradation products affect all the microbial populations in the anaerobic bioreactor . Our results imply that high levels of DEHP or other recalcitrant PAEs in wastewater sludge are likely to compromise methanogenesis and removal of biodegradable PAEs in sludge digesters.

Indian J Exp Biol, 2002 Aug, 40(8), 930 - 3
Solid state cultivation of Curvularia lunata for transformation of rifamycin B to S; Rasalkar AA et al.; Biotransformation of rifamycin B to rifamycin S using two strains of C . lunata namely NCIM 716 and NMU grown on various solid substrates viz., grass, paper, jowar/wheat straw, bran and bagasse was studied . Almost complete biotransformation efficiency of rifamycin B at 0 . 06 mM concentration was observed within 24 hr . Among these two strains, C . lunata NMU showed 90% of biotransformation and higher rate of cellulose utilization on solid substrates vis-a-vis reference strain . Cellulase activity of both strains was also studied for exoglucanase, endoglucanase and beta-glucosidase . Column bioreactor studies with bagasse revealed further improvement in biotransformation efficiency of C . lunata NMU.

J Biomech Eng, 2002 Dec, 124(6), 742 - 9
Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering; Altman GH et al.; Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues . To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control . We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function . Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation . A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port . Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture . The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).

Biomaterials, 2003 May, 24(10), 1771 - 80
Microcapsules with improved mechanical stability for hepatocyte culture; Yin C et al.; Packed-bed or fluidized-bed bioreactor filled with microencapsulated hepatocytes has been proposed as one of the promising designs for bioartificial liver assist device (BLAD) because of potential advantages of high mass transport rate and optimal microenvironment for hepatocyte culture . Recently, we have developed a microcapsule system for the encapsulation of hepatocytes . The microcapsules consist of an inner core of modified collagen and an outer shell of terpolymer of methyl methacrylate, methacrylate and hydroxyethyl methacrylate . Cells encapsulated in these microcapsules exhibit enhanced cellular functions . Improving the mechanical stability of the microcapsules to withstand the shear stress induced by high perfusion rate would be crucial to the success of BLAD applications . In this study, we investigated the effects of terpolymer molecular weight (M(w)) on the mechanical property of these microcapsules and the differentiated functions of encapsulated hepatocytes . Six terpolymers with different M(w) were synthesized using radical polymerization in solution by adjusting the reaction temperature and the initiator concentration . All the terpolymers formed microcapsules with the methylated collagen . While the terpolymer M(w) had little effect on the capsule membrane thickness and permeability of serum albumin, the mechanical property of the microcapsules was significantly improved by the higher M(w) of the terpolymer . Differentiated functions of the hepatocytes cultured in the microcapsules, including urea synthesis, albumin synthesis and cytochrome P450 metabolic activity, were not significantly affected by the terpolymer M(w) .

Anal Chem, 2003 Feb 1, 75(3), 388 - 93
Creation of an on-chip enzyme reactor by encapsulating trypsin in sol-gel on a plastic microchip; Sakai-Kato K et al.; Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection . Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip . Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip . The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated . The sol-gel in the SR did not alter the separation efficiency of each peak . With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes . This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments . Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.

Biotechnol Appl Biochem, 2003 Feb, 37(Pt 1), 83 - 90
Effects of fermentation strategy on the characteristics of plasmid DNA production; O'Kennedy RD et al.; The synthesis of supercoiled plasmid DNA (SC-pDNA) for therapeutic use will involve large-scale production in bioreactors . The success of these fermentations will be dependent on the interactions between the host organism, the recombinant plasmid vector and the growth environment . In the present study, the recombinant host, Escherichia coli DH5 alpha bearing the recombinant plasmid pSV beta, was grown in shake flasks, batch and exponentially fed-batch bioreactors . Specific and volumetric pDNA yields were increased 8- and 25-fold respectively using exponentially fed-batch cultures in comparison with shake-flask cultures . The percentage of SC-pDNA as a proportion of total plasmid DNA decreased over time in batch cultures, but remained relatively constant during fed-batch cultures . The relative merits of different modes of fermentation and their effects on the quality of alkaline lysate extracts of pDNA with respect to genomic contamination and the percentage of SC-pDNA are discussed.

Water Sci Technol, 2003, 47(1), 319 - 24
A comparative study of anaerobic digestion of food waste in a single pass, a leachate recycle and coupled solid/liquid reactors; Xu HL et al.; A single pass reactor (R1), a leachate recycle reactor (R2) and a coupled solid/liquid bioreactor (R3-Rm) for anaerobic digestion of food waste were comparatively investigated in terms of digestion process and treatment efficiency . The coupled solid/liquid bioreactor is an enhanced two-phase system and distinctive from a traditional two-phase process with an upflow anaerobic sludge blanket (UASB) reactor as the methanogenic phase and a circulation of treated leachate between the acidification and methanogenic phases . In comparison with R1 and R2, R3-Rm enhanced the digestion process and increased the methane content of biogas . 100% of the R3-Rm methane yield was from the methanogenic phase with average methane content of 71% . The significant enhancement was also confirmed by the removal of 79% of total organic carbon (TOC), 60% of volatile solids (VS) and 80% of total COD in 12 days running of R3-Rm . However, no active methane fermentation was detected in R1 and R2 during 60 days operation . The results in this laboratory-scale study show that the rapid accumulation of volatile fatty acids (VFAs) due to the rapid acidification of food waste inhibits the development of effective methane fermentation in single pass and leachate recycle reactors . The coupled solid/liquid bioreactor is more efficient in converting food waste into methane and carbon dioxide.

Water Sci Technol, 2003, 47(1), 121 - 6
Operational factors of submerged inorganic membrane bioreactor for organic wastewater treatment: sludge concentration and aeration rate; Hwang EJ et al.; Various sludge concentrations and aeration rates were evaluated to find the optimal operation condition of a submerged ceramic membrane filtration system . 5.6 g/L of sludge was diluted with water to concentrations of 2.8 g/L and 1.4 g/L, and the three sludge concentrations were compared in terms of titration characteristics such as pressure and filtrate flux . Flux was at the highest value of about 30 L/m2 x hr at 50 kPa when sludge concentration was 1.4 g/L . In contrast, when sludge concentrations increased to 2.8 g/L and 5.6 g/L, the flux at 50 kPa decreased significantly to 18 L/m2 x hr and 10 L/m2 x hr, respectively . It was concluded that the sludge concentration directly affected the filtration efficiency, and low sludge concentration was suitable for improving filtration efficiency . Adjusting the aeration rate from 2 L/min to 4 L/min at 5.6 g/L of sludge and 50 kPa of pressure increased flux from 10 L/m2 x hr to 13 L/m2 x hr . It was obvious that the vigorous aeration improved the filtration efficiency, but the aeration rate did not seem to be high enough to maintain flux lower than critical flux.

Water Sci Technol, 2003, 47(1), 1 - 5
Membrane bioreactors for wastewater treatment and reuse: a success story; Ben Aim RM et al.; This paper briefly reviews our current understanding of membrane bioreactor processes (including conventional and immersed membrane designs) resulting from three decades of development, highlighting process advantages, applications and areas for continuing research.

Trends Biotechnol, 2003 Feb, 21(2), 89 - 93
Sonobioreactors: using ultrasound for enhanced microbial productivity; Chisti Y; Enhanced metabolic productivity of microbial, plant and animal cells in bioreactors can greatly improve the economics of biotechnology processes . Ultrasound is one method of intensifying the performance of live biocatalysts . Ultrasonication is generally associated with damage to cells but evidence is emerging for beneficial effects of controlled sonication on conversions catalyzed by live cells . This review focuses on the productivity enhancing effects of ultrasound on live biological systems and the design considerations for sonobioreactors required for ultrasound-enhanced biocatalysis.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 202 - 8
PH, pCO2, and temperature effect on R-adenovirus production; Jardon M et al.; The effects of pH, carbon dioxide vapor pressure, pCO(2), and temperature on E1 and E3 deleted recombinant adenovirus vector (rAV) production with HEK293S cells have been studied in the ranges of pH = 6.7-7.7, pCO(2) = 0.05-0.20 atm, and T = 32-39 degrees C, respectively . The experiments were performed in four 500-mL bioreactors in parallel, which make possible the reduction of inter-run variability . Cell concentration and viability, relative oxygen uptake rate (OUR), fluorescence, and viral titer were measured . It was found that, although pH and pCO(2) did not affect significantly cell viability in the range studied, they had an important effect on virus titer . pCO(2) allowed the maximum production of rAV at 0.05 atm, and pH showed a very sharp optimum at 7.2 . Temperature had an effect on both cell metabolism and virus titer . Low temperature prolonged cell viability and high OUR . Most of all, a 3-fold increase in virus yield was found at 35 degrees C compared to that at 37 degrees C, while 32 degrees C was not as beneficial (1.5-fold increase) . This finding could have an important impact on large-scale production . This phenomenon was modeled using a simple 3-parameter synthesis-decay model . This model shows how the optimum gain in virus production at 35 degrees C is due to a balance between the production and decay processes at that temperature.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 158 - 62
Improvement of monoclonal antibody production in hybridoma cells by dimethyl sulfoxide; Ling WL et al.; Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation . However, these cultures typically have low volumetric and specific productivities . In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures . From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity . This timing coordinated with high cell viability and density . Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control . We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2-fold increase in specific mAb production . This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement . Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 98 - 108
Metabolic screening of mammalian cell cultures using well-plates; Balcarcel RR et al.; For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High-Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells . This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering . First, HTMS makes use of a simplified metabolic network for metabolic flux analysis . Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network . Second, HTMS makes use of microtiter well-plate technology and adaptations of well-known enzymatic assays to increase precision and throughput for cell culture experiments . Multireplicate, multiparallel cultures in the sub-milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T-flasks, spinner flasks, or bioreactor systems . The simplicity of the network and the well-plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses . The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 45 - 51
Strategies for improved dCO2 removal in large-scale fed-batch cultures; Mostafa SS et al.; Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity . In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity . The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale . The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process . A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor . dCO(2) was measured using a fiber optic based probe . dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed . Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.) . Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value . A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures . Thus, this strategy was not used at pilot scale . Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold . Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal . Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position . The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult . Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less . Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg) . Titer at day 10 of culture improved by 1.5-fold . Specific productivity improved by 41% . Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 30 - 6
Optimization of an acoustic cell filter with a novel air-backflush system; Gorenflo VM et al.; Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes . An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies . A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality . A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s . This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line . The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation . Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1) . The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3% . This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively . Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.

Biotechnol Prog, 2003 Jan-Feb, 19(1), 2 - 8
Fluid mechanics, cell distribution, and environment in CellCube bioreactors; Aunins JG et al.; Cultivation of MRC-5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine (1), is performed in the CellCube reactor, a laminar flow fixed-bed bioreactor with an unusual diamond-shaped, diverging-converging flow geometry . These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation (2, 3) . Early testing of the CellCube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase . Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained . Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment.

Appl Environ Microbiol, 2003 Feb, 69(2), 852 - 60
Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments; Stamper DM et al.; The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters . The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass . Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S . Navy ships, was collected offsite . Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day . Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products . DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure . Long-term similarity between the communities was 0 to 25%, depending on the method of analysis . In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2002 Sep, 16(5), 325 - 8
{Advances in research and development of tissue engineering}; Yao KD et al.; OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue . METHODS: The advances in tissue engineering technologies were introduced based on the recent literature . RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro . CONCLUSION: Biomaterials play an important role in the tissue engineering . They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.

Biotechnol Bioeng, 2003 Apr 5, 82(1), 103 - 10
Enhancement of 2-phenylethanol productivity by Saccharomyces cerevisiae in two-phase fed-batch fermentations using solvent immobilization; Serp D et al.; The bioconversion of L-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae in fed-batch experiments has shown that concentrations of 2-phenylethanol of >2.9 g/L have a negative impact on the oxidative capacity of the yeast . Without tight control on ethanol production, and hence on the feed rate, ethanol rapidly accumulates in the culture media, resulting in complete inhibition of cell growth before the maximal 2-phenylethanol concentration of 3.8 g/L, obtained in the absence of ethanol production, could be achieved . This effect was attributed to a cumulative effect of ethanol and 2-phenylethanol, which reduced the tolerance of the cells for these two products . To enhance the productivity of the bioconversion, a novel in situ product recovery strategy, based on the entrapment of an organic solvent (dibutylsebacate) into a polymeric matrix of polyethylene to form a highly absorbent and chemically and mechanically stable composite resin, was developed . Immobilization of the organic solvent successfully prevented phase toxicity of the solvent and allowed for an efficient removal of 2-phenylethanol from the bioreactor without the need for prior cell separation . The use of the composite resin increased the volumetric productivity of 2-phenylethanol by a factor 2 and significantly facilitated downstream processing, because no stable emulsion was formed . The 2-phenylethanol could be backextracted from the composite resin, yielding a concentrated and almost cell-free solution . In comparison to two-phase extractive fermentations with cells immobilized in alginate-reinforced chitosan beads, the use of a composite resin was extremely inexpensive and simple . In addition, the composite resin was found to be insensitive to abrasion and chemically stable, such that sterilization with 2 M NaOH or heat was possible . Finally, the composite resin could be produced on a large scale using commercially available equipment .

Biotechnol Bioeng, 2003 Apr 5, 82(1), 86 - 92
Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization; Piletsky S et al.; Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer . The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability . The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization . The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI . The storage conditions for PANI/PP membranes containing immobilized HRP were studied . HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5 degrees C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes .

Biotechnol Bioeng, 2003 Apr 5, 82(1), 1 - 11
Development of a novel process for the biological conversion of H2S and methanethiol to elemental sulfur; Sipma J et al.; The feasibility of anaerobic treatment of wastewater containing methanethiol (MT), an extremely volatile and malodorous sulfur compound, was investigated in lab-scale bioreactors . Inoculum biomass originating from full-scale anaerobic wastewater treatment facilities was used . Several sludges, tested for their ability to degrade MT, revealed the presence of organisms capable of metabolizing MT as their sole source of energy . Furthermore, batch tests were executed to gain a better understanding of the inhibition potential of MT . It was found that increasing MT concentrations affected acetotrophic organisms more dramatically than methylotrophic organisms . Continuous reactor experiments, using two lab-scale upflow anaerobic sludge bed (UASB) reactors (R1 and R2), aimed to determine the maximal MT load and the effect of elevated sulfide concentrations on MT conversion . Both reactors were operated at a hydraulic retention time (HRT) of about 7 hours, a temperature of 30 degrees C, and a pH of between 7.3 and 7.6 . At the highest influent MT concentration applied, 14 mM in R1, corresponding to a volumetric loading rate of about 50 mM MT per day, 87% of the organic sulfur was recovered as hydrogen sulfide (12.2 mM) and the remainder as volatile organic sulfur compounds (VOSCs) . Upon decreasing the HRT to 3.5 to 4.0 h at a constant MT loading rate, the sulfide concentration in the reactor decreased to 8 mM and MT conversion efficiency increased to values near 100% . MT conversion was apparently inhibited by the high sulfide concentrations in the reactor . The specific MT degradation rate, as determined after 120 days of operation in R1, was 2.83 +/- 0.27 mmol MT g VSS(-1) day(-1) . During biological desulfurization of liquid hydrocarbon phases, such as with liquefied petroleum gas (LPG), the combined removal of hydrogen sulfide and MT is desired . In R2, the simultaneous addition of sodium sulfide and MT was therefore studied and the effect of elevated sulfide concentrations was investigated . The addition of sodium sulfide resulted in enhanced disintegration of sludge granules, causing significant washout of biomass . Additional acetate, added to stimulate growth of methanogenic bacteria to promote granulation, was hardly converted at the termination of the experimental period .

J Biotechnol, 2003 Mar 6, 101(2), 165 - 72
Estimation of viable biomass in aerobic biodegradation processes of organic fraction of municipal solid waste (MSW); Liwarska-Bizukojc E et al.; 2-(p-Iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) dehydrogenase test and RNA assay were introduced to evaluate biomass in the processes of aerobic biodegradation of the organic fraction of municipal solid waste (MSW) in bioreactors . It was found that RNA quantification by KOH/UV method delivered reliable and repeatable results . Relative standard deviation (RSD) for INT test was significantly higher than for RNA assay and achieved values of 3-15% . Moreover, it occurred that the optimum temperature for the growth of autochthonic biomass, which takes part in the biodegradation process, was in the range from 25 to 37 degrees C .

J Biotechnol, 2003 Mar 6, 101(2), 147 - 55
Adhesion of plant roots to poly-L-lysine coated polypropylene substrates; Towler MJ et al.; The ability to immobilize plant tissue in a bioreactor is an important process tool . We have shown that roots of several species rapidly attach to poly-L-lysine coated polypropylene mesh in a liquid environment . Using transformed roots of Artemisia annua as a model, the attachment process was found to be enhanced by sheep serum, but not BSA and inhibited by excess Mn(2+), but unaffected by Ca(2+) or Mg(2+) . Attempts to characterize the molecule(s) responsible for binding using lectins and antibodies showed that the binding site does not appear to be glycosylated or vitronectin-like . This method of rapid attachment should prove useful for controlled immobilization of roots in bioreactors .

Environ Sci Technol, 2003 Jan 15, 37(2), 402 - 8
Activated carbon as an electron acceptor and redox mediator during the anaerobic biotransformation of azo dyes; van der Zee FP et al.; Activated carbon (AC) has a long history of applications in environmental technology as an adsorbent of pollutants for the purification of drinking waters and wastewaters . Here we describe novel role of AC as redox mediator in accelerating the reductive transformation of pollutants as well as a terminal electron acceptor in the biological oxidation of an organic substrate . This study explores the use of AC as an immobilized redox mediator for the reduction of a recalcitrant azo dye (hydrolyzed Reactive Red 2) in laboratory-scale anaerobic bioreactors, using volatile fatty acids as electron donor . The incorporation of AC in the sludge bed greatly improved dye removal and formation of aniline, a dye reduction product . These results indicate that AC acts as a redox mediator . In supporting batch experiments, bacteria were shown to oxidize acetate at the expense of reducing AC . Furthermore, AC greatly accelerated the chemical reduction of an azo dye by sulfide . The results taken as a whole clearly suggest that AC accepts electrons from the microbial oxidation of organic acids and transfers the electrons to azo dyes, accelerating their reduction . A possible role of quinone surface groups in the catalysis is discussed.

Environ Sci Technol, 2003 Jan 15, 37(2), 379 - 85
Electron-beam treatment of aromatic hydrocarbons that can be air-stripped from contaminated groundwater . 2 . Gas-phase studies; Prager L et al.; The electron-beam (EB) degradation of volatile aromatics (benzene, toluene, ethylbenzene, xylenes: BTEX) in groundwater strip gas, which in the present work has been modeled by the introduction of the desired aromatic(s) to a stream of air or another gas, such as oxygen, is initiated essentially by the addition of *OH radicals to the aromatic ring, giving rise to hydroxycyclohexadienyl radicals, which form the corresponding peroxyl radicals upon addition of oxygen . As studied in some detail with benzene as a BTEX representative, various reactions of these lead to numerous oxidation products in a cascade of reactions, including the decomposition of products under the prevailing conditions of high turnover of the initial aromatic . Importantly, hydroxycyclohexadienylperoxyl radical formation is partly reversible, and the reactions of the hydroxycyclohexadienyl radicals, which thus have a significant presence in these systems, must therefore also be taken into consideration . In the gas phase, in contrast to the aqueous phase (see Part 1), the reactions of the hydroxycyclohexadienyl radicals lead to oligomeric products that appear to contribute, in addition to ionic clusters, to nucleation for the aerosols observed . Various nitrated products, among them nitrophenols, are observed when air is used for the stripping . However, these studies did not clear the pilot plant stage, since BTEX degradation using a bioreactor carried out in parallel was so successful that the EB technology was judged to be noncompetitive . As for the latter, expensive equipment consisting of a stripper, the EB machine, and an aerosol precipitator would be required . The condensed aerosols are biorefractory and would require further treatment for detoxification.

Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 93 - 8
{The kinetic characteristics of mycelium growth of medicinal mushroom Ganoderma sinense in liquid-submerged cultivation}; Gong J et al.; The study was carried out to investigate the kinetic characteristics of Ganoderma sinense mycelium growth in liquid-submerged cultivation, using a air-lift bioreactor ALR/ff specially designed for the cultivation of mushrooms' suspending mycelia cultures . Mycelia of G . sinense grew in the range of 25 degrees C-35 degrees C, and at the different temperature in which the specific growth rate showed by the mycelia much differed . The specific growth rates with the maximum of 0.0444(h-1) at 1.64 vvm of aeration in the cultivation were more than at 0.93 vvm, also more than at 2.50 vvm . In comparison with the less glucose concentration, the culture of 2.80 g glucose/100 mL in the medium had a shorter lag-period, and the more concentration of glucose favoured mycelia growth in the latter cultivation course with the denser pellets of mycelia . Then the culture of the less concentration of glucose in the medium had the higher conversion-rates of glucose to G . sinense biomass all along the cultivation course, than of the more concentration of glucose . Futhermore, in the continuous cultivations with growth-limiting substrate of carbon source the mycelia displaying different growing activities, demonstrated the varied forms of mycelium-gathering . In that cases the relationships among mycelia biomass, growth-limiting substrate and productivity of mycelium biomass obviously differed from the ones in the case of bacteria . Also in the range of dilution rates 0.010-0.220 (h-1) the growing of G . sinense mycelia followed the function of Contois equation.

Biochem Cell Biol, 2002, 80(6), 777 - 88
Secretion of foreign proteins mediated by chicken lysozyme gene regulatory sequences; Lampard GR et al.; Exploitation of the insulating properties of the complete chicken lysozyme gene domain may facilitate the production of transgenic chicken bioreactors with the capacity to deposit valuable proteins in the egg white . Chimeric genes consisting of the chicken lysozyme gene regulatory sequences and sequences encoding foreign proteins could be inserted randomly into the chicken genome and retain appropriate expression levels . The research reported here established that chicken lysozyme gene regulatory sequences can be used to direct the production and secretion of green fluorescent protein (used as a reporter protein) in transiently transfected chicken blastodermal cells . Attempts to verify these findings in transgenic hens are currently in progress . To provide a rapid means of generating constructs encoding other foreign proteins under the control of lysozyme gene regulatory sequences that can facilitate the secretion of heterologous proteins in vivo, a generic lysozyme gene regulatory scaffold was created using a poxvirus-mediated gene targeting system.

Wei Sheng Wu Xue Bao, 1999 Aug, 39(4), 367 - 72
{Industrial scale-up of continuous ethanol fermentation using yeast flocs}; Xie J et al.; Inducing yeast cells to self-flocculate as a cell immobilization method, an industrial scale-up plant composed of 4 air-lift suspended-bed bioreactors in parallel and with a total volume of 400 m3 was established and operated . During a more than 6 months continuous operation, the effluent contained ethanol of 70-80 g/L and residual sugar less than 5 g/L and the ethanol productivity of 7-8 g/L.h-1 was achieved.

Water Res, 2003 Mar, 37(5), 1192 - 7
Characteristics of microfiltration membranes in a membrane coupled sequencing batch reactor system; Kang IJ et al.; Factors affecting filtration performance were investigated in a sequencing batch reactor (SBR) coupled with a submerged microfiltration module . Special bioreactors for aerobic and anoxic phases were specifically designed in order to differentiate the effect of dissolved oxygen (DO) from that of mixing intensity, on membrane filterability . When the filterability of a submerged microfilter was examined at each SBR phase, DO concentration, as well as mixing intensity proved to have a major influence on the membrane performance regardless of the SBR phase . A higher DO concentration resulted in a slower rise in TMP, corresponding to less membrane fouling, which was investigated in depth through a series of analyses including resistance measurements and compressibility of the cake layer as well as particle sizes as a functions of DO for both aerobic and anoxic phases in SBR.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 1999 Jun, 16(2), 143 - 6
{Study on the biologic function of extracorporeal bioartificial liver support system}; Wang Y et al.; In this study, a total of 2 x 10(8) hepatocytes and nonparenchymal liver cells cultured as aggregate spheroids and a hollow fiber bioreactor were used to set up the extracorporeal bioartificial liver support system (EBLSS) . Its function was determined throungh the analysis of some cells production . After 6 hours circulating of EBLSS, the total protein, albumin, alpha-fetoprotein and urea were markedly increased in the circulating media, and the DNA synthesis of rat hepatocytes cultured in the media was significantly increased (compared with control group, P < 0.01) . This suggests that the EBLSS which consists of cultured human liver cells and hollow fiber bioreactor has the biologic function of liver and has a stimulative action on the proliferation of hepatocytes.

Wei Sheng Wu Xue Bao, 2001 Jun, 41(3), 367 - 71
{High concentration ethanol continuous fermentation using yeast flocs}; Liu C et al.; Continuous ethanol fermentation using yeast flocs was carried out in 4 air-lift suspended-bed bioreactors operated in series . Drafted by CO2, with complete recycle of ethanol distilled effluent broth and at the dilution rate of 0.2/h, the average ethanol concentration of the fermentation broth was 96.6 g/L, while the average concentration of residual total sugar was 4.1 g/L and residual reducing sugar was 1.2 g/L.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 415 - 9
{L-lactic acid fermentation by immobilized Rhizopus oryzae in a three-phase fluidized-bed}; Chen Y et al.; L-lactic acid fermentation in a three-phase fluidized-bed bioreactor was studied by using immobilized Rhizopus oryzae cells with polyurethane particles as supporter . The results indicated that the immobilized cells were stable after used for 8 batches repeatedly . The rate and the yield of lactic acid fermentation with glucose as carbon source were 19.1 g h-1.L (bead)-1 and 82.5%, respectively . With the hydrolyzate of corn cob residue from xylose manufacturer by cellulase hydrolysis, the yield of lactic acid reached 71.9%.

Shi Yan Sheng Wu Xue Bao, 1999 Sep, 32(3), 227 - 32
{The development of mouse bioreactor expressing human tissue plasminogen activator (tPA) in mammary gland by transfecting spermatozoa in testicular duct}; Huang WM et al.; The most established methods for development of transgenic animals are the microinjection of DNA into the fertilized eggs, but it is still a procedure of certain complexity and high cost . Therefore, the idea of using sperm as a vehicle to carry exogenous DNA into eggs is very attractive, and there have been some successful reports . Though the methods are rather simple they sometimes have low reproducibility . To improve the technique we transinfected the spermatozoa in testicular duct, not in vitro, to produce mice which expressed human tissue plasminogen activator (tPA) in mammary gland . The results demonstrated that: (1) 5 transinfected mice mated 10 female mice in 10 days after operation, (2) 79 founders were developed and 42 survived, (3) using PCR to detect foreign DNA integrated into the genome of founders, 7 out of 42 founders were positive (16.67%), (4) The expression level of tPA was 48-80 ng/ml in the milk of 5 PCR positive founders and (5) the foreign DNA integrated into the genome was detected in 2 out of 4 1st offspring by PCR technique.

Ann N Y Acad Sci, 2002 Dec, 979, 10 - 26; discussion 35-8
An overview of the pathology and approaches to tissue engineering; Ochoa ER et al.; In tissue engineering, there is an attempt to culture living tissues for surgical transplantation . In vitro and in vivo approaches have produced vascular and cardiovascular components, cartilage, bone, intestine, and liver . Attempts to microdesign cell-culture support scaffolds have used a new generation of biocompatible and bioabsorbable polymers . Suspensions of donor cells are seeded onto protein-coated polymer scaffolds and grown to confluence in dynamic bioreactors . In vitro techniques produce monolayers of tissues . Denser masses are achieved by implanting monolayers onto a host, or by culturing cell/polymer constructs in vivo . Existing techniques have produced functioning heart valves from sheep endothelial cells and myofibroblasts . Cultured ovine arterial cells have replaced 2-cm segments of pulmonary artery in lambs . Chondrocyte cultures have produced a human-ear-shaped construct, temporo-mandibular joint discs, meniscal replacement devices, and human-phalange-shaped constructs, complete with a joint . The culture of composite tissue types has recently been reported . Intestinal organoid units containing a mesenchymal core with surrounding polarized epithelia have been used in lieu of an ileal pouch in Lewis rats, and the long-term culture of rat hepatocytes has revealed cellular differentiation and neomorphology resembling elements of a biliary drainage system . To sustain the in vitro culture of dense tissues prior to implantation, micro-electro-mechanical systems (MEMS) fabrication technologies have been adapted to create wafers of polymer containing sealed, branching, vascular-type spaces . After seeding with rat lung endothelial cells, followed by 5 days of bioreactor culture, the result is an endothelial network with controlled blood flow rates, pressure, and hematocrit . When these customized vascular systems can be used to support in vitro culture, a new generation of dense, composite, morphologically complex tissues will be available for clinical development.

Tissue Eng, 2002 Dec, 8(6), 1083 - 1088
A Novel Pulsatile, Laminar Flow Bioreactor for the Development of Tissue-Engineered Vascular Structures; Thompson CA et al.; Exposure of vascular cell-seeded, tubular, biodegradable polymers to pulsatile flow conditions has been proposed as a method to develop tissue-engineered blood vessels by "maturing" structural integrity, and increasing collagen content, suture retention, burst pressure, and tissue formation . These in vitro tissue-engineered arteries demonstrate contractile responses to pharmacologic agents and express markers of vascular differentiation . Current methods to induce pulsatile flow in a bioreactor system are limited by the creation of nonphysiologic pressure waveforms and noncompliant reservoirs to house the tissue-engineered vascular constructs . We have developed a novel method for the in vitro development of tubular vascular structures by using a mechanical ventilator to induce pulsatile, laminar flow into a fluid column, resulting in pressurized waveforms similar to mammalian physiology . The vascular constructs are housed in semicompliant tubing to facilitate an additional variable of circumferential stretch as a potential signaling mechanism . This approach more closely approximates mammalian physiology and we hypothesize that it will facilitate mechanical signaling necessary for the development of tissue-engineered vessels for clinical applications.

Tissue Eng, 2002 Dec, 8(6), 1009 - 1016
Potential Use of Chitosan as a Cell Scaffold Material for Cartilage Tissue Engineering; Nettles DL et al.; One of the most important factors in any tissue-engineering application is the cell substrate . The purpose of this study was the initial evaluation of chitosan, a derivative of the abundant, naturally occurring biopolymer chitin, as a cell scaffold for cartilage tissue engineering . Chitosan scaffolds having an interconnecting porous structure were easily fabricated by simple freezing and lyophilization of a chitosan solution . After rehydration of scaffolds, porcine chondrocytes were seeded onto scaffolds and cultured for up to 28 days in a rotating-wall bioreactor . Chitosan scaffolds supported cell attachment and maintenance of a rounded cell morphology . After 18 days, cells within the scaffolds had synthesized extracellular matrix in which proteoglycan and type II collagen were detected by toluidine blue staining and immunohistochemistry, respectively . Abundant extracellular matrix was found almost exclusively in the periphery of the scaffolds, as scaffold microstructure prevented cells from penetrating to interior regions . Nonetheless, the results suggest that chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.

Cytometry A, 2003 Feb, 51(2), 87 - 96
Automated flow cytometry for acquisition of time-dependent population data; Abu-Absi NR et al.; BACKGROUND: The implementation of flow cytometry in many experimental settings can be limited by the extensive amount of sample handling and preparation required for analysis . We describe a system that automatically performs sample handling and flow cytometric analysis, thus allowing one to construct detailed pictures of changes in cell population distributions as a function of time . METHODS: Cell samples from bioreactors were loaded into a microchamber designed to perform all sample preparation steps including washing, fixation, staining, and dilution . The sample was then transported into a sample loop of known volume from which it was injected into the flow cell for determination of cell counts and measurement of scattering and fluorescence parameters . The apparatus was fully automated and controlled with a personal computer equipped with a data acquisition card . An inexpensive mechanism that continuously replenished the sheath fluid was implemented to ensure continuous and uninterrupted operation of the flow cytometer for several days . The device was interfaced with a FACSCalibur equipped with CellQuest software for data acquisition and analysis . RESULTS: The set-up was tested with batch cultures of Saccharomyces cerevisiae expressing the green fluorescent protein (GFP) . On-line cell counts showed close agreement with off-line measurements throughout the exponential growth of a yeast culture . The time course of light scattering, GFP fluorescence, and viability distributions provided a detailed description of changes occurring in growing cell cultures based on sampling approximately every 15 min for more than 40 consecutive hours . Therefore, the device could be used to obtain descriptions of the dynamic behavior of cell populations with no user intervention required for several days . CONCLUSIONS: The system significantly expanded the utility of flow cytometry by eliminating cumbersome and time-consuming steps that make the application of flow cytometry impractical in certain situations . It is anticipated that the described set-up will find utility in biotechnology applications such as monitoring of cell cultures, screening of biologically active compounds, and in functional genomics efforts for phenotypic characterizations of cells .

Vaccine, 2003 Jan 30, 21(7-8), 803 - 8
Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives; Sala F et al.; Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g . tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration . This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants . The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours . They also offer a convenient tool against the threat of bio-terrorism . State of the art, experimental strategies, safety and perspectives are discussed in this article.

Water Res, 2003 Feb, 37(4), 902 - 8
Anaerobic treatment of wastewater with high organic content using a stirred tank reactor coupled with a membrane filtration unit; Fuchs W et al.; Using a cross-flow membrane bioreactor, high anaerobic conversion rates of three different types of wastewater with varying organic content were achieved . Loading rates obtained were as follows: 20 g CODL(-1) x d(-1) for artificial wastewater, approximately 8 g CODL(-1) x d(-1) from vegetable processing industry (sauerkraut brine) and 6-8 g CODL(-1) x d(-1) for wastewater from an animal slaughterhouse . At stable conditions, COD-removal rates in all three wastewaters were higher than 90% . Methane yields from the treatment of artificial wastewater, sauerkraut brine, and animal slaughterhouse wastewater were in the range of 0.17-0.30, 0.20-0.34, and 0.12-0.32 L(n) x g(-1) COD(-1) fed, respectively . The complete retention of biomass and suspended solids is a unique feature of this treatment process, which combines a high loading capacity and at the same time, high COD removal rates even for complex wastewater containing high concentrations of particulate matter.

Anal Chem, 2003 Jan 1, 75(1), 22 - 6
A microfluidic bioreactor based on hydrogel-entrapped E . coli: cell viability, lysis, and intracellular enzyme reactions; Heo J et al.; Viable E . coli cells were entrapped in hydrogel micropatches photopolymerized within microfluidic systems . The microfluidic channels and the micropatches have sizes on the order of 100-500 microm . Small molecules, such as dyes and surfactants, present in the solution surrounding the hydrogel, are able to diffuse into the gel and encounter the cells, but the cells are sufficiently large to be retained . For example, sodium dodecyl sulfate is a lysis agent that is able to penetrate the hydrogel and disrupt the cellular membrane . Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification . Hydrogel-immobilized cells are able to carry out chemical reactions within microfluidic channels . Specifically, a nonfluorescent dye, BCECF-AM, is able to penetrate both the hydrogel and the bacterial membrane and be converted into a fluorescent form (BCECF) by the interior cellular machinery . These results suggest that cells immobilized within microfluidic channels can act as sensors for small molecules and as bioreactors for carrying out reactions.

J Math Biol, 2003 Jan, 46(1), 48 - 70
Feedback control for chemostat models; De Leenheer P et al.; It is shown that a chemostat with two organisms can be made coexistent by means of feedback control of the dilution rate . Remaining freedom in the feedback law can be used to guarantee robustness or improve particular performance indices . Unfortunately a topological property prevents coexistence by feedback control for chemostats with more than two organisms . We apply our results to control bioreactors aimed at producing commercial products through genetically altered organisms . In all our results the coexistence takes its simplest form: a global asymptotically stable equilibrium point in the interior of the non-negative orthant.

J Oral Maxillofac Surg, 2003 Jan, 61(1), 94 - 100
Formation of a mandibular condyle in vitro by tissue engineering; Abukawa H et al.; PURPOSE: Mandibular reconstructive procedures often produce significant donor site morbidity . Recently, the use of minimally invasive techniques has been reported for mandibular reconstruction with decreased morbidity at the primary operative site . To date, these techniques have not addressed the graft donor site . We hypothesize that tissue-engineering techniques may be used to fabricate bone and thereby eliminate donor site morbidity . METHODS: Porcine mesenchymal stem cells (pMSCs) were isolated from the bone marrow of 3 Yucatan minipigs and grown in standard culture flasks . When they became near-confluent, cells were detached and replated with the addition of osteogenic supplements . A model of a porcine mandibular condyle was made and used to fabricate porous polymer scaffolds from biodegradable poly DL-lactic-co-glycolic acid (PLGA) . Differentiated osteoblasts were transferred to the PLGA scaffold and cultured for 6 weeks in a rotational oxygen-permeable bioreactor system . The cultured constructs, consisting of scaffold and cells, were evaluated by gross, radiologic, and histologic examinations . RESULTS: The engineered constructs were white and hard and had a shape that closely resembled that of the model condyle . Plain radiographs demonstrated that the radiodensity of the construct was between that of the normal condyle and that of control scaffolds . Histologically, bone was observed on the entire surface of the PLGA scaffolds with an average thickness of 0.03 mm . Bone was not observed in the control scaffolds . CONCLUSION: In this pilot study, autologous tissue-engineered bone constructs were successfully made by combining biodegradable polymers and pMSCs .

Environ Sci Technol, 2002 Dec 1, 36(23), 5245 - 51
Characteristics of a self-forming dynamic membrane coupled with a bioreactor for municipal wastewater treatment; Fan B et al.; A self-forming dynamic membrane (SFDM) method that used the biomass layer formed on a coarse mesh to effect solid-liquid separation was proposed . A 100-microm Dacron mesh material was used to make the SFDM modules . A SFDM coupled bioreactor (SFDMBR) was tested to treat actual municipal wastewater, and the performance and mechanisms of the SFDM were investigated . The SFDMBR worked by gravity filtration, and the water head drop was generally <5 cm . The effluent suspended solid concentrations were undetectable in most cases, and the COD and NH3 N removal efficiencies averaged 84.2% and 98.03%, respectively . An aeration in situ was adequate for cleaning the clogged modules, and the SFDM was readily and quickly re-formed . The biomass layer of the SFDM consisted of a cake layer and a gel layer . The gel layer had structures like conventional membranes and acted the key role in the SFDM . The permeability of the used mesh attached to the gel layer could be improved because the gel layer made the filtration surface more hydrophilic.

J Biomater Sci Polym Ed, 2002, 13(11), 1175 - 87
Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads; Simsek-Ege FA et al.; The enzyme bovine carbonic anhydrase (BCA) has been immobilized in the chitosan-alginate system for the first time, to catalyze the conversion of CO2 to HCO3- . Chitosan-coated alginate beads are a biodegradable and environmentally benign matrix, chosen for application of the enzyme in a novel biomimetic CO2 sequestration system . The feasibility of the system and immobilization of the enzyme were demonstrated in our earlier studies . Optimization of the matrix to improve the retention time of the enzyme in an encapsulated form is the subject of the present study . The improvement in the molecular weight cut-off of the beads was accomplished by adjusting the cross-linking conditions, coating composition, and molecular weight of the system . The quantity of enzyme released from the system was measured by a Bio-Rad protein assay . Poly-L-lysine was also used as a coating reagent for comparison purposes . The presence of a coating on the alginate beads was verified by Kjeldahl analyses . The difference in the microstructures of alginate and chitosan/alginate beads was demonstrated by SEM studies . Mineralization of the chitosan/alginate matrix in the presence of CaCO3 was also studied by FT-IR, to assess the possibility of using the beads continuously in a bioreactor.

Biotechnol Bioeng, 2003 Mar 5, 81(5), 518 - 24
Synthesis of 3-tert-butylcatechol by an engineered monooxygenase; Meyer A et al.; Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol . The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1 . The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme . The biotransformation was performed in a 3-L bioreactor for 24 h . To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed . Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth . In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution . The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated . Down-stream processing consisted of two steps . First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol . Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate . The final purity of 3-tert-butylcatechol was greater than 98% .

Appl Environ Microbiol, 2003 Jan, 69(1), 252 - 7
Large-scale cultivation of acidophilic hyperthermophiles for recovery of secreted proteins; Worthington P et al.; An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins . Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system . These alterations provided accurate temperature and pH control . The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus . Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components . Substrate induction of synthesis of the S . solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes . An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant . These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.

Vopr Virusol, 2002 Nov-Dec, 47(6), 4 - 11
{Cultural influenza vaccines}; Gendon IuZ; Recent achievements in development of influenza vaccines using long-term cell cultures MDSK or Vero are reviewed . Cell cultures were grown in bioreactors (fermenters) filled with microcarriers using serum-free culture media . Inactivated cultural influenza vaccines were clinically tested in many countries . The results of these trials showed that cultural influenza vaccines are as safe and immunogenic as conventional vaccines prepared using chicken embryos . It is emphasized that cultural influenza vaccines prepared using serum-free culture media have a number of advantages over embryonic influenza vaccines: standard preparation technology, intactness of all hemagglutinin antigen domains, absence of protein allergens and possible viral contamination inherent in chicken embryos and blood serum, and possibility of easy intensification of vaccine production in case of influenza pandemic . Taking into consideration these advantages, it may be suggested that cultural influenza vaccines will soon be widely used in medical practice.

J Infect Dis, 2003 Jan 1, 187(1), 159 - 62 Epub 2002 Dec 13.
A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities; Li T et al.; Plasmodium falciparum replicates poorly in erythrocyte densities greater than a hematocrit of 20% . A new method to culture the major malaria parasite was developed by using a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100% . P . falciparum replicated equally well at all densities studied . This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof), because high yields ( approximately 10(10) in 4 days) could be prepared with less cost and labor . Concomitantly, secreted proteins were concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8%-12% with the 3D7 strain . The finding that P . falciparum can replicate at packed erythrocyte densities suggests that this system may be useful for study of the pathogenesis of fatal cerebral malaria, of which one feature is densely packed blood cells in brain microvasculature.

J Recept Signal Transduct Res, 2002 Feb-Nov, 22(1-4), 229 - 40
Semliki forest virus-based expression for versatile use in receptor research; Lundstrom K; Semliki Forest virus (SFV) vectors have been generated for highly efficient studies on gene expression in a variety of mammalian host cells, including immortalized cell lines as well as primary cells in culture . Moreover, SFV expression has been scaled up for mammalian suspension cultures in spinner flasks and bioreactors for production of large quantities of recombinant proteins for drug screening and purification . The strong preference of expression in neuronal cells in primary cell cultures, in organotypic hippocampal slices and in vivo has made SFV vectors attractive for neurobiological studies . Additionally, the engineering of novel, less cytotoxic and temperature-sensitive SFV mutant vectors has further increased their application range.

Water Res, 2003 Jan, 37(2), 289 - 96
The enzymology of sludge solubilisation utilising sulphate-reducing systems: the properties of lipases; Whiteley CG et al.; The first stage in the degradation and recycling of particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the sulphidogenic environment . An investigation into the enzymology of these processes has shown that lipase enzyme activities were found predominantly associated with the organic particulate matter of the sewage sludge . Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant . pH and temperature optimisation studies showed optima between 6.5 and 8 and 50-60 degrees C, respectively . All the lipase enzymes from the methanogenic bioreactors indicated extensive stability for at least an hour at their respective optimum temperatures and pH; sulphidogenic lipases reflected limited stability at these temperatures and pH during this time period . Though sulphate showed inhibitory properties towards lipases both sulphide and sulphite appeared to enhance the activity of the enzymes . It is argued that these sulphur species, liberated at different times during the sulphate reduction process, disrupt the integrity of the organic particulate floc by neutralising acidic components on the surface . The release of further entrapped enzymes from the organic particulate matter result in a subsequent enhancement of hydrolysis of polymeric material.

Appl Biochem Biotechnol, 2003 Jan, 104(1), 1 - 11
Continuous oxygen monitoring of mammalian cell growth on space shuttle mission STS-93 with a novel radioluminescent oxygen sensor; Reece JS et al.; A compact, flow-through oxygen sensor device based on luminescence quenching was used to monitor dissolved oxygen levels during mammalian cell growth on the STS-93 mission of the Columbia space shuttle . Excitation of an oxygen-sensitive ruthenium complex was provided by a radioluminescent light source (0.9 mm in diameter, 2.5 mm long), and the intensity of the resulting luminescence was measured by a simple photodiode detector . The use of radioluminescence for the excitation light source is a unique approach that provides many features important for long-term and remote monitoring applications . For the spaceflight experiment, human lung fibroblast cells (WI-38) were grown in hollow-fiber bioreactors . Oxygen concentration was measured in the flow path both before and after the bioreactor cartridge in order to gain information about the metabolism of the cells . The sensor was found to be nonperturbing to cell growth and withstood the challenging physical conditions of shuttle launch and landing while maintaining a stable calibration function . In addition, the sensor provided physically meaningful oxygen predictions.

J Environ Sci (China), 2002 Oct, 14(4), 501 - 7
Determination and discussion hydraulic retention time in membrane bioreactor system; Zhang SY et al.; Based on the microorganism kinetic model, the formula for computing hydraulic retention time in a membrane bioreactor system (MBR) is derived . With considering HRT as an evaluation index a combinational approach was used to discuss factors which have an effect on MBR . As a result, the influencing factors were listed in order from strength to weakness as: maximum specific removal rate K, saturation constant Ks, maintenance coefficient m, maximum specific growth rate mu m and observed yield coefficient Yobs . Moreover, the formula was simplified, whose parameters were experimentally determined in petrochemical wastewater treatment . The simplified formula is theta = 1.1(1/beta-1) (Ks + S)/KX0, for petrochemical wastewater treatment K and Ks equaled 0.185 and 154.2, respectively.

J Pediatr Surg, 2002 Dec, 37(12), 1720 - 5
Engineered fetal cartilage: structural and functional analysis in vitro; Fuchs JR et al.; BACKGROUND/PURPOSE: This study was aimed at characterizing the structure and function of engineered fetal cartilage in vitro . METHODS: Chondrocytes from ovine specimens of fetal elastic, fetal hyaline, and adult elastic cartilage were expanded in culture and their growth rates determined . Cells were seeded onto synthetic scaffolds, which were then maintained in a bioreactor . Matrix deposition was determined by specific staining and quantitative assays for glycosaminoglycans (GAG), type II collagen (CII), and elastin, as well as compared with native tissue . Statistical analysis was by analysis of variance (ANOVA) and Students' t test, with significance set at P less than.01 . RESULTS: Fetal elastic chondrocytes grew significantly faster than all other cell types . All fetal constructs resembled hyaline cartilage, regardless of the cell source . There were significantly higher levels of GAG and CII in fetal versus adult constructs, but no significant difference between fetal constructs from different sources . Unlike their adult counterparts, fetal constructs had GAG and CII levels similar to native tissues . CONCLUSIONS: Fetal chondrocytes can be rapidly expanded in culture . Compared with adult constructs, matrix deposition is enhanced in engineered fetal cartilage, which closely resembles native tissue, regardless of the cell source . Engineered fetal cartilage may be a preferable option during surgical reconstruction of select congenital anomalies .

Life Support Biosph Sci, 2002, 8(3-4), 181 - 9
Enzyme-based CO2 capture for advanced life support; Ge J et al.; Elevated CO2 levels in air can lead to impaired functioning and even death to humans . Control of CO2 is critical in confined spaces that have little physical or biological buffering capacity (e.g., spacecraft, submarines, or aircraft) . A novel enzyme-based contained liquid membrane bioreactor was designed for CO2 capture and certain application cases are reported in this article . The results show that the liquid layer accounts for the major transport resistance . With addition of carbonic anhydrase, the transport resistance decreased by 71% . Volatile organic compounds of the type and concentration expected to be present in either the crew cabin or a plant growth chamber did not influence carbonic anhydrase activity or reactor operation during 1-day operation . Alternative sweep method studies, examined as a means of eliminating consumables, showed that the feed gas could be used successfully in a bypass mode when combined with medium vacuum pressure (-85 kPa) to achieve CO2 separation comparable to that with an inert sweep gas . The reactor exhibited a selectivity for CO2 versus N2 of 1400:1 and CO2 versus O2 is 866:1 . The CO2 permeance was 1.44 x 10(-7) mol m-2 Pa-1 s-1 (4.3 x 10(-4) cm3 cm-2 s-1 cmHg-1) at a feed concentration of 0.1% CO2 . These data show that the enzyme-based contained liquid membrane is a promising candidate technology that may be suitable for NASA applications to control CO2 in the crew or plant chambers.

Trends Biotechnol, 2003 Jan, 21(1), 14 - 9
Avian transgenesis: progress towards the promise; Ivarie R; The hen has long held promise as a low cost, high-yield bioreactor for the production of human biopharmaceuticals in egg whites . A typical egg white contains 3.5-4.0 grams of protein, more than half of which comes from a single gene (ovalbumin) . Harnessing the power of the gene to express a recombinant protein could yield up to a gram or more of the protein in the naturally sterile egg . Accordingly, a major effort has been underway for more than a decade to develop robust methods for modification of the chicken genome . This effort intensified in the mid-1990s when several avian transgenic companies entered the scene . Progress has been made in that time but much remains to be done.

Biotechnol Bioeng, 2003 Feb 5, 81(3), 305 - 15
Dilution of solar radiation through "culture" lamination in photobioreactor rows facing south-north: a way to improve the efficiency of light utilization by cyanobacteria (Arthrospira platensis); Carlozzi P; Efficient utilization of solar radiation for the photoautotrophic production of cyanobacterium biomass was achieved, using small pipes (ID = 0.01 m) arranged in rows in two photobioreactors facing south-north . A high Arthrospira yield of 47.7 g m(-2) (installation area) d(-1) was attained under outdoor conditions in a tubular undulating row photobioreactor (TURP-10r) . During the summer, under a semicontinuous culture regime, the optimal biomass concentration (OBC) in TURP-5r was 6.0 g L(-1): it was 5.0 g L(-1) in TURP-10r . These OBCs made it possible to produce a biomass output rate of 2.7 +/- 0.2 g L(-1) d(-1) in the former and 2.1 +/- 0.1 g L(-1) d(-1) in the latter . When Arthrospira was grown at a preset dilution rate (0.3 d(-1)), sunrise cell density (SrCD) variations were not proportional to the drop of solar radiation . The SrCD was comparatively high at high solar radiation and decreased abruptly with decreasing solar radiation . There was a tendency to stabilize at low solar radiation . In both photobioreactors, the chlorophyll content of the Arthrospira biomass (% of the dry weight) was higher at sunrise than at sunset . A comparison of the chlorophyll biomass content in the TURPs showed no significant differences . Night biomass losses were very high (> 30% of the daylight productivity) when the culture temperature was kept constant at 31 +/- 1.0 degrees C: these losses fell to < 20% of the daylight productivity, when the night temperature of the cultures decreased according to the environmental temperature . Dilution of solar radiation was carried out using two quasi-laminated bioreactors . The rows of S-N facing bioreactors showed a very high growth yield in TURP-10r {about 2.1g (d.w.) MJ(-1)} . In TURP-10r, the high photic ratio (R(f) = 6), the high surface-to-volume ratio (S(ill)/V = 400 m(-1)) and the S-N facing of the rows (better than an E-W orientation) allowed for good results .

Biotechnol Bioeng, 2003 Feb 5, 81(3), 269 - 78
Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm; Marteijn RC et al.; Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media . With respect to the culture method, the fed-batch culture mode is often found to give the best yields . However, optimization of the feed composition is usually a laborious task . In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described . A feed was developed from 11 different medium components, each used at a wide range of concentrations . The feed was optimized within four sets of 20 experiments . The optimized feed was tested in bioreactors and the addition scheme was further improved . The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 x 10(6) cells/mL compared to a control fermentation in an optimized commercial medium . No accumulation of waste products was found, and none of the amino acids was depleted . Glucose was depleted, which suggests that even further improvement is possible . We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism .

Water Environ Res, 2002 Sep-Oct, 74(5), 450 - 61
Microbial community structure and activity in a compartmentalized, anaerobic bioreactor; Angenent LT et al.; The objective of this study was to evaluate staging and its effects on reactor performance in a compartmentalized bioreactor, designated the anaerobic migrating blanket reactor (AMBR) . The AMBR was operated by reversing the flow several times per day, which allowed for substantial biomass migration without biomass accumulation in the final compartment . During reactor startup, the structures of the microbial communities in the five compartments were quite similar despite substantial differences in substrate types and concentrations in the different compartments . During the rest of the operational period, biomass migration was reduced by changing operating conditions and, as a result, a larger difference in the structures of the microbial communities developed for the different compartments (biomass staging) . For example, after changing operating conditions, rRNA levels for the acetate-utilizing methanogen Methanosaeta concilii were approximately 35 and 10% of the total rRNA in the middle and outside compartments, respectively (before changing operating conditions these levels were approximately 20 and 12% of the total rRNA, respectively) . Promoting larger differences in the structures of the microbial communities for the different compartments did not improve reactor performance as lower levels of M . concilii in the outside compartments hindered acetate removal and compromised effluent quality.

Semin Cell Dev Biol, 2002 Dec, 13(6), 447 - 54
Improving the next generation of bioartificial liver devices; Allen JW et al.; Several extracorporeal bioartificial liver (BAL) devices are currently being evaluated as an alternative or adjunct therapy for liver disease . While these hybrid systems show promise, in order to become a clinical reality, BAL devices must clearly demonstrate efficacy in improving patient outcomes . Here, we present aspects of BAL devices that could benefit from fundamental advances in cell and developmental biology . In particular, we examine the development of human hepatocyte cell lines, strategies to stabilize the hepatocyte phenotype in vitro, and emphasize the importance of the cellular microenvironment in bioreactor design . Consideration of these key components of BAL systems will greatly improve next generation devices.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1408 - 13
On-line monitoring of adhesion and proliferation of cultured hepatoma cells using optical waveguide lightmode spectroscopy (OWLS); Hug TS et al.; Monitoring of cell adhesion, cell spreading, and cell proliferation opens attractive perspectives in the on-line control of monolayer cell cultures in toxicity tests, in bioreactors as used for the serial production of skin grafts, or in extracorporeal liver devices . In this study the hepatoma Hep G2 cell adhesion and proliferation was monitored using an integrated optical method, optical waveguide lightmode spectroscopy (OWLS) . This method is based upon refractive index measurements within a 100-nm thin layer above a Si(Ti)O(2) surface on which the cells were cultured and exposed to cytotoxic and cytostatic agents . The OWLS signal was proportional to cell density during the spreading period (4 h), and in long-term experiments (46 h) the OWLS signal correlated on a logarithmic scale with cell density . After administration of the protein synthesis inhibitor cycloheximide (4 microg/mL) to fully spread hepatoma cells, cell growth was arrested and change of the OWLS signal became noticeable within 6 h after drug administration . For exposure to increasing concentrations of the anticancer drug cyclophosphamide (2.5-20 mM) a concentration-dependent reduction of the OWLS signal was found . For cycloheximide and cyclophospamide the OWLS signal was also confirmed by cell viability measurements using the neutral red assay, the thiazolylblue tetrazoliumbromide assay, total protein measurements, and cell morphology . It was demonstrated that the OWLS signal detects minor changes in cell adhesion, which serve as indicators of metabolic state and growth behavior . OWLS is thus a quantitative tool to characterize impaired cell growth mediated by culture medium, by extracellular matrix, or after exposure to a toxin.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1387 - 91
On-line determination of biomass in a microalga bioreactor using a novel computerized flow injection analysis system; Meireles LA et al.; A flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor . The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash-free dry weight, during the entire culture time . Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user-friendly software . The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1282 - 6
Continuous production of high-content fructooligosaccharides by a complex cell system; Sheu DC et al.; A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process . The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity . Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate . Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module . The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate . The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1233 - 9
Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge bioreactor; Zandvoort MH et al.; The effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics . A UASB reactor (30 degrees C; pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT) . The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD) x L reactor(-1) x d(-1) to 7.8 g COD x L reactor(-1) x d(-1) . Cobalt deprivation had a strong impact on the methanogenic activity of the sludge . In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt . The sludge had an apparent K(m) for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run . Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55% . Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100% . In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 microg x g TSS(-1) . Upon omission of the cobalt addition, cobalt washed-out at a stable rate of 0.1 microg x g VSS(-1) x d(-1) . At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.

Water Res, 2003 Jan, 37(1), 121 - 7
Characteristics and fates of soluble microbial products in ceramic membrane bioreactor at various sludge retention times; Shin HS et al.; The formation and fate of soluble microbial products (SMP) in membrane bioreactor (MBR) was investigated at various sludge retention times (SRT) for 170 days . The SMP concentration was estimated by feeding glucose, which could be completely degraded, and by measuring the dissolved organic carbon (DOC) of the effluent from MBR . Under the conditions of SRT of 20 days, influent DOC of 112 mg/l and HRT of 6 h, the produced SMP was 4.7 mg DOC/l of which 57% was removed or retained by the membrane . DOC of MBR supernatant increased during 100 days and then gradually decreased . Specific UV absorbance showed that the accumulated compounds had a portion of larger, more aromatic, more hydrophobic and double-bond-rich organics, which originated from the decayed biomass . Molecular weight distributions of SMP in MBR supernatant showed that the acclimated microorganisms in a long SRT could decompose high molecular weight organics, it caused the shift of molecular weight distributions of SMP to a lower range . During the operation period, enumeration of active cells in the MBR showed that microbial inhibitions by accumulated SMP was not observed.

Biomed Tech (Berl), 2002, 47 Suppl 1 Pt 2, 798 - 9
{Realization of a measurement module for determining pH value in perfusion cultures}; Dencks S et al.; For cultivating animal cells in bioreactors the maintenance of a fixed pH-value is of elementary importance . In a perfused cell culture system the pH-value is measured by electrochemical flow rate sensors . For the acceptance of a technical solution the integration in complex workplaces is necessary, thereby small size and low costs are the main features . For that a special measurement module was developed, consisting of measuring amplifier and microcontroller component with CAN-Bus-interface.

Biomed Tech (Berl), 2002, 47 Suppl 1 Pt 2, 596 - 7
Applications of autofluorescence for characterisation of biological systems (biomonitoring); Plettenberg HK et al.; We describe a system capable of measuring the laser induced fluorescence emission of two coenzymes (NADH and FAD) via an optical fibre for in vivo monitoring of anatomical, physiological or pathological changes in biological systems . This optical technique permits the measurement of changes in the metabolism of cells in real-time and is nearly non-destructive . We present results of examinations on the localisation of transitions between different tissue and the monitoring of the oxygenation level (ischemia) in muscle or changes in the cellular activity of bacteria in a bioreactor.

Tissue Eng, 2002 Oct, 8(5), 863 - 70
Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering; Sodian R et al.; One approach to the tissue engineering of vascular structures is to develop in vitro conditions in order ultimately to fabricate functional vascular tissues before final implantation . In our experiment, we aimed to develop a new combined cell seeding and perfusion system that provides sterile conditions during cell seeding and biomechanical stimuli in order to fabricate autologous human vascular tissue in vitro . The cell seeding and perfusion system is made of Plexiglas and is completely transparent (Berlin Heart, Berlin, Germany; University Hospital Benjamin Franklin, Berlin, Germany) . The whole system consists of a cell seeding chamber that can be incorporated into the perfusion system and an air-driven respirator pump connected to the bioreactor . The cell culture medium continuously circulates through a closed-loop system . We thus developed a cell seeding device for static and dynamic seeding of vascular cells onto a polymeric vascular scaffold and a closed-loop perfused bioreactor for long-term vascular conditioning . The cell seeding chamber can be easily connected to the bioreactor, which combines continuous, pulsatile perfusion and mechanical stimulation to the tissue-engineered conduit . Adjusting the stroke volume, the stroke rate, and the inspiration/expiration time of the ventilator allows various pulsatile flows and different levels of pressure . The whole system is a highly isolated cell culture setting, which provides a high level of sterility and a gas supply and fits into a standard humidified incubator . The device can be sterilized by ethylene oxide and assembled with a standard screwdriver . Our newly developed combination of a cell seeding and conditioning device provides sterile conditions and biodynamic stimuli for controlled tissue development and in vitro conditioning of an autologous tissue-engineered vessel.

Vet Microbiol, 2003 Feb 2, 91(2-3), 157 - 68
Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili; Prieto CI et al.; Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry . M . bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease . The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components . In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M . bovis cell surface . A sequential competitive bact-ELISA was developed using highly piliated M . bovis cells as antigen . Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M . bovis . This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M . bovis cells, and represents an important tool for cellular piliation monitoring daburing M . bovis cells production in stirred bioreactors.

Pharmacol Res, 2002 Dec, 46(6), 511 - 7
Urea synthesis and cyclosporin a biotransformation in a laboratory scale hepatocyte bioreactor model; Farghali H et al.; Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture . The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal . For the perifusion of cells, they must be immobilized to prepare a bioreactor model . The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture . Hepatocytes were isolated, immobilized and perifused with William's E culture medium containing 1mM NH(4)Cl and CsA (20 microM) . Hepatocytes in the culture were treated in the same way . CsA disappearance from the perifusion or culture media was determined by a HPLC method . Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5+/-0.05 mg h(-1) vs 0.33+/-0.03 mg h(-1), respectively) . ALT leakage was lower in the bioreactor model (60 Ul(-1)) as compared to hepatocyte culture (125 Ul(-1)) . The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively) . The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture . Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes . In addition, the cellular model described here inherits economic and ethical potentials.

Contemp Top Lab Anim Sci, 1998 Sep, 37(5), 61 - 66
Comparison of In Vitro Monoclonal Antibody Production Methods with an In Vivo Ascites Production Technique; Peterson NC et al.; Economic, technical, legislative, and ethical issues influence decisions concerning alternatives to the use of animals in biomedical research . Since the development of cell hybridoma technology, production of ascites in mice has been a popular technique for generating high concentrations of monoclonal antibodies . However, the availability of several in vitro methods that can be performed in most laboratories make these alternative methods of monoclonal antibody production an attractive option . To evaluate the practicality of the use of in vitro techniques, three tissue culture methods and a technique for production of ascites were compared on the basis of yield, material costs, and time requirements . Analysis of results revealed that the time and material costs to produce 100 mg of monoclonal antibody 7.16.4 by inducing ascites in irradiated mice was similar or slightly more than that for tissue culture in standard plastic flasks and hollow fiber cartridge bioreactors; however, tissue culture in gas permeable bags was slightly less expensive than these methods in terms of cost and time . When ascites production in irradiated mice was compared with tissue culture in plastic flasks or gas permeable bags for monoclonal antibody 225, the in vitro methods were approximately five times more expensive than the ascites production technique . Information reported here outlines factors that should be considered and evaluated when choosing a monoclonal antibody production method . Furthermore, these results documented that in vitro technology for the production of monoclonal antibodies can be adapted by most conventional laboratories to provide sufficient resources for the most commonly performed experimental protocols.

Int J Artif Organs, 2002 Oct, 25(10), 1013 - 8
Extracorporeal liver support: porcine or human cell based systems?
Gerlach JC, Zeilinger K, Sauer IM, Mieder T, Naumann G, Grunwald A, Pless G, Holland G, Mas A, Vienken J, Neuhaus P.
Initial results of the clinical use of primary porcine liver cells for extracorporeal liver support are being reviewed as the cell source is controversial . According to Eurotransplant data 20-25% of explanted donor livers are not transplanted, due to factors such as steatosis or cirrhosis . This number corresponds to the number of patients with acute liver failure who require bridging therapy to transplantation . Primary human liver cells from transplant discards can be isolated, purified and maintained in bioreactors and provide an alternative for cell-based extracorporeal liver support therapy . A four-compartment bioreactor enables recovery from preservation and isolation injury in a three-dimensional network of interwoven capillary membranes with integrated oxygenation, rendering the liver cells from these discarded donor organs viable for clinical utilization . Patient contact with additional animal-derived biomatrix and fetal calf serum can be avoided . The initiation of an in vitro cultivation phase allows cell stabilization, quality control, and immediate availability of a characterized system without cryopreservation . The hypothesis of this paper is that with appropriate logistics and four-compartment bioreactor technology, cells from human liver transplant discards can serve the demand for cell-based therapy, including extracorporeal liver support.

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