Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

A novel packed-bed bioreactor operating under isothermal and non-isothermal conditions

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

Toluene removal from waste air using a flat composite membrane bioreactor

In this report, gaseous toluene biodegradation results in a flat composite membrane reactor inoculated with Pseudomonas putida TVA8 are presented. Preliminary abiotic experiments showed that transport of toluene through the membrane was linearly and negatively correlated with the gas residence time (tau). During a 339-day biofiltration experiment, the influence of gas residence time (2-24 sec) and mass loading rate (B(v); 10-483 g x m(-3) h(-1)) on the toluene elimination capacity was investigated. A maximum elimination capacity (EC(max)) of 397 g x m(-3) h(-1) was achieved at tau = 24 sec and B(v) = 473 g x m(-3) h(-1). Expressed per unit membrane area, the EC(m,max) was 0.793 g x m(-2) h(-1), which is five times higher than results obtained with other membrane bioreactor experiments in the same range of loading rates. At low gas residence times, reactor performance was limited by mass transfer. Toluene concentration profiles along the membrane were measured for several biotic and abiotic conditions. For inlet concentrations (C(in)) up to 1 g x m(-3), more than 90% was eliminated at 15 cm from the reactor inlet. For C(in) > 1.65 g x m(-3), longer membranes are necessary to obtain these high removal efficiencies.     

Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.     

Occurrence of circadian rhythms in hairy root cultures grown under controlled conditions

Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media. Fundamental knowledge of hairy root physiology is essential to develop and control its culture. In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data logging provides an efficient tool to study rapid physiological variations in hairy root cultures. Datura innoxia hairy roots were grown in a bioreactor equipped with on-line data analyses of pH, dissolved oxygen (pO2), conductivity, oxygen, and carbon dioxide. The experiments were done at a constant temperature and in the absence of light cues. The results obtained showed that the carbon dioxide evolution rate (CER) presented regular oscillations during the culture. Similar oscillations were also observed for the oxygen uptake rate (OUR). These signals were treated mathematically to look for the existence of a rhythm. An autocorrelation function was used to detect any periodic components. The results demonstrate that hairy root respiration exhibited peaks of 1 day. These oscillations, having a period of about 24 h, were also observed in pH and conductivity signals, although not for the pO2 signal. The data acquired in the absence of hairy roots showed that the observed periodic behavior was not an artifact. No effect on rhythms was observed by the imposition of an external "day/night" cycle. The fact that oscillations persisted in the absence of external stimuli, with a free-running period of 24 h, suggests that a circadian rhythm exists in hairy roots of D. innoxia.     

Multienzyme mevalonate pathway bioreactor

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.     

Increased rate of chondrocyte aggregation in a wavy-walled bioreactor

A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.     

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.     

Comparative life cycle assessment of two landfill technologies for the treatment of municipal solid waste

Goal and Scope  The potential environmental impacts associated with two landfill technologies for the treatment of municipal solid waste (MSW), the engineered landfill and the bioreactor landfill, were assessed using the life cycle assessment (LCA) tool. The system boundaries were expanded to include an external energy production function since the landfill gas collected from the bioreactor landfill can be energetically valorized into either electricity or heat; the functional unit was then defined as the stabilization of 600 000 tonnes of MSW and the production of 2.56x10⁸ MJ of electricity and 7.81x10⁸ MJ of heat. Methods  Only the life cycle stages that presented differences between the two compared options were considered in the study. The four life cycle stages considered in the study cover the landfill cell construction, the daily and closure operations, the leachate and landfill gas associated emissions and the external energy production. The temporal boundary corresponded to the stabilization of the waste and was represented by the time to produce 95% of the calculated landfill gas volume. The potential impacts were evaluated using the EDIP97 method, stopping after the characterization step. Results and Discussion  The inventory phase of the LCA showed that the engineered landfill uses 26% more natural resources and generates 81% more solid wastes throughout its life cycle than the bioreactor landfill. The evaluated impacts, essentially associated with the external energy production and the landfill gas related emissions, are on average 91% higher for the engineered landfill, since for this option 1) no energy is recovered from the landfill gas and 2) more landfill gas is released untreated after the end of the post-closure monitoring period. The valorization of the landfill gas to electricity or heat showed similar environmental profiles (1% more raw materials and 7% more solid waste for the heat option but 13% more impacts for the electricity option). Conclusion and Recommendations  The methodological choices made during this study, e.g. simplification of the systems by the exclusion of the identical life cycle stages, limit the use of the results to the comparison of the two considered options. The validity of this comparison could however be improved if the systems were placed in the larger context of municipal solid waste management and include activities such as recycling, composting and incineration.     

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor

BACKGROUND: One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. METHODS: In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. RESULTS: A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). CONCLUSIONS: MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.