Fermentation coupled with microfiltration: kinetics of ethanol fermentation with cell recycle

Cell recycle by microfiltration was used in yeast alcoholic fermentation in continuous operation. Toxins were proved to be washed by increasing dilution rate. — Specific ethanol production rate followed an exponential inhibition equation, which is function of both biomass concentration and dilution rate. — Productivity is shown to be 40 times greater than in conventional continuous operation.     

Continuous cultivation of the yeastSaccharomyces cerevisiae at different dilution rates and glucose concentrations in nutrient media

The influence of glucose concentration in nutrient media on the specific growth rate and biomass yield in the course of continuous fermentation ofSaccharomyces cerevisiae was investigated. An increase of glucose content in media decreased the specific growth rate and the biomass yield. Glucose concentration had significant effects on protein and phosphate contents of cells. However, an increased glucose concentration increased the fermentative power ofS. cerevisiae (SJA-method). An increase of the dilution rate decreased the cell concentration in the fermentor. Specific growth rate approached the values of the dilution rate. The best agreement has been obtained at a dilution rate of 0.20/h. This dilution rate proved to be most convenient for the investigated microorganism and cultivation conditions (media composition, pH, aeration intensity and temperature). Biomass yield proved to be decreased by an increase of the dilution rate.     

Effect of the growth conditions on the synthesis of a recombinant beta-1,4-endoglucanase in continuous and fed-batch culture

Continuous culture and fed-batch fermentations were used to test the behavior of the system Bacillus subtilis DN1885(pCH7) that synthesizes a recombinant beta-1,4-endoglucanase. Continuous culture experiments were focused on the study of the instability aspects of the system as well as determination of the biomass growth rate range at which the recombinant enzyme synthesis was improved. Fed-batch fermentations were carried out to study the possibility of enhancing recombinant enzyme synthesis through the control of medium addition. It was found that, in continuous culture fermentations, the culture is less unstable at low dilution rates (dilution rate < 0.1 h(-)(1)). Also, low dilution rates give a higher specific recombinant enzyme concentration (10 times more than that obtained at high dilution rates). In fed-batch fermentation, the final recombinant enzyme concentration can be manipulated through the medium addition strategy. To increase the recombinant enzyme concentration, the carbon source has to be fed slowly, otherwise enzyme synthesis is impaired due to catabolite repression. Therefore, an increase in the biomass concentration does not necessarily imply an increase in the recombinant enzyme concentration. Higher recombinant enzyme concentrations were found in fed-batch fermentations compared to those obtained in continuous culture.     

High production of acetone-butanol-ethanol with high cell density culture by cell-recycling and bleeding

A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).     

Cultivation of the yeast Candida lipolytica on hydrocarbons. II. One-stage continuous cultivation on gas oil

Continuous cultivation of the yeast Candida lipolytica on gas oil was studied from the viewpoint of biomass production and oil deparaffination. Optimum conditions wore found at the dilution rate D = 0.16–0.19 when biomass productivity 1.7 g/l/hr and yield coefficient. y = 0.92 were achieved. At deparaffination to the same freezing point, more than double the production of biomass and deparaffined oil during a given time unit was achieved in a continuous process than in batch cultivation. Consumption of substrate was followed in both cultivation processes and it was confirmed that individual n-alkanes of gas oil were degraded at various rates and yields. Results proved optimum cultivation conditions to depend on concentration and composition of the paraffinic fraction of gas oil used. To achieve these conditions the continuous process may be controlled by choice; of suitable dilution rate and concentration of gas oil.     

Improvement of Process Stability of Microbiological Quinoline Degradation in a Three‐Phase Fluidized Bed Reactor

The biological degradation of quinoline by suspended and immobilized Comamonas acidovorans was studied under continuous and discontinuous operating conditions in a three‐phase fluidized bed reactor. C. acidovorans degrades quinoline into biomass and carbon dioxide. Quinoline and the intermediates of its metabolic pathway are found only by quinoline shockloads. The continuous degradation of quinoline by suspended biomass was only possible, if the dilution rate was less than the growth rate (μ_max =0.42 h^–1) and the concentration of a shockload was less than 1 kg/m³. A concentration greater than 1 kg/m³ led to an irreversible damage of the cells. Hence, two different carrier materials were used for immobilization by attachment, to increase the stability of the process. Using immobilization of biomass on carriers decouples the hydrodynamic retention time and the growth rate of the microorganisms. A comparison of the carrier material showed no differences with respect of activity and stability of the biofilm. The process stability of a three‐phase fluidized bed reactor was increased by immobilized biomass. The degradation of toxic shockloads was only possible with immobilized biomass. A dynamic model has been developed to describe the concentration profile of quinoline, 2‐hydroxyquinoline as metabolite and the suspended biomass. A comparison of the measured and calculated values showed good agreement.     

Influence of dilution rate and induction of cloned gene expression in continuous fermentations of recombinant yeast

The effects of growth rate on cloned gene product synthesis in recombinant Saccharomyces cerevisiae have been studied in continuous culture. The plasmid employed contains a yeast GAL10-CYC1 hybrid promoter directing expression of the E. coli lacZ gene. beta-Galactosidase production was therefore controlled by the yeast galactose regulatory circuit, and the induction process and its effects were studied at the various dilution rates. At all dilution rates plasmid stability decreased with induction of lacZ gene expression. In some instances, two induced "steady states" were observed, the first 10-15 residence times after induction and the second after 40-50 residence times. The second induced steady state was characterized by greater biomass concentration and lower beta-galactosidase specific activity relative to the first induced "steady-state." beta-Galactosidase specific activity and biomass concentration increased as dilution rate was reduced, and despite lower flow rate and plasmid stability, overall productivity (activity/L/hr) was substantially higher at low dilution rate. Important factors influencing all of the trends were the glucose and galactose (inducer) concentrations in the vessel and inducer metabolism.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Specific ATP and specific oxygen uptake rate in immobilized cell aggregates: Experimental results and theoretical analysis using a structured model of immobilized cell growth

In this work the quality and activity of immobilized Beneckea natriegens have been measured using Specific ATP (SATP) [mg (ATP) g(-1) (dry biomass)] and Specific Oxygen Uptake Rate (SOUR) [mg O(2) g(-1) (dry biomass) h(-1)]. The cells were grown in a 3 L Three Phase Air Lift Bioreactor (TPALB) and were immobilized on diatomaceous earth (silica) support particles; sterile conditions were employed during the experiment, with n-propanol as the sole carbon source. Two sets of experiments were performed, one with varying dilution rate and the other with varying inlet substrate concentration. The average SATP and SOUR of the immobilized biomass was 3-4 times lower than the values obtained for suspended Beneckea natriegens growing at its maximum growth rate. The suspended biomass present in the TPALB was generated primarily through attrition from the outer layer of the biofilm, and maintained higher levels of SATP and SOUR than the immobilized biomass. This result indicates that the immobilized biomass quality/activity is higher at the external layer of the biofilm. A structured model in which biomass is divided into two compartments, active and inert, was used to describe the experimental results. This model predicts the biomass quality/activity and substrate concentration distributions in the biofilm. These distributions were integrated to give overall values of SATP and SOUR for the immobilized biomass which compared favorably with experimental data. The primary implication of the results is that the location of immobilized biomass in the biofilm affects its biocatalytic activity, and should be taken into account when modeling immobilized biomass bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 660-673, 1997.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Continuous limonin degradation by immobilizedRhodococcus fascians cells in K-carrageenan

Limonin can be effectively degraded byRhodococcus fascians cells. These bacteria can be entraped in -carrageenan, and used in a continuous stirred tank reactor to degrade limonin in a continuous process. The effects of temperature limonin concentration, dilution rate, and aeration on the reactor behaviour have been tested, and the results correlated with changes in limonin conversion, substrate degradation rate, and free and immobilized biomass. Results showed that the immobilized cells were able to debitter limonin-containing media and the immobilized biomass was quite stable throughout the operational conditions tested. A population of free biomass was present in the reactor, the quantity of which was dependent on dilution rate. The immobilized bacteria increased its limonin-degrading capability when the substrate concentration was increased. The aeration was not strictly necessary for limonin degradation. Additionally, the immobilized cells were active and stable for more than 2 months of continuous operation, and were able to recover their limonin-degrading capability when used intermittently. Finally, none of the main components of a juice was noticeably altered during limonin degradation, so the reactor response was good enough to consider its application.     

Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

Production of 2,3-butanediol in a membrane bioreactor with cell recycle

Summary The production of 2,3-butanediol by Enterobacter aerogenes DSM 30053 was studied in a cell recycle system with a microfiltration module. Emphasis was put on the influence of oxygen supply, cell residence time, dilution rate, and pH. Under optimal conditions a productivity as high as 14.6 g butanediol + acetoin/l per hour was achieved with a product concentration of 54 g/l and a product yield of 88%. This productivity is three times higher than that of an ordinary continuous culture. The achievable final product concentration of a cell recycle system was limited by the accumulation of the inhibiting by-product acetic acid, which increased very rapidly at low dilution rate. To maximize product concentration a fed-batch fermentation was carried out with stepwise pH adaption at high cell density. A final product concentration of 110 g/l was obtained with a productivity of 5.4 g/l per hour and a yield of 97%.     

The use of a mixed culture of Geotrichum candidum,Candida krusei and Hansenula anomala for microbial protein production from whiskey distillery spent wash

Summary The use of a mixed culture of Geotrichum candidum, Hansenula anomala and Candida krusei for microbial protein production and treatment of whiskey distillery spent wash was investigated in both batch and continuous culture. Although capable of assimilating the same substrate constituents in pure culture the organisms used different constituents for growth in mixed continuous culture, allowing a stable mixed population to be established. The relative proportions of the three organisms in the population was dependent on the dilution rate.The mixed culture did not give superior yields or productivities, but did give proportionally greater COD reduction and would be expected to bemore stable and resistant to contamination.At a dilution rate of 0.10 h^–1 the mixed culture gave a biomass yield of 4.8 g l^–1, containing 55% crude protein, with a COD reduction of 31.5%, while in mixed batch culture a biomass yield of 12.5 g l^–1, containing 48% crude protein, was obtained with a COD reduction of 54.9%.     

Effect of furfural on the growth of lactose-utilizing Candida Blankii 35

The effects of furfural on the growth of the lactose-utilizing yeast Candida blankii 35 were investigated using the method of continuous cultivation under conditions of carbon limitation and at dilution rates of 0.1 and 0.25 h⁻¹. The data obtained at dilution rate 0.1 h⁻¹ and 0.04% furfural showed a decrease in the yield of biomass by 6% and in the RNA content, but the synthesis of cell protein increased with 11.6% compared to the control. Treatment with 0.08% furfural induced significant changes in growth and biosynthesizing ability. A strong inhibitory effect of furfural was observed: the biomass yield decreased by half at 48 h and the culture the reached the control level of protein content. The effect of 0.02% furfural at 0.25 h⁻¹ dilution rate caused a significant reduction of biomass yield (34.4%) and the substrate utilization rate reached values higher by 52.4% at 48 h, but the protein-synthesizing ability of the cells slightly increased. The results showed that a treatment with 0.04 or 0.08% furfural caused significant disturbances of cell functions, the yields of biomass and protein drastically decreased and the culture was washed out. Data showed that the inhibitory effect of furfural on the growth and protein-synthesizing ability of Candida blankii 35 depends on the inhibitor concentration as well as the dilution rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biomass and lipid enhancement in Ankistrodesmus sp. cultured with reused and minimal nutrients media

Microalgae are a promising feedstock for biofuel production. Lipid content in microalgae could be enhanced under nutrient depletion. This work investigated the effect of the nutrient on lipid accumulation in Ankistrodesmus sp. culture. Batch cultures were carried out using fresh BG11 medium, and after the harvest, the medium was reused for the next culture; this method was repeated two times. The maximum lipid productivity of 29.75 mg L^?1 day^?1 was obtained from the culture with the second reuse medium. In continuous cultures, Ankistrodesmus sp. was cultured in both fresh and modified BG11 mediums. The modified BG11 medium was adjusted to resemble the content of the first reuse medium. As a comparison between batch and continuous cultures, it was proven that the productivity in the continuous culture was better than in the batch, where the achievable maximum biomass and lipid were 188.30 and 38.32 mg L^?1 day^?1. The maximum lipid content of 34.22% was obtained from the continuous culture at a dilution rate of 0.08 day^?1, whereas the maximum saturated and unsaturated fatty acid productivities of 79.96 and 104.54 mg L^?1 day^?1 were obtained at a dilution rate of 0.16 day^?1.     

Extractive fermentation by Zymomonas mobilis and the control of oscillatory behavior

Summary Extractive fermentation is shown to greatly improve the performance ofZymomonas mobilis in continuous culture during the conversion of concentrated substrates to ethanol, and it is also used to eliminate the oscillatory behavior often exhibited byZ. mobilis in conventional fermentations. An ethanol productivity of 15.6 g/Lh is achieved with the near-conversion of a 295 g/L glucose feed at a medium dilution rate of 0.11 h^–1 and solvent dilution rate of 1.5 h^–1. This is more than triple the productivity obtained during conventional fermentation of a 135 g/L glucose feed at the same medium dilution rate.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

Dynamics of anaerobic growth of Saccharomyces cerevisiae in the chemostat.

A kinetic Monod model has been used to describe the dynamic response of a continuous stirred tank fermentor (CSTF) to changes in dilution rate. A general analytical solution of a linearized model was obtained. Experimental results (Vairo et al. 1977) of continuous anaerobic culture of Saccharomyces cerevisiae have verified the model quantitatively. For step disturbances on the dilution rate the responses of biomass concentration and the outlet substrate concentration were calculated on a digital computer and compared with the experimental data.