Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed-batch fermentors

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h(-1), a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L . h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell . h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L . h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. (c) 1995 John Wiley & Sons, Inc.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor

A recombinant Escherichia coli HB101(pPAKS2) producing penicillin acylase was cultured in a membrane cell recycle fermentor. The strain was very stable throughout the whole experiment. The main inhibitory by-product was acetic acid, and cell growth ceased when its concentration was above 14 g/L Cell density could be increased up to 145 g/L dry weight without experiencing by-product inhibition by regulating glucose concentration in the fermentor and by using total membrane recycle. Acetic acid formation was negligible not only when cells were cultured in medium containing no glucose but also when glucose was limited. Dissolved oxygen control as well as glucose limitation was an indispensable condition for minimizing acetic acid formation when the medium contained glucose. Low concentrations of accumulated acetic acid were reused when glucose was limited. Use of highly concentrated medium reduced the membrane surface area required for cell recycle greatly. The recycle fermentor could be operated in various operational modes including partial bleed and repeated recycle culture to give high productivity. Productivity of a repeated recycle system was over 10 times higher than that of a simple batch system.     

Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

Spent sulfite liquor (SSL), a waste product of the paper pulping industry, is produced at a rate of 1 ton (dry basis) per ton of pulp. The sugar content of SSL is about 30 g/L. To reduce the biological oxygen demand (BOD) of SSL before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L h, with cell concentration up to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.     

Rapid ethanol fermentations using vacuum and cell recycle

Cell recycle and vacuum fermentation systems were developed for continuous ethanol production. Cell recycle was employed in both atmospheric pressure and vacuum fermentations to achieve high cell densities and rapid ethanol fermentation rates. Studies were conducted with Saccharomyces cerevisiae (ATCC No. 4126) at a fermentation temperature of 35°C. Employing a 10% glucose feed, a cell density of 50 g dry wt/liter was obtained in atmospheric-cell recycle fermentations which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. This permitted the rapid and complete fermentation of concentrated sugar solutions. At a total pressure of 50 mmHg and using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentations. In order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile compounds. It was also necessary to sparge the vacuum fermentor with pure oxygen to satisfy the trace oxygen requirement of the fermenting yeast.     

Experimental evaluation of liquid film resistance in oxygen transport to microbial cells

A membrane probe was used to monitor the dissolved oxygen concentrations in continuous cultures of Candida utilis and Micrococcus roseus growing at low dissolved oxygen concentrations and various agitation levels. For the yeast fermentations, increasing the agitation level within the range of 0.1 to 0.3 w per liter lowered steady-state dissolved oxygen concentrations in the fermentor. The steady-state dissolved oxygen concentration in the fermentor was not influenced by the agitation level within the range of 0.3 to 1.8 w per liter. With M. roseus, no effect of agitation on steady-state dissolved oxygen concentrations in the fermentor was observed within the range of 0.1 to 1.8 w per liter. It was concluded that, under the conditions used, a measurable transfer barrier from the liquid to the yeast cells existed at agitation levels below 0.3 w per liter and that this barrier did not exist at agitation levels above 0.3 w per liter. The transfer barrier from the liquid to the yeast surface could be represented by a stagnant film of liquid 0.6 × 10^-4 cm thick surrounding the cell at an agitation level of 0.10 w per liter. This film represented an oxygen concentration drop of 1.3 × 10^-7 M from the bulk of the medium to the cells under the experimental conditions.     

Urokinase bound to fibrocollagenous tubes: an in vitro kinetic study

Urokinase (UK) has been immobilized to the inner surfaces of fibrocollagenous tubes (FCT) in an attempt to develop a fibrinolytic biomaterial which may be suitable for use as a small diameter vascular prosthesis. The enzyme was bound by adsorption followed by glutaraldehyde crosslinking. An in virto kinetic study of immobilized urokinase was conducted by employing the tubular material as a flow through reactor operated in a batch recycle mode in which the esterolysis of the model substrate, N-alpha-acetyl-L-lysine methyl ester (ALME), was monitored as a function of substrate concentration, recycle flow rate, and temperature. Results were compared with data from the soluble enzyme reaction, which was conducted in the presence and absence of 10% swine skin gelatin, in order to identify the specific effects of a collagenous microenvironment. Observed rates for the UK-FCT catalyzed reaction were observed to be dependent on recycle flow rates below 12 mL/min (Re = 107). Apparent Michaelis-Menten rate parameters were determined by a nonlinear search technique for two flow rates: one above the critical point for external diffusion effects (Re = 282) and one within the mass-transfer-limited region (Re = 71). When the latter data were corrected for external diffusion by applying the Graetz correlation for laminar flow in tubes to estimate themass transfer coefficient, the corrected K(m) of 6.45 +/- 0.38 mM agreed very closely with the diffusion free parameter (i.e. 6.13 +/- 0.63). Furthermore, this value was observed to be an order of magnitude higher than that of the soluble enzyme but approximately equal to the K(m) of the soluble enzyme in a 10% gelatin environment (8.13 +/- 1.53 mM). It is postulated that the difference in kinetic parameters between soluble and collagen immobilized UK is due to an inherent interaction between collagen and enzyme rather than to mass transfer effects. Such aninteraction is supported by the effects of collagen on thermal stability and energy of activation.     

Vacuum fermentation for ethanol production using strains of Zymomonas mobilis

Summary Using strains of Z.mobilis, a vacuum fermentation system has been evaluated. The system was designed with the fermentor at atmospheric pressure and an external vacuum vessel (50 mm Hg). Sequential operation of the vacuum vessel was under microprocessor control. The use of Z.mobilis together with the two-stage design of the vacuum system has been found to overcome the problems of oxygen addition and the possibility of contamination reported previously for vacuum fermentations with yeasts. The productivity of 85 g/1/h found in the continuous cell recycle experiments was similar to that reported previously for a strain of S.cerevisiae.     

Performance of a two-stage fermentor with cell recycle for continuous production of lactic acid

The performasnce of a recycle two-stage fermentor with cell separators after each stage is analyzed numerically for continuous production of lactic acid. In this system, the bleed broth withdrawn from the first stage is provided to the second fermentor to reuse viable cells in the bleed. Biological rate expressions and parametric values are taken from the literature. The effects of operating parameters on the concentrations of total and viable cells, substrate and product in each stage, the lactic acid productivity and the substrate conversion are examined and discussed. With respect to overall productivity and conversion, it is found that the present fermentor system is more efficient than a conventional chemostat fermentor with cell recycle.     

Cell retention culture with an internal filter module: continuous ethanol fermentation

A new internal filter feedback system with a stainless steel filter was introduced and its application for continuous ethanol fermentation was investigated. The filter performance was highly influenced by agitation speed and yeast concentration. Retention coefficient with a filter of 2 mum pore size was found more than 97.5%, and the filter was suitable for yeast separation. Maximum yeast concentration was 157 g/L and the best operable cell concentration was between 90 and 150 g/L. Which was similar to that obtained in the external membrane cell recycle culture. The cell concentration in the fermentor was maintained by manipulation of dilution rate and bleed ratio with the growth rate. The internal filter feedback system was successfully operated for more than 10 days. This study shows that the internal filter feedback system with a stainless steel filter can be used high-density cell culture and ethanol fermentation. Furthermore, it can be scaled up more easily than the external cell recycle system. (c) 1993 John Wiley & Sons, Inc.     

Use of an exchange filtration technique to obtain synchronous sporulation in an extended batch fermentation

A fermentor system with an external filtration loop has been developed to control the growth and sporulation of yeast in a single vessel. Excess growth medium, instead of being removed by centrifugation, is removed by filtration and replaced with acetate sporulation medium. The technique did give 80% sporulation after 20 hr, greatly improving the rate and degree of synchrony of sporulation and it also eliminated the contamination hazard of the conventional harvest technique, centrifugation, and resuspension of vegetative cells in sporulation medium. Furthermore it permits proper control of the environmental conditions throughout the growth, exchange, and sporulation phase. In this technique 100% recycle of biomass is achieved without any packing of the cells on the filter. This technique has wide application in the study of industrial fermentation that involves microbial differentiation such as the production of ergot alkaloids, bacitracin, and cephalosporin.     

Improvements in ethanol concentration and fermentor ethanol productivity in yeast fermentations using whole soy flour in batch,and continuous recycle systems

Summary Ethanol concentrations and fermentor productivities were increased 20.2 and 15.5% at 90 and 95% recycle, respectively, when whole soy flour was added to the feed (2 g/ at 90% recycle, and 1 g/ at 95% recycle) of a continuous yeast fermentation system with recycle of cells and soy flour (soy flour concentrations were 2% at steady state in the fermentor) by UF membranes as compared to controls. The improvements were primarily due to increases in cell concentrations. Similar results were obtained for batch cultures.     

Performance evaluation of ethanol fermentor systems using a vector-valued objective function

It is shown that the performance evaluation using a vector-valued objection function whose components are the product productivity, the product concentration, and the substrate conversion is quite useful in getting deeper insight into the development of new processes and in determining the operating point. Particular attention is focused on the ethanol fermentation using variety of systems such as the conventional chemostat system, multiple fermentor system, cell recycle system, extractive fermentor system, cell recycle system, extractive fermentor system, and immobilized cell system. The contour map and the projection of the noninferior set are used in investigating the performance improvement and the trade-offs among performance indexes.     

Improved Bioreaction Kinetics for the Simulation of Continuous Ethanol Fermentation by Saccharomyces cerevisiae

A kinetic model for the production of ethanol by Saccharomyces cerevisiae has been developed from semiempirical analysis. The values for the parameters in this model were then determined by nonlinear multiple regression using the data of Bazua and Wilke ( 1977). The final equations were μ=0.427s(1-(p/101.6)^1.95)/(0.245+s), Y_X/p=0.291, and Y_X/s=0.152(1-p/302.3). This model was then used to simulate a continuous stirred tank fermentor (CSTF) and compared to other models using the same experimental data but different kinetics. The equations required to use these kinetics in a CSTF with recycle were then developed. From this simulation, it was found that, for a CSTF with recycle, the best configuration to operate is an external recycle, with a low bleed and recycle ratio.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

The rotorfermentor. II. Application to ethanol fermentation.

The kinetics of microbial growth and product formation are described as applied to the high cell concentration scheme of the rotorfermentor. A bench scale pilot plant was designed and built in order to demonstrate the operational feasibility of the rotorfermentor. The fermentation of glucose to ethanol by Saccharomyces cerevisiae ATCC 4126 was used. When the rotorfermentor was used with a glucose feed concentration of 104 g/liter almost 100% glucose utilization was obtained and the ethanol productivity rate was 27.3 g ethanol/liter hr which was found to be about 10 times greater than the ethanol productivity obtained from an ordinary continuous stirred tank (CST) fermentor. The ethanol experimental results obtained from the rotorfermentor and an ordinary CST fermentor were used as a basis to assess the economic feasibility of the rotorfermentor. The economics of an industrial scale ordinary CST fermentor with and without cell recycle is compared with a rotorfermentor unit for the same ethanol production throughput. For the process conditions considered in this case, calculations showed that the rotorfermentor may replace both a CST fermentor and cell centrifuge resulting in lower capital equipment costs and lower power consumption requirements.     

Citrate production from hydrocarbons by use of a nonsterile,semicontinuous cell recycle system

In order to minimize product toxicity, decrease fermentation cost, prolong the effective production phase, and shorten the lag phase before production in the citrate-hydrocarbon fermentation by Candida lipolytica ATCC 8661, the use of a nonsterile semicontinuous cell recycle system was investigated. Model experiments demonstrated that portions of the fermentor broth could periodically be removed and centrifuged under nonaseptic conditions with the cells being added to fresh medium and returned to the fermentor. Citrate production continued, however with repeated semicontinuous cell recycle, acid production gradually decreased. It was postulated that this decrease could be attributed largely to physiological trauma and that a truly continuous cell recycle system would minimize these effects and permit maintenance of higher citrate production rates.     

Batch Production of Protein from Ethane and Ethane-Methane Mixtures

A culture of Graphium grows upon natural gas and a mineral salt solution. Ethane is the preferred substrate but methane is co-utilized. A stirred-tank type fermentor was used to study batch growth. Maximum production rate of biomass was 80 mg/liter.hr, at pH 4, using simple synthetic supporting medium with ammonium sulfate as a nitrogen source. This rate was observed after 40 hr of fermentation. A doubling time of 3.7 hr was observed. The corresponding specific growth rate was 0.187 per hr. A magnetic drive fermentor was used to study the effect of continuous recycle of gases in a gas-tight system. The rate of oxygen utilization is approximately 2.1 times higher than for ethane. Oxygen must not be allowed to become limiting in recycle gases. The calculated efficiency of overall biomass synthesis averages 30%. Hyphal and unicellular tissue of Graphium contains 52% protein. It compares favorably with standard FAO protein in its content of amino acids.     

Continuous cultivation ofLactobacillus rhamnosus with cell recycling using an acoustic cell settler

Continuous production of lactic acid from glucose byLactobacillus rhamnosus with cell recycling using an acoustic cell settler was carried out. The performance of the system, such as the concentration of cell and product were compared with the control experiment without recycling. The acoustic settler showed cell separation efficiency of 67% during the continuous operation and the cell concentration in the fermentor with recycle exceeded that of the control by 29%. Compared with the control, lactic acid production was increased by 40%, while glucose consumption was only increased by 8%. The higher value of lactic acid production to substrate consumption (Y _P/S, product yield coefficient) achieved by cell recycling is interpreted to indicate that the recycled cell mass consumes less substrate to produce the same amount of product than the control. Within system environmental changes due to the longer mean cell residence time induced the cells maintaining the metabolic pathways to produce less by-product but more product, lactic acid.