Continuous acetone-butanol fermentation: a global approach for the improvement in the solvent productivity in synthetic medium

Summary The present chemostat culture studies were performed in a partial gas recycle system using Clostridium acetobutylicum ATCC 824. Initiation of chemostat cultures at two different levels of vitamins has demonstrated a more than 3 fold improvement in the solvent productivity. The production of extracellular autobacteriocin was increased markedly, when strict anaerobic conditions of the feed vessel were not maintained. When pH was regulated at 4.4, very little variation was seen in the concentration of acids. The duration of solvent production was reduced significantly when NH₄OH was replaced by NaOH for pH regulation, whereas a marked increase in the production of extracellular autobacteriocin was observed. In the optimised medium conditions, a solvent productivity of 2.09 g l^-1 h^-1 (highest ever) could be obtained in the synthetic medium.     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

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.     

By-product formation in cell-recycled continuous culture of Lactobacillus casei

While the volumetric productivity of lactic acid increased in continuous culture of Lactobacillus casei with cell recycle, enhanced formation of by-products such as acetate, formate, ethanol, and D-lactate was observed in the cell-recycled fermenter compared with a simple chemostat at a similar range of dilution rate. The increased formation of by-product which was significantly dependent on substrate limitation resulted from a lower dilution rate rather than a high cell concentration in the cell-recycled fermenter.     

Inulin enrichment by fermentation in a flocculating yeast reactor

Summary Simultaneous production of ethanol and fructose enriched syrups was obtained from Jerusalem artichoke extract using a Saccharomyces diastaticus flocculating yeast in a continuous gas-lift reactor with internal biomass recycle. This allowed the production of 42 g/L of ethanol and 70 g/L of inulin containing up to 92% fructose (fructose/glucose ratio of 11). These results can be compared to the batch and chemostat fermentations which gave a higher ethanol concentration but a lower fructose enrichment. Mass transfert limitations can explain both the productivity decrease and the selectivity improvement in the gas-lift reactor.     

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.     

Optimization of conditions and cell feeding procedures for alcohol fermentation

Alcohol fermentation was studied with an emphasis on the separation of cell growth and alcohol production stages. Experiments were conducted to establish the optimal conditions for alcohol production in batch fermentations and to simulate continuous fermentations with cell feeding at various stages. It was found that the glucose concentration should be kept under 10% (w/v), and the temperature should be between 40 and 42.5 degrees C for maximum specific alcohol productivity. If the cell concentration is increased, a decrease in specific alcohol productivity is observed. Higher cell concentrations are needed for higher final alcohol concentrations. Among the cell feeding procedures into alcohol production stages, a cocurrent design was found to be better than recycle and countercurrent designs.     

Biomass recycling and species competition in continuous cultures

A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate dissolved in the inflow, a gas bubbled directly into the reactor, and light. The effects of recycle are distinct in each case, because the relationships between hydraulic detention time and nutrient inflow are different for each type of nutrient, Effluent recycle, in which the recycle stream is more dilute than the reactor, is discussed in terms of cell detention time and nutrient limitation. Results from chemostat cultures of the blue-green alga, Spirulina geitleri, demonsrtat cell feedback under light limitation. Maximum Productivity is fixed by the incident light intensity. At a particular dilution rate recycling increases or decreases productivity by taking cell density closer or further from the optimum density. Cell recycle with heterogeneous populations can change the outcome of species competition. Selective recycling of one species can reverse this outcome or stabilize coexistence by its selective effect on cell detention time. Experimental results from light-limited mixed cultures of S. geitleri and a Chlorella sp. verify this.     

Vancomycin production is enhanced in chemostat culture with biomass-recycle

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.     

Production of catalytic cells for formaldehyde production and alcohol oxidase by a catabolite repression-insensitive mutant of a methanol yeast Candida boidinii A5

A catabolite repression-insensitive mutant of Candida boidinii A5, strain ADU-15, was investigated as to alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production. The mutant strain ADU-15 showed higher cell productivity and higher alcohol oxidase activity when grown on mixed substrates (glucose-methanol), especially with a high concentration of glucose in the medium. Thus, even under substrate (glucose-methanol)-limited chemostat conditions, where the glucose concentration was low, partial derepression of alcohol oxidase by glucose in mutant strain ADU-15 was detected. The chemostat culture conditions with the glucose-methanol medium were optimized for alcohol oxidase production and the production of cells exhibiting the maximum catalytic activity for formaldehyde production, respectively. By means of chemostat culturing on mixed substrates, we improved the alcohol oxidase productivity 5.0-fold and the productivity of cells exhibiting the maximum catalytic activity for formaldehyde production 3.8-fold, in comparison with the parent strain chemostat cultured with methanol as the single substrate.     

A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity

A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.     

Improved ε‐poly‐l‐lysine productivity partly resulting from rapid cell growth in cultures using a glucose–glycerol mixed carbon source

Productivity enhancements with mixed carbon sources are usually accompanied by simultaneous improvement of cell growth. However, whether the enhanced cell growth in mixed carbon sources influences the biochemical productivity of ε‐poly‐l ‐lysine (ε‐PL) still remains unclear. In this study, we investigated the effect of growth rate on the ε‐PL productivity in a glucose–glycerol mixed carbon source. Based on the typical ε‐PL fed batch fermentation, chemostat culture and relevant physiological analyses were carried out. The ε‐PL productivity was positively correlated to the growth rate ranging from 0.02 to 0.06 h^?1. The primary metabolism activity was enhanced at higher growth rate, providing sufficient precursor l ‐lysine and energy for ε‐PL production. Meanwhile, these two key elements were equally important for biomass production, which could be quickly produced when the cells were fast growing. In addition, rapid growth also strengthened the antioxidant capacity of cells to defend potential oxidative stress. The positive correlation between the growth rate and ε‐PL productivity indicated that the improvement of ε‐PL productivity in the mixed carbon source was partly attributed to the simultaneously enhanced cell growth. Information obtained may provide references for further studies on other secondary biochemicals’ production using mixed carbon sources.     

Investigation of physiological limits and conditions for robust bioprocessing of an extreme halophilic archaeon using external cell retention system

High biological activity and volumetric productivity are considered as prerequisites for efficient bioprocesses, extreme halophilic Archaea have, however, lower growth rates, for which reason halophilic Archaea are so far not used in industrial bioprocesses. To overcome this physiological limit and to achieve increased volumetric productivity, the produced biomass must be retained in a bioreactor, for example equipped with an external cell retention system. In this study, the characterization and parameterization of a bioreactor setup with cell retention was carried out with an extreme halophilic archaeon. Bioprocess quantification was used to demonstrate the process controllability. Focussing on maximizing the volumetric productivity; 10-fold productivity increase was achieved compared to chemostat continuous cultures. Circulation of the broth between the bioreactor and the membrane unit can be however challenging from physiological points-of-view. Hence, operating the system with external cell retention at optimal cross flow rate is physiologically essential: at lower cross flow rates, higher extracellular protein concentrations were measured due to oxygen limitation. In turn, at higher cross flow rates, shear stress reasoned higher concentrations of DNA fragments. This work contributes in a pioneering way to the bioprocess development of extreme halophilic Archaea by optimizing continuous laboratory scale processes regarding robustness and scalability.     

Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris

Simple cyclic fed-batch culture (cfbc), consisting of a constant medium feed with periodic withdrawals of culture, resulted in a product yield (13.4 mg protein per gram biomass) similar to that obtained using the complex multiphase industrial production strategy (13.7 mg protein per gram biomass). In cfbc, productivity was ultimately limited by the rate at which the cells could assimilate methanol. Glycerol was inhibitory to growth at high concentrations. However, product yield continued to increase as the glycerol concentration was increased. In chemostat culture, dissolved oxygen concentration influenced product yield independently of any detectable influence on cell growth.     

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%.     

Design of metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli. Integration of the metabolic and bioreactor levels

In this work metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli based on the Biochemical System Theory and the Indirect Optimization Method are presented. The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of L-(-)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in L-(-)-carnitine production (74% increase in production rate), in close agreement with the model's predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the L-(-)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.