Cell-recycle Bioreactor Research Articles

Novel Technologies for Butyric Acid Fermentation: Use of Cellulosic Biomass, Rapid Bioreactor, and Efficient Product Recovery

Butyric acid, a four-carbon fatty acid, is an important industrial chemical and feedstock. To produce this chemical, a control fermentation was run with a 126.5 g.L−1 glucose concentration in the feed medium. In this medium, the strain produced 44.8 g.L−1 total acid with a productivity of 0.23 g.L−1h−1 and a yield of 0.41 g.g−1. The strain (Clostridium tyrobutyricum ATCC 25755) was also able to utilize glucose and xylose simultaneously with similar fermentation performance. The culture was also used to produce butyric acid from wheat straw hydrolysate (WSH) employing a hot water pretreatment. In a batch system, the strain resulted in a productivity and yield of 0.27 g.L−1h−1 and 0.44 g.g−1, respectively, which was an improvement over the use of glucose or xylose alone or mixtures of both. To improve reactor productivity, a membrane cell recycle bioreactor was used which resulted in a productivity of 1.89 g.L−1h−1. This productivity was 822% of that achieved in the glucose or xylose batch fermentation. Furthermore, a butyric acid recovery method was developed using XAD-4 adsorbent resin. In this system, up to 206.1 g.L−1 of butyric acid was used in the feed and, as a result of the quick adsorption, the residual butyric acid concentration was 29.5 g.L−1. In this experiment, the rate of acid removal of 1059.4 g.L−1h−1 was achieved.

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600 of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.

Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Conventional acetone-butanol-ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L(-1) of ABE (17.6 g L(-1) butanol, 10.5 g L(-1) ethanol, and 4.4 g L(-1) acetone) from 85.2 g L(-1) glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell-recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L(-1) h(-1) , respectively, could be achieved at the dilution rate of 0.85 h(-1) . Further cell recycling experiments were carried out with controlled cell-bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h(-1) with the bleeding rate of 0.04 h(-1) . Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L(-1) h(-1) , and the yields of 0.17 and 0.34 g g(-1) , respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known-processes.

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h−1 dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.

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.

Modeling, optimization and experimental assessment of continuous L‐(−)‐carnitine production by Escherichia coli cultures

In a previous paper Cánovas et al. (Biotechnol Bioeng 2002;77:764-775) presented a model for L-(-)-carnitine production using Escherichia coli O44 K74, in a cell-recycle bioreactor for the biotransformation of crotonobetaine into L-carnitine. In this work we optimize this biotechnological setup and experimentally verify the predicted optimal parameter profiles. Provided with a reliable and robust S-system description of the cell-bioreactor combined system, we applied the Indirect Optimization Method described by Torres et al. (Biotechnol Bioeng 1997;55(5):758-772; Food Technol Biotechnol 1998;36(3):177-184). This optimization approach provides different parameter value profiles, all of which are compatible with the cell physiology and the bioreactor operating conditions, that yield increased rates of L-(-)-carnitine production. Three parameters were seen to be of critical importance for maximizing L-(-)-carnitine production: the dilution rate, the initial crotonobetaine concentration, and the carnitine dehydratase activity. When the first two were changed in the experimental setup, there was a 74% increase in the L-(-)-carnitine production rate, performance that was in close agreement with the predictions of the model. In accordance with the optimized solution, a further improvement (90% increase in the L-(-)-carnitine production rate) could be attained by over-expressing up to 5 times the carnitine dehydratase basal activity. Thus the optimization approach shown herein provides experimental evidence of a new strategy which demonstrates the possible variables that can be subjected to modifications compatible with the cell physiology and bioreactor operating conditions, and which are able to yield increased rates of L-(-)-carnitine production.

High-level production of D-mannitol with membrane cell-recycle bioreactor.

Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2 g l(-1) h(-1)) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l(-1) resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters.

High-level production of D-mannitol with membrane cell-recycle bioreactor

High-level production of D-mannitol with membrane cell-recycle bioreactor

Production of acetic acid and glycerol from salted and dried whey in a membrane cell recycle bioreactor

The use of salted whey (liquid by-product from the dairy industry containing 7.5% NaCl) as a substrate for either acetic acid or glycerol production was investigated using two yeast strains. One was Kluyveromyces fragilis. The other organism (strain L) can utilize whey lactose that was isolated from waste whey (disposal stream). 8% NaCl, 3% Na 2SO 3 and pH 8.5 at 32°C were the optimum operating conditions for maximum acetic acid production (25.84 g l −1) from salted whey using immobilized cells of yeast strain L. Also, strain L gave a higher yield of acetic acid (0.497 g acetic acid per gram lactose) as compared with K. fragilis (0.322 g acetic acid per gram lactose). 1% Na 2SO 3 at pH 7 and whey supplemented with peptone and malt extract were the optimal conditions for maximum batch glycerol production (13.2 g l −1) from salted whey using immobilized cells of K. fragilis. The extreme values, 18.7 g l −1 for glycerol concentration and 39.78 for the percent yield of glycerol (based on sugar concentration) in a membrane cell recycle bioreactor were higher than those obtained for the immobilized cell batch reactors (13.2 g l −1 and 28, respectively).

Enhanced production of cis,cis-muconate in a cell-recycle bioreactor

For enhanced production of cis,cis-muconate via biocatalytic conversion from benzoate, a high cell density culture of a mutant Pseudomonas putida strain, BM014 in a cell-recycle bioreactor has been investigated. The volumetric productivity in a continuous bioconversion process was dependent on the biomass concentration, specific growth rate, and environmental conditions including dissolved oxygen tension (DOT) and cis,cis-muconate concentration. Biotransformation was started in a cell-recycle continuous culture mode for obtaining a high cell density of 40 g/ l and then carried out in a fed-batch mode for accumulation of cis,cis-muconate to up to 12 g/ l at which induction of catechol 1,2-dioxygenase, a key enzyme catalyzing the rate-determining step in the biosynthetic pathway, was maximized. Since in the biosynthesis of cis,cis-muconate growing cells were utilized as catalysts, cell growth should be continued, but a low specific growth rate achieved under phosphate-limited conditions was indispensable for higher productivity. When the concentration of phosphate in the feed was lowered from 150 to 10 mM, the productivity could be enhanced three fold. It was also found that cobalt ions markedly increased the specific productivity and that DOT should be kept at above 20% of the saturation level for achieving optimum rates of cell growth and product formation. After a period of fed-batch operation continuous production with cell recycling was attempted with phosphate-limited minimal medium supplemented with cobalt ions, and benzoate medium. Enhanced productivity of 5.5 g/( l·h) and a cis,cis-muconate concentration of 13.5 g/ l in the culture broth were obtained and continuous production in a cell-recycle bioreactor was maintained for more than 7 d.

Effect of dissolved O concentration on the Escherichia coli in a cell-recycle bioreactor

In the fermentation of Escherichia coli, a ceramic membrane filter was used in conjunction with a fermenter for cell-recycle operation. The metabolic behaviour, especially the acetic acid production and the specific growth rate, under different concentrations of dissolved O was investigated. An unstructured model was constructed, and it was shown that the observed lower cell density under low dissolved O concentrations was accompanied by large acetic aicd production. However, the concentration of acetic acid was not high enough to cause the observed growth inhibition, and inhibition by other metabolites under low O environment is suspected.

Cell recycling studies for ?-amylase production by Bacillus amyloliquefaciens

Production of α-amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased α-amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on α-amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of α-amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of α-amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h−1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.

Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.

Concentrated starter productions in a two stage cell-recycle bioreactor plant

Continuous production of concentratedLactobacillus acidophilus andBifidobacterium bifidum cells is described in two successive cell-recycle bioreactors. This process allows the study of the relationships between two microorganisms without mixed cultures.

Extractive ethanol production in a membrane cell recycle bioreactor

Ethanol production kinetics of free Saccharomyces cerevisiae cells was compared in a continuous reactor and a cell recycle membrane reactor, with and without solvent extraction. In the continuous culture the in-situ extraction using dibutyl phthalate increased glucose conversion from 45% to 91%, the cell concentration from 10.2 g l −1 to 23.6 g l −1 and the ethanol productivity from 8.2 g l −1 h −1 to 20 g l −1 h −1 when a 270 g l −1 glucose solution was fed to the reactor at a dilution rate of 0.16 h −1. In the membrane cell recycle culture with the solvent extraction the ethanol productivity increased to 45 g l −1 h −1 and 70% of the ethanol produced was extracted to the solvent stream. The mathematical modeling of the extractive bioconversion predicted ethanol concentration and productivity of the system reasonably well.

Development of an ethanol/sorbitol process using a ceramic membrane cell recycle bioreactor

The properties of a ceramic microfiltration membrane unit have been investigated as a component of a cell recycle bioreactor used for the production of ethanol/sorbitol. The conversion is carried out by a glucose/fructose oxidoreductase (and bound co-factor NADP) using cells of Zymomonas mobilis and an equimolar mixture of glucose and fructose. With 100 g/l each of glucose and fructose, a product stream containing 40 g/l sorbitol and 75 g/l ethanol was obtained. The potential economic benefits of such a process are discussed.