Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle

Summary To increase the solvent productivity of the acetone-butanol fermentation, a continuous culture of Clostridium acetobytylicum with cell recycling was used. At a dry cell mass concentration of 8 g l^-1 and a dilution rate of D=0.64 h^-1, a solvent productivity of 5.4 g l^-1 h^-1 was attained. To prevent degeneration of the culture, which occurs with high concentrations of solvents (acetone, butanol and ethanol), different reactor cascades were used. A two-stage cascade with cell recycling and turbidostatic cell concentration control turned out to be the best solution, the first stage of which was kept at relatively low cell and product concentrations. A solvent productivity of 3 and 2.3 g l^-1 h^-1, respectively, was achieved at solvent concentrations of 12 and 15 g l^-1.Symbols D Dilution rate (h^-1) - r _p solvent productivity (g l^-1 h^-1) - s residual glucose concentration (g l^-1) - V _R reactor volume (l) - V _O overall volume (l) - x (dry) cell mass concentration (g l^-1) - Y _P/S solvent yield (g g^-1)     

Protease production by Clostridium perfringens in batch and continuous culture

A single covalently closed circular plasmid isolated from Selenomonas ruminantium HD4 migrated at 3–5 kilobase pairs (kb). A second band migrating at 23 kb could not be confirmed as plasmid DNA. The plasmid was digested by HindIII. Extraction of plasmid DNA from S. ruminantium HD4 was facilitated by the use of a carbonate buffer wash during cell harvest that allowed for rapid and complete lysis by lysozyme and markedly improved the release of DNA.     

Production of acetic acid by Clostridium thermoaceticum in batch and continuous fermentations

Batch and continuous fermentations with Clostridium thermoaceticum (ATCC 39073) using automatic pH control were conducted. The value of mu(max) obtained from batch fermentation was about 0.14 h(-1); acetate yield, which was both growth and non-growth associated, was about 2 mole of acetic acid/mole of glucose, compared with a theoretical maximum value of 3. This low yield, compared with literature data, may be explained by glucose loss through a combination of degradation routes. Continuous fermentation could be sustained for 1600 h or more without contamination problems. Continuous fermentation at high dilution rates indicates that mu(max) may be well above 0.17 h(-1) when fresh feed medium is used. Acetate yields in continuous fermentation were about 77% of theoretical or 2.3 mole of acetic acid/mole of glucose.     

Non-production of acetone and butanol by Clostridium acetobutylicum during glucose- and ammonium- limitation in continuous culture

Normal amounts of acetone and butanol were formed during the fermentation of glucose in a defined minimal medium in pH-regulated batch cultures (pH 5.0). No solventogenesis occurred during glucose- or ammonium-limited growth in a chemostat. Moreover, the capacity to produce solvents appeared to be lost during growth in continuous culture, apparently together with the ability to form endospores.     

Production of gramicidin S in batch and continuous culture

A mathematical model for the production of gramicidin S in batch and continuous culture is proposed. It is based on the division of the age of a cell into two phases—an immature and a mature one. A nongrowth associated product, such as an antibiotic, is assumed to be produced when the organism is in the older of these two phases, the mature state. The parameters describing the model were evaluated from batch and single stage transient continuous culture of Bacillus brevis, which produces the antibiotic gramicidin S. The predictive value of the model was studied in steady-state single stage continuous culture and in a transient two stage system. Good agreement between the theoretical curves and the experimental results was found in the transient response of both the first and second stage systems, although at high dilution rates (0.34 hr^?1) in the first stage, deviations from the predicted response were observed in the second stage. These may have been due to chemostat instability at dilution rates close to washout, lags in cell growth, and a metabolic lag on going from stage one to stage two.     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

Production of L- AND D-lactate by Pediococcus pentosaceus in batch and steady-state continuous culture

The fast growth and acid production of a strain of Pediococcus pentosaceus , used as a starter culture in the production of dry sausages, was dependent on the presence of acetate. In a batch culture on a mixture of glucose and sucrose both sugars were consumed simultaneously. Similar growth rates and product yields were obtained on glucose and sucrose, d - AND l -lactate were produced via a D- and L-lactate dehydrogenase (LDH), respectively, and no racemase was present. In batch cultures about 15% of the lactic acid produced was the D-isomer, whereas in a sucrose-limited, continuous culture the fraction of D-lactic acid increased with decreasing dilution rate. The results are discussed in relation to the two LDH activities.     

Production of thermostable xylanases in batch and continuous culture by Thermomonospora fusca KW 3

Summary The actinomycete Thermomonospora fusca KW 3 produced novel thermostable xylanases in batch and continuous cultures in media containing insoluble xylan. The production of xylanases could be induced with oat spelt or beech xylan. Very low activities were detected when the strain was grown on glucose or xylose. In continuous cultivations, mycelial wall growth could be prevented using a stirrer speed controller. Homogeneous mixing of the insoluble substrate was obtained by vibrating the flexible tubes. T. fusca KW 3 could be grown on insoluble xylan at growth rates as high as 0.23 h^–1, equivalent to a doubling time of 3 h. Xylanase activity decreased from maximum levels of 2.5 units (U) ml^–1 with increasing dilution rate and was nearly constant at a level of 0.5 U ml^–1 with dilution rates greater than 0.1 h^–1. Correspondence to: P. Röthlisberger     

Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture

Summary When Clostridium acetobutylicum was grown in continuous culture under glucose limitation at neutral pH and varying dilution rates the only fermentation products formed were acetate, butyrate, carbon dioxide and molecular hydrogen. The Y _glucose ^max and (Y _ATP ^max ) _gluc ^exp values were 48.3 and 23.8 dry weight/mol, respectively. Acetone and butanol were produced when the pH was decreased below 5.0 (optimum at pH 4.3). The addition of butyric acid (20 to 80 mM) to the medium with a pH of 4.3 resulted in a shift of the fermentation from acid, to solvent formation.A preliminary report of part of this work was presented at a symposium Trends in the Biology of Fermentations for Fuels and Chemicals held December 7–11, 1980, at Brookhaven National Laboratory, Upton, New York; Gottschalk and Bahl 1981     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Continuous production of acetone and butanol by Clostridium acetobutylicum growing in turbidostat culture

Turbidostat cultures of Clostridium acetobutylicum were analysed with respect to their fermentation products after steady states were obtained at various cell densities. It was found that at low densities the fermentation of glucose was essentially acidogenic in nature, whereas acetone and butanol were the major end-products when the cultures were maintained at a high cell density.     

Enhancement of butanol production and reducing power using a two-stage controlled-pH strategy in batch culture of Clostridium acetobutylicum XY16

The effects of pH control on the process of acetone/butanol/ethanol (ABE) production in batch cultures of Clostridium acetobutylicum XY16 have been investigated. Based on the specific acid- and solvent-forming rates in batch fermentation at different pH values (from 4.3 to 6.0), a two-stage controlled-pH strategy was developed in which the pH was shifted from 5.5 to 4.9 after a dry cell weight of 0.5 g L(-1) was achieved. By applying this strategy, the maximum ABE concentration and productivity reached 20.3 g L(-1) and 0.63 g L(-1) h (-1), and were significantly improved by 12.2 and 40.1 %, respectively, compared with the process with no pH control. In addition, reducing power capability was significantly enhanced by this strategy. The two-stage controlled-pH strategy was a convenient and rapid method for high intensity ABE production.     

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.     

Growth of Nitrosomonas europaea in batch and continuous culture

Summary A clear medium has been used to grow pur cultures of Nitrosomonas europaea in flasks and in a continuous culture apparatus.Of several metallic ions examined in flask cultures of Nitrosomonas, Fe at 2 ppm and Co, Mn and Zn at 1 ppm were not toxic, Ni and Cr at concentrations greater than 0.25 ppm inhibited growth and Cu stopped growth completely at 0.5 ppm and inhibited at 0.1 ppm. Stainless steel of the specification EN58 B did not affect growth.In the continuous culture vessel, Nitrosomonas showed a growth response to Fe only when the population exceeded about 500×10⁶ organisms/ml. The minimum doubling time was about 8 hours in flasks and 11 hours in the culture vessel. With effective aeration and automatic P_H control, cultures of Nitrosomonas were grown successfully in continuous culture and gave a yield of 2.14 g dry weight of bacteria from 30 litres of culture in 5 days.     

Effect of glucose flow on the acetone butanol fermentation in fed batch culture

Summary Clostridium acetobutylicum was grown in fed-batch cultures at different feeding rates of glucose. The sugar converted to butanol and acetone increased with increasing the glucose flow, on the contrary the conversion to butyric acid was highest at slow glucose feeding rate. The acetic acid concentration was constant at the different flows of glucose. The solventogenesis was not inhibited at high flow of sugar.