Effect of growth supplements and whey pretreatment on butyric acid production by Clostridium butyricum

Improved production of butyrate (up to 19 g/l) from whey by Clostridium butyricum was achieved by adding either yeast extract (5 g/l) or biotin (50 g/l). Hydrolysed lactose and proteolysed whey were less effective even with added biotin.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, Radlinského 9, Bratislava 812 37, Slovakia     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

Effect of controlled substrate feeding on butyric acid production byClostridium tyrobutyricum

Summary Production of butyric acid from wheat flour hydrolysate was studied withClostridium tyrobutyricum. The mode of substrate supply was found a key parameter for fermentation performance as large improvements were obtained by feeding with a non-limiting supply of substrate. With this procedure, increases in product concentration and productivity but also in selectivity and yield for butyrate were obtained. Substrate feeding controlled by the rate of gas production was found preferable to constant rate feeding for reason of convenience and flexibility. In these conditions, a butyrate concentration of 62.8 gl^–1 was obtained with a productivity of 1.25 gl^–1 h^–1, a selectivity of 91.5% and a yield of 0.45 g per g of glucose.     

Influence of acetic and butyric acid addition on polysaccharide formation byClostridium acetobutylicum

Summary The production of granulose (an intracellular reserve polygranule), capsule and exopolysaccharide was investigated in a synthetic medium in which the oxido-reduction level was modified by the addition of acetic or butyric acid. After addition of the acids, granulose synthesis increased from 150 to 300 mg glucose equivalents ·1^–1 and capsular synthesis decreased by 25%. Exopolysaccharide production was unchanged under these conditions. A hypothesis that attributes a role to the polymer in the oxido-reduction sequences is discussed.     

Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus

Summary Investigations have been carried out on lactic acid production by Lactobacillus helveticus CNRZ 303 in whey ultrafiltrate. Addition of beet molasses was investigated with good results, although yeast extract proved to be more effective. The size of inoculum and the preculture medium also played a significant role in determining the amount of lactic acid produced during the fermentation process. High lactose consumption (94.09%), together with good lactic acid production (26.09 g/l) and yield (0.90%), were obtained in whey ultrafiltrate supplemented with 1% (w/v) beet molasses (WUM), with a 10% (w/v) inoculum and peptonized milk as preculture medium. Although these results were similar to those obtained when yeast extract was used as supplement, the maximum volumetric productivities proved to be quite different, and were definitely higher with yeast extract. Offprint requests to: L. Chiarini     

Influence of different vitamin-nitrogen sources on cell growth and propionic acid production from sucrose by Propionibacterium shermanii

Cell growth and organic acid production by Propionibacteria are dependent on the vitamin-nitrogen source in the culture medium. Final cell and propionic acid concentrations produced by Propionibacterium shermanii, using corn-steep liquor, were higher than those obtained utilizing yeast extracts. Since corn-steep liquor is much cheaper than yeast extract, the process becomes more attractive. By calculating the specific growth rates, it was observed that the critical propionic acid concentration, that prevents all growth (μ_X = 0), is different depending on the vitamin-nitrogen source used and its concentration. For example, for 5.0 and 15.0 g/l Oxoid yeast extract, those critical propionic acid concentrations were 16.0 and 27.0 g/l, respectively. Such propionic acid concentrations inhibit the cell growth, but not the formation of acid. The specific propionic acid production rate also indicates that the critical concentration for metabolic activity, when propionic acid is no longer produced (μ_P = 0), varies according to the vitamin-nitrogen source and its concentration in the medium. For 5.0 and 15.0 g/l Oxoid yeast extract, those concentrations were 22.1 and 30.1 g/l, respectively.     

H2 production from chemostat fermentation of glucose byClostridium butyricum andClostridium pasteurianum in ammonium- and phosphate limitation

Summary In ammonium-limitation (4.55 mM NH₄ ⁺) at a dilution rate (D)=0.081 h^–1,Clostridium butyricum produced 2 mol H₂ per mol glucose consumed at pH 5.0, but at a low fermentation rate. At higher pH, important amounts of extracellular protein were produced. Phosphatelimitation (0.5 mM PO₄ ^–3) at D=0.061 h^–1 and pH 7.0 were the best conditions tested for hydrogen gas production (2.22 mol H₂ per mol glucose consumed) at a high fermentation rate. Steady-state growth at lower pH and with 0.1 mM PO₄ ^–3 resulted in proportional higher glucose incorporation into biomass and lower H₂ production. C. pasteurianum in NH₄ ⁺ limitation showed higher fermentation rates thanC. butyricum and a stabilized H₂ production around 2.08 (±0.06) mol per mol glucose consumed at various defined pH conditions, although the acetate/butyrate ratio increased to 1 at pH 7.0. The latter was also observed in phosphate-limitation, but here H₂ production was maximal (1.90 mol. per mol glucose consumed) at the lowest pH (5.5) tested.     

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta⁻, buk⁻, ctfB⁻ and adhE1⁻) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta⁻, buk⁻, ctfB⁻, adhE1⁻ and hydA⁻) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.     

Biosynthesis of butyric acid from cabbage stem and molasses by the strain Clostridium butyricum VKPM B-9619

The acidogenesis of the strain Clostridium butyricum VKPM B-9619 on synthetic medium SOL, a fermentative hydrolyzate of cabbage stem, and molasses as cheap sources of carbohydrate nutrition of micro-organisms, has been studied. The yield of butyric acid was no less than 43%. It was found that, in addition to hexoses, the strain ferments pentoses (xylose and arabinose), which enables the processing of wastes of pentosan- and cellulose-containing plant raw materials with the addition of molasses. The fermentation liquid of the strain can be used for the isolation of butyric acid by the ion exchange method.     

Advances in biotechnological production of butyric acid

The review is focused on several aspects of butyric acid production: butyric acid-producing bacterial strains, the characteristics of the genus Clostridium (the bacterium most used for butyrate production), and alternative methods of obtaining butyric acid by alcohol biotransformation. Further, the main metabolic pathways of butyrate production, and possibilities for their control are outlined. Batch, fed-batch or continuous fermentation combined with cell recycle or immobilization are applicable for anaerobic fermentations using Clostridium as the production strain. The best process comprises a combination of high cell concentration and slowly growing biomass, in addition to high production selectivity and low inhibitory effects of the end-product. Inhibitory effects may be avoided by on-line removal of the end-product. Extraction alone or extraction combined with simultaneous stripping of the organic phase (liquid membrane) into the second aqueous phase (pertraction) seem to be the most suitable methods for on-line butyrate removal. The biocompatibility and the distribution coefficient of the organic phase under fermentation conditions should be considered before designing a fermentation apparatus. Journal of Industrial Microbiology & Biotechnology (2000) 24, 153–160. Received 12 August 1999/ Accepted in revised form 03 December 1999     

Effect of iron on growth and toxin production byClostridium botulinum type A

The kinetics of growth and toxin production by the Hall strain ofClostridium botulinum type A was examined in the presence of various concentrations of iron (0.1 to 10.1 g/ml, 1.8 to 182 M) in a chemically defined medium. At concentrations below 0.5 g/ml, iron insufficiency limited the growth of the organism. The maximum amount of toxin produced varied by only twofold (6×10⁵ to 1.2×10⁶ mouse median lethal doses/ml per A₅₄₀ unit) over the 100-fold range of iron concentrations used. High concentrations of iron did not reduce the elaboration of botulinum toxin, in contrast with its marked inhibitory effects on the production of many bacterial toxins. Iron is unlikely to be a regulatory effector for the formation of botulinum toxin by the Hall strain of type A.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

Effect of pH on organic acid production byClostridium propionicum in test tube and fermentor cultures

Summary Clostridum propionicum is a chemical autotroph that metabolizes alanine to propionic acid (reduction product) and acetic acid (oxidation product). The ratio of propionate/acetate predicted by the electron balance is 2:1. This study reports the effect of pH on growth and organic acid production by this organism when grown in both test tube cultures initially buffered from pH 7.0 to 5.0, and in fermentors maintained at pH 7.0 and 6.5. Highest growth and organic acid production was found at pH 7.0 in both cases. HPLC analysis showed that at pH 7.0, the ratios of propionate to acetate were 0.45:1 (stationary tube, 24 h). The highest ratio observed was 1.8:1 (stationary tube, pH 6.0, 24h). This tube produced 8.5% of the acids produced in the pH 7.0 culture tube. The identify of the major portion of the reduction products of the organism remains unknown.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

The effect of novobiocin on solvent production byClostridium acetobutylicum

Cells ofClostridium acetobutylicum treated with novobiocin, a DNA gyrase inhibitor, produced higher butyrate levels and lower solvent levels with acetone being the most affected. Seven enzyme activities involved in acid and solvent production were analyzed. Among them, only CoA transferase, required for acetone formation and acid uptake, experienced a significant decrease in activity. As inEscherichia coli andBacillus subtilis, DNA fromC. acetobutylicum became less negatively supercoiled in the early stationary phase (solventogenic stage), as shown by analysis of linking number of a reporter plasmid by agarose gel electrophoresis in the presence of chloroquine.     

Itaconic acid production by immobilizedAspergillus terreus on sucrose medium

Summary The itaconic acid production by immobilizedAspergillus terreus TTK 200-5-3 mycelium was optimized in shake flask fermentations using statistical experimental design and empirical modelling. The maximum itaconic acid concentration was calculated to be 13.3 g/l in the investigated experimental area when initial sucrose concentration was 10%, ammonium nitrate concentration 0.275% and initial pH 3. The itaconic acid product concentration using immobilized mycelium was about double of that obtained with the free mycelium.     

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.     

Influence of pH and undissociated butyric acid on the production of acetone and butanol in batch cultures of Clostridium acetobutylicum

Summary The kinetics of growth and acid and solvent production are examined in batch fermentation of Clostridium acetobutylicum at pH between 4.5 and 6.0. At the lower pH, growth occurs in two consecutive phases and solvents are the main excreted metabolites. At the higher pH, there is a single growth phase with only acid formation. The influence of the pH can be correlated with a critical role of the concentration of undissociated butyric acid in the medium: cellular growth is inhibited above 0.5 g/l and solvent production starts at an undissociated acid level of 1.5 g/l. Reducing the intracellular acid dissociation by lowering the intracellular pH also favours the production of acetone and butanol.