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.     

Impact of pH and butyric acid on butanol production during batch fermentation using a new local isolate of Clostridium acetobutylicum YM1

The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (Y_P/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1.     

Improvements of metabolites tolerance in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.     

Extracellular glycosyl hydrolase activity of the Clostridium strains producing acetone, butanol, and ethanol

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% wheat flour medium amounted to 12.7–15 g/l with butanol constituting 51.0–55.6%. Activities of these strains towards xylan, β-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acetobutylicum 6, C. acetobutylicum 7, and C. acetobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824 in lab-scale butch cultures. It was demonstrated that starch in the medium could be partially substituted with plant biomass.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Controlled-ph batch butanol-acetone fermentation by low acid producing Clostridium acetobutylicum B18

Summary C. acetobutylicum B18 produced a large amount of butanol over a wide range of pH (4.5–6.0). At pH 6.0 fermentation and cell growth were most active at pH 6.0, and the highest values of glucose consumption rate (4.37 g/L-h), butanol productivity (1.0 g/L-h), butyric acid recycle rate (0.31 g/L-h), and cell growth rate (0.2 h^-1) were obtained. There existed a critical pH between 6.0 and 6.5 above which cells switched to organic acid producing mode. Clostridial stage appeared essential for solvent production by strain B18 but sporulation was not necessary for solvent formation.     

Enhancement of ABE fermentation through regulation of ammonium acetate and D–xylose uptake from acid-pretreated corncobs

Clostridium acetobutylicum TISTR 1462 and Clostridium beijerinckii TISTR 1461 were chosen to optimize acetone–butanol–ethanol (ABE) fermentation by using glucose as a carbon source. The enhancement in its productivity by adding various concentrations of ammonium acetate was studied. Then, the variation of glucose/xylose ratios in the pre-grown medium was investigated. The results showed that both increased ammonium acetate in the production medium and D–xylose in the pre-grown medium could produce more ABE. With these conditions, using corncob hydrolysate as a substrate, 20.58 g/L ABE was produced from C. beijerinckii TISTR 1461 with 0.44 g/L/h and 0.45 of ABE productivity and yield, respectively.     

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.     

Enhanced butanol production by optimization of medium parameters using Clostridium acetobutylicum YM1

A new isolate of the solvent-producing Clostridium acetobutylicum YM1 was used to produce butanol in batch culture fermentation. The effects of glucose concentration, butyric acid addition and C/N ratio were studied conventionally (one-factor-at-a-time). Moreover, the interactions between glucose concentration, butyric acid addition and C/N ratio were further investigated to optimize butanol production using response surface methodology (RSM). A central composite design was applied, and a polynomial regression model with a quadratic term was used to analyze the experimental data using analysis of variance (ANOVA). ANOVA revealed that the model was highly significant (p < 0.0001) and the effects of the glucose and butyric acid concentrations on butanol production were significant. The model validation experiment showed 13.82 g/L butanol was produced under optimum conditions. Scale up fermentation in optimized medium resulted in 17 g/L of butanol and 21.71 g/L of ABE. The experimental data of scale up in 5 L bioreactor and flask scale were fitted to kinetic mathematical models published in the literature to estimate the kinetic parameters of the fermentation. The models used gave the best fit for butanol production, biomass and glucose consumption for both flask scale and bioreactor scale up.     

ABE production from yellow poplar through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation

ABE (acetone-butanol-ethanol) was produced through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation using yellow poplar as a raw material. In alkaline pre-hydrolysis, 51.1% of the biomass remained as a residue. In the main woody components, the degrees of lignin and xylan removal were 94.3 and 62.0%, respectively. A yield of 80.9% for cellulose-to-glucose and 81.2% for xylan-to-xylose were obtained by enzymatic hydrolysis. The sugar composition of enzymatic hydrolysate was 95.1 g/L of glucose and 21.4 g/L of xylose. The enzymatic hydrolysate also contained 0.5 g/L of acetic acid and 0.5 g/L of total phenolics. Furfural and 5-hydroxymethylfurfural (5-HMF) were not detected in this hydrolysate. The yellow poplar hydrolysate (YPH) from enzymatic saccharification was used for the production of ABE using Clostridium acetobutylicum and C. beijerinckii. In YPH fermentation, C. acetobutylicum produced 18.1 g/L total ABE (productivity 0.38 g/L h, and yield 0.42), and C. beijerinckii produced 12.1 g/L (productivity 0.25 g/L h, and yield 0.37). Although the ABE productivity by C. beijerinckii was slightly low, the general performance of ABE fermentation in YPH was similar to or higher than those reported previously. Therefore, alkaline pre-hydrolysis could be a very effective pretreatment step prior to enzymatic hydrolysis.     

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.     

Improvement of butanol production in <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> through enhancement of NAD(P)H availability

Clostridium acetobutylicum is a natural producer of butanol, butyrate, acetone and ethanol. The pattern of metabolites reflects the partitioning of redox equivalents between hydrogen and carbon metabolites. Here the exogenous genes of ferredoxin-NAD(P)⁺ oxidoreductase (FdNR) and trans-enoyl-coenzyme reductase (TER) are introduced to three different Clostridium acetobutylicum strains to investigate the distribution of redox equivalents and butanol productivity. The FdNR improves NAD(P)H availability by capturing reducing power from ferredoxin. A butanol production of 9.01 g/L (36.9% higher than the control), and the highest ratios of butanol/acetate (7.02) and C₄/C₂ (3.17) derived metabolites were obtained in the C acetobutylicum buk^- strain expressing FdNR. While the TER functions as an NAD(P)H oxidase, butanol production was decreased in the C. acetobutylicum strains containing TER. The results illustrate that metabolic flux can be significantly changed and directed into butanol or butyrate due to enhancement of NAD(P)H availability by controlling electron flow through the ferredoxin node.     

Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation

As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.     

Characteristics of butanol fermentation by a low-acid-producing Clostridium acetobutylicum B18

Fermentation characteristics of Clostridium acetobutylicum B18 were studied in batch experiments with and without pH control. This strain is shown to be potentially useful in simultaneous acetone-butanol-ethanol fermentation-separation systems because of its low acid production. In a pH-uncontrolled batch culture this strain produced mostly solvents, including 15 g/l of butanol. Ethanol production was low. Strain B18 recycled organic acids more efficiently than other strains. In particular, butyric acid was completely recycled when glucose was not limiting. Yield of liquid products (solvents plus organic acids) and carbon recovery in total products (gas plus liquid) were 33.1–36.4 wt% and 90–91 mol%, respectively, for 20–80 g/l of initial glucose. Glucose consumption and the percentage of butanol among solvents were higher at 32°C than at 37°C. Strain B18 required approximately 0.4 g/l of undissociated butyric acid at the onset of solvent production in pH-uncontrolled batch culture. The low undissociated butyric acid requirement enabled this strain to produce 13.8 g/l of butanol at a controlled pH of 6.0.Contribution no. 19998 of the Minnesota Agricultural Experiment Station Correspondence to: C.-H. Park     

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH?=?6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L?×?h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L?×?h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.     

Metabolic engineering of Candida utilis for isopropanol production

A genetically-engineered strain of the yeast Candida utilis harboring genes encoding (1) an acetoacetyl-CoA transferase from Clostridium acetobutylicum ATCC 824, (2) an acetoacetate decarboxylase, and (3) a primary–secondary alcohol dehydrogenase derived from Clostridium beijerinckii NRRL B593 produced up to 0.21 g/L of isopropanol. Because the engineered strain accumulated acetate, isopropanol titer was improved to 1.2 g/L under neutralized fermentation conditions. Optimization of isopropanol production was attempted by the overexpression and disruption of several endogenous genes. Simultaneous overexpression of two genes encoding acetyl-CoA synthetase and acetyl-CoA acetyltransferase increased isopropanol titer to 9.5 g/L. Moreover, in fed-batch cultivation, the resultant recombinant strain produced 27.2 g/L of isopropanol from glucose with a yield of 41.5 % (mol/mol). This is the first demonstration of the production of isopropanol by genetically engineered yeast.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Influence of pH on butyrate uptake and solvent fermentation by a mutant strain of Clostridiumacetobutylicum

The effect of pH (between 4.4 and 6.6) on butyrate uptake by the mutant strain of Clostridium acetobutylicum was studied using the fermentation broth from fermentor-2 (solventogenic stage) of a two-fermentor continuous system. Low pH (?1 butyrate, under batch incubation at 30?°C, was not inhibited at pH?>?5.2, however, at pH??1. Batch incubation at relatively higher temperatures (35° and 37?°C) indicated a similar trend i.e., a pH of >5.5 was required for uptake of >8?g l^?1 butyrate. Optimization studies for butyrate uptake by C. acetobutylicum suggested a direct correlation between minimum pH and butyrate concentration or temperature. The role of undissociated butyric acid appears to be critical in regulation of butyrate uptake.     

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.     

Continuous acetone-butanol fermentation with high productivity by cell ultrafiltration and recycling

To increase the productivity of the acetone-butanol fermentation, a hollow-fiber ultrafilter is used to separate and recycle cells in a continuous fermentation ofClostridium acetobutylicum. Under partial cell recycling and at a dilution rate of 0.5 hr^–1, a cellular concentration of 20 g/l and a solvent productivity of 6.5 g/l.hr is maintained for several days at a total solvent concentration of 13 g/l.