Fermentative production of succinic acid from glucose and corn steep liquor byAnaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production. It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD₆₆₀ of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

Improvement of glycerol production by Candida krusei in batch and continuous cultures using corn steep liquor

No fermentation parameter was affected at phosphate concentration above 0.4 g l^–1 when KH₂PO₄ was used as phosphate source and the glucose consumption rate was difficult to control when corn steep liquor (CSL) was adopted as the phosphate source. However, if CSL was supplemented as a source of growth factors instead of as the phosphate source, not only glucose uptake and glycerol was improved, but also fermentation became easy to control and a steady state of continuous culture was easily obtained.     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli

AIM: Development and optimization of an efficient and inexpensive medium for succinic acid production by Escherichia coli under anaerobic conditions. METHODS AND RESULTS: Initially, 0.8 gl(-1) of succinic acid was produced in 60 h in 300-ml medium. On optimization, glucose and peptone were replaced by cane molasses and corn steep liquor. Three hundred ml of this medium was inoculated with 4% (v/v) of seed inoculum, incubated at 39 degrees C for 72 h, resulted in 7.1 gl(-1) of succinic acid in 36 h. Scale up in a 10-l fermentor under conditions of controlled pH and continuous CO2 supply in this medium resulted in 17 gl(-1) of succinic acid in 30 h. CONCLUSIONS: A ninefold increase in succinic acid production was obtained in 500-ml anaerobic bottles with optimized medium having cane molasses and corn steep liquor as against initial medium containing glucose and peptone. However, a subsequent scale up in a 10-l fermentor resulted in a 2.5-fold increase in succinic acid production as against optimized medium used in 500-ml anaerobic bottles. SIGNIFICANCE AND IMPACT OF THE STUDY: Succinic acid production was enhanced in medium consisting of inexpensive carbon and nitrogen sources in a shorter span of time.     

Anaerobic fermentative production of lactic acid using cheese whey and corn steep liquor

Cheese whey was the most suitable substrate for production of lactic acid under anaerobic conditions by Entercoccus flavescens which, on supplementating with corn steep liquor (5% v/v) and 10 mM CaCO₃ at pH 5.5, 37°C, yielded 12.6 g lactic acid/l in 36 h. Production was scaled up to a 10 l bioreactor under controlled pH and continuous CO₂ supply and gave 28 g lactic acid/l in 30 h resulting in a net 8.7-fold increase in production as compared to unoptimized conditions.     

Brewer's spent grain and corn steep liquor as substrates for cellulolytic enzymes production by Streptomyces malaysiensis

Aims:  To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme.
Methods and Results:  Maximum cellulase production by Streptomyces malaysiensis (720 U l⁻¹) occurred within 4 days incubation when using a growth medium containing BSG 0·5% (w/v) and CSL1·2% (w/v). CMCases activity showed to be stable over an acidic pH range (2·0–7·0) and in temperatures of 40–60°C. Zymogram indicated three bands of CMCase activity, with different molecular masses.
Conclusion:  S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas.
Significance and Impact of the Study:  To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.     

以玉米浆和木薯为原料机械搅拌发酵制备细菌纤维素的研究

本文以玉米浆和木薯为原料,用机械搅拌式发酵罐制备细菌纤维素（BC）,对发酵过程的纤维素产量、还原糖消耗、溶氧变化和茵浓变化进行了监测,并以葡萄糖一蛋白胨-酵母粉培养基为对照进行了比较。实验得出玉米浆作氮源时不溶BC的产量为9．2g／L,而氮源成本只是对照组的15％;木薯水解液作碳源时的不溶BC产量达到11．7g／L,比对照组（10．8g／L）高8％;而用玉米浆搭配木薯水解液发酵生产BC,产量也达到10．1g／L,验证了这两种天然原料的廉价高效性,用于工业生产细菌纤维素具有良好的前景。     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract

The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more ethanol (1.7 g L⁻¹) was produced with 20 g L⁻¹ CSL media in 250-mL bottle fermentations compared to media with 1 g L⁻¹ YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a 7.5-L fermentor with 10 and 20 g L⁻¹ CSL media were 8.6 and 9.6 g L⁻¹, respectively, which represent 57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L⁻¹ of ethanol was obtained in the medium with 1 g L⁻¹ YE after 360 h, which represents 53% of the theoretical ethanol yield from CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermentations. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11.     

Batch and continuous cultures of<Emphasis Type="Italic"> Mannheimia succiniciproducens</Emphasis> MBEL55E for the production of succinic acid from whey and corn steep liquor

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce a large amount of succinic acid in a medium containing glucose, peptone, and yeast extract. In order to reduce the cost of the medium, whey and corn steep liquor (CSL) were used as substrates for the production of succinic acid by M. succiniciproducens MBEL55E. Anaerobic batch cultures of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in the production of succinic acid with a yield of 71% and productivity of 1.18 g/l/h, which are similar to those obtained in a whey-based medium containing yeast extract (72% and 1.21 g/l/h). Anaerobic continuous culture of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in a succinic acid yield of 69% and a succinic acid productivity as high as 3.90 g/l/h. These results show that succinic acid can be produced efficiently and economically by M. succiniciproducens MBEL55E from whey and CSL.     

Effects of corn steep liquor on production of 2,3-butanediol and acetoin by Bacillus subtilis

The initial concentration of corn steep liquor (CSL) have remarkable effects on not only 2,3-butanediol (2,3-BD) and acetoin (metabolic precursor) production, but also on the ratio of 2,3-BD to acetoin. When a high concentration of CSL was supplemented, cell growth was improved, acetoin reductase (ACR) was stimulated, the concentration of 2,3-BD increased by 78.6%, acetoin decreased by 61.9%, and the ratio of 2,3-BD to acetoin increased by 3.69-fold. The acr gene, encoding ACR, was over-expressed in Bacillus subtilis. Compared to the control (parent strain), low levels of CSL in the engineered strain increased 2,3-BD concentration and the ratio 2,3-BD to acetoin by 13.9% and 39.5%, respectively, and decreased acetoin titer by 18.3%. Acetoin became a major product under low levels of CSL. Also, a knockout strain carrying an acr::cat insertion mutation was constructed. As expected, the loss of ACR activity led to an accumulation of acetoin in the supernatants of acr:: cat mutant cultures. Additionally, the productivity of acetoin was improved by high concentration of CSL. The results above demonstrate the feasibility of using B. subtilis for the production of not only 2,3-BD but also acetoin as a major product.     

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO₂ into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO₂ sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO₂ sparging.The authors are with the Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860, Japan     

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.     

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.     

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mol·min^-1·mg^-1) of FTHFS in cell free extracts of CRL47 were 28–89 when assayed at 50°C and pH8. This was from 3–10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.Abbreviations FTHFS formyltetrahydrofolate synthetase - kb kilobase - H₄-folate tetrahydrofolate - SDS sodium dodecyl sulfate A preliminary account of this work was presented at the annual meeting of the American Society for Microbiology, Atlanta, GA, 1987 (C. R. Lovell, A. Przybyla and L. G. Ljungdahl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, K126, p. 223).     

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.