Properties of Allyl Alcohol-Resistant Mutants of Clostridium butyricum Grown on Glycerol

Mutants of Clostridium butyricum E5 exhibiting resistance to allyl alcohol which produced the same quantities of 1,3-propanediol as the wild-type strain but more acetate than butyrate were isolated. The acetate-butyrate formation plays a major function in the regulation of the internal redox balance. Allyl alcohol resistance can be attributed not to the loss of 1,3-propanediol dehydrogenase but to a shift in the reductive properties of the enzyme. The data support the view that cellular regulation is modified to avoid intracellular accumulation of 3-hydroxypropionaldehyde.     

Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

羊源丁酸梭菌HDRyYB1发酵工艺的优化

【目的】目前,国内外鲜有关于羊源丁酸梭菌的报道。本课题选用羊源丁酸梭菌HDRy YB1为研究对象,对其发酵工艺进行优化,为该菌株作为饲料添加剂应用于畜牧业生产奠定基础。【方法】采用Plackett-Burman(PB)试验设计法和响应面法分析并优化显著影响HDRy YB1菌株发酵液中芽胞数的培养基成分。【结果】发酵培养基中的面粉浓度、鱼粉浓度和米粉浓度显著影响发酵液中的芽胞数,优化后的发酵培养基组分(质量体积比)为:面粉3.72%、鱼粉0.90%、米粉3.96%、酵母粉0.60%、Na Cl 0.19%、Mg SO4·7H2O 0.19%、KH2PO4 0.01%、Na HCO3 0.01%、Ca CO3 0.48%;培养参数为:37°C,初始p H为7.2-7.4,瓶装量100/250,接种量3%。在此条件下,HDRy YB1菌株发酵完全(18 h)的芽胞数为1.478×108 CFU/m L,是优化前的2.7倍。【结论】HDRy YB1菌株发酵培养基得到了优化,优化后的培养基可用于后期的扩大发酵试验,验证其在实践生产中的应用价值。     

Glycerol fermentation of 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat

Summary The fermentation of glycerol to 1,3-propanediol, acetate, and butyrate by Clostridium butyricum was studied with respect to growth inhibition by the accumulating products. The clostridia were grown in a pH-auxostat culture at low cell density and product concentration and near maximum growth rate. The products were then added individually to the medium in increasing concentrations and the resulting depression of growth rate was used as a quantitative estimate of product inhibition. Under these conditions growth was totally inhibited at concentrations of 60 g/l for 1,3-propanediol, 27 g/l for acetic acid and 19 g/l for butyric acid at pH 6.5. Appreciable inhibition by glycerol was found only above a concentration of 80 g/l. In a pH-auxostat without added products but with high cell density as well as in batch cultures the product proportions were different. The 1,3-propanediol concentration may approach the value of complete inhibition while the concentrations of acetic and butyric acids remained below these values by at least one order of magnitude. It was therefore concluded that 1,3-propanediol is the first range inhibitor in this fermentation.     

Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431

S. ABBAD-ANDALOUSSI, E. GUEDON, E. SPIESSER AND H. PETITDEMANGE. 1996. Glycerol catabolism by Clostridium butyricum DSM 5431 into acetate, butyrate and 1,3-propanediol (1,3-PD) was studied in chemostat culture. The fact that the intracellular concentrations of NADH (18–22 μUmol g^-1dry cell mass) were extremely high suggested that the dehydratase activity was the rate limiting step in 1,3-PD formation. This limitation was proved by the addition of propionaldehyde, another substrate of propanediol dehydrogenase, into the culture medium. This resulted in an increase in (i) glycerol utilization, (ii) biomass formation and (iii) product biosynthesis.     

Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum

Clostridium formicoaceticum ferments fructose labeled with (14)C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of (14)CO(2), acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B(12) to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO(2) to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO(2) via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.     

Fermentation of mannitol by Clostridium butyricum: role of acetate as an external hydrogen acceptor

Summary The continuous fermentation of mannitol (pH 6, dilution rate (D)=0.087 h^-1) by Clostridium butyricum LMG 1213t1 was investigated under several conditions. Mannitol was readily fermented when glucose or acetate were added in the in-flow medium as co-substrate. Butyrate, CO₂ and H₂ were the major fermentation products. In mannitol-glucose mixtures (ratios 4 or 8) the amount of mannitol fermented depended upon the amount of glucose in the in-flow medium. In mannitol-acetate mixtures, 1 mol of acetate was needed for the fermentation of approximately 5.5 mol mannitol. We detected d-mannitol-1-phosphate dehydrogenase activity, responsible for the generation of supplementary reduced nicotine adenine dinucleotide (NADH) as a source for extra H₂ gas. Fermentation of mannitol-acetate in the presence of [¹⁴C]-labelled acetate revealed butyrate as the only labelled fermentation end-product.     

Evidence of Leu-Val-Leu Hydrolysis by Clostridium butyricum

The ability of Clostridium butyricum cultures in synthetic medium to hydrolyse the tripeptide Leu-Val-Leu was demonstrated using gas chromatography coupled with mass spectrometry. Electrospray mass spectrometry was used to confirm this unknown activity for this C -heterotrophic species listed saccharolytic, non-proteolytic clostridia. The oligopeptide hydrolysis activity is characterised by the detection of the released amino acids and the products of their bioconversion, the corresponding 2-hydroxyacids. This observation opens new aspects in the study of C. butyricum and raised questions about the peptide metabolism by this species. The enzymes implied in the oligopepitide hydrolysis can be regarded as potential virulence factors and being an element in the comprehension of certain pathologies.     

Cleavage of L-leucine-containing dipeptides by Clostridium butyricum

The ability of Clostridium butyricum cultures to hydrolyze three L-leucine-containing dipeptides (Leu-Leu, Leu-Gly and Gly-Leu) in a synthetic minimal medium is demonstrated by using gas chromatography coupled with mass spectrometry. The ¹³C nuclear magnetic resonance and a labeled dipeptide L-[1-¹³C]Leu-Gly were used to confirm this activity.     

Mutagenesis of Clostridium butyricum

Mutagenesis of Clostridium butyricum ATCC 19398 was best accomplished by u. v. irradiation (254 nm) and methyl methane sulphonate. Nitrosoguanidine was only slightly mutagenic for this organism. Procedures are described which yielded a variety of auxotrophic and antibiotic-resistant mutant strains.     

Fermentation of native and processed starches by the porcine caecal anaerobe Clostridium butyricum (NCIMB 7423)

Starch fermentation by the porcine caecal anaerobe Clostridium butyricum was examined using gas and volatile fatty acid production as determinants of activity. Potato starch and amylopectin were studied in their native form as well as after retrogradation, which should render them resistant to pancreatic α-amylase digestion. Fermentation of both substrates was enhanced by pancreatin digestion of the native material, possibly due to the removal or disruption of part of the structure of the starch by the pancreatic enzymes. However, pancreatic digestion of retrograded potato starch apparently reduced the amount available for bacterial fermentation, whereas no significant effect was observed with amylopectin. The data suggests that starches which are high in amylopectin would be more likely to influence fermentation in the large intestine in monogastric animals, and that the presence of residual pancreatic enzymes in the lower gut could potentially enhance starch fermentation by this micro-organism.     

Photoreactivating capacity in Clostridium butyricum and Clostridium acetobutylicum

No difference in survival was observed when u.v.-irradiated clostridial cells were subsequently incubated in the dark or exposed to photoreactivating light. This suggests that photoreactivation does not occur in Clostridium butyricum and in Clostridium acetohutylicum.     

原材料对甘油发酵影响的研究

以木薯干片,木薯淀粉,玉米淀粉的水解糖为原料,采用好氧深层发酵法进行甘油发酵。以木薯干片为原料时,甘油产率最高为１１－１２％,全糖转化率为４７－４９％,发酵周期为６０－６６ｈ;而以玉米淀粉为原料,甘油产率仅８－９％,全糖转化率３８－４０％,发酵周期却需７２小时以上     

Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate

The levels of seven intermediary enzymes involved in acetate and butyrate formation from acetyl coenzyme A in the saccharolytic anaerobe Clostridium acetobutylicum were investigated as a function of time in solvent-producing batch fermentations. Phosphate acetyltransferase and acetate kinase, which are known to form acetate from acetyl coenzyme A, both showed a decrease in specific activity when the organism reached the solvent formation stage. The three consecutive enzymes thiolase, β-hydroxybutyrylcoenzyme A dehydrogenase, and crotonase exhibited a coordinate expression and a maximal activity after growth had ceased. Only low levels of butyryl coenzyme A dehydrogenase activity were found. Phosphate butyryltransferase activity rapidly decreased after 20 h from 5 to 11 U/mg of protein to below the detection limit (1 mU/mg). Butyrate no longer can be formed, and the metabolic flux may be diverted to butanol. Butyrate kinase showed a 2.5- to 10-fold increase in specific activity after phosphate butyryltransferase activity no longer could be detected. These results suggest that the uptake of acetate and butyrate during solvent formation can not proceed via a complete reversal of the phosphate transferase and kinase reactions. The activities of all enzymes investigated as a function of time in vitro are much higher than the metabolic fluxes through them in vivo. This indicates that none of the maximal activities of the enzymes assayed is rate limiting in C. acetobutylicum.     

Solvent production by an aggregate-forming variant of Clostridium butyricum

Summary When an aggregate-forming variant of Clostridium butyricum was grown in continuous culture under glucose-limited conditions (pH 5.2, dilution rate = 0.1 h^–1, 30° C), glucose was mainly fermented to isopropanol and butanol. Production of solvents was pH dependent and initiated above a threshold concentration of acetic and butyric acid, which probably means that it was triggered by the undissociated concentration of these acids. This was confirmed by studies with a second reactor. Solvent productivity by this variant was found to be stable and the highest ever reported for Clostridium sp. in argued that, due to the aggregated growth mode, this variant is able to reach the threshold concentration of acids at which solventogenesis is initiated at high dilution rates.Offprint requests to: G. R. Zoutberg     

Production of 1,3-Propanediol from Glycerol by Clostridium acetobutylicum and Other Clostridium Species

Glycerol was fermented with the production of 1,3-propanediol as the major fermentation product by four strains of Clostridium acetobutylicum, six of C. butylicum, two of C. beijerinckii, one of C. kainantoi, and three of C. butyricum. 1,3-Propanediol was identified by its retention times in gas chromatography and high-pressure liquid chromatography and by its mass spectrum. During growth of C. butylicum B593 in a chemostat culture at pH 6.5, 61% of the glycerol fermented was converted to 1,3-propanediol. When the pH was decreased to 4.9, growth and 1,3-propanediol production were substantially reduced.