Kinetic Analysis of Bifidobacterial Metabolism Reveals a Minor Role for Succinic Acid in the Regeneration of NAD+ through Its Growth-Associated Production

Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD⁺, in addition to the regeneration through the production of lactic acid and ethanol.     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.     

In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F2 and F3) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F4, GF4, F5, GF5, and F6) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.     

In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria

In this Study the effects of both pH and organic acids on Helicobacter pylori NCTC 11637 were tested. Lactobacillus acidophilus, Lact. casei, Lact. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were assayed for their lactic acid production, pH and inhibition of H. pylori growth. A standard antimicrobial plate well diffusion assay was employed to examine inhibitory effects. Lactic, acetic and hydrochloric acids demonstrated inhibition of H. pylori growth in a concentration-dependent manner with the lactic acid demonstrating the greatest inhibition. This inhibition was due both to the pH of the solution and its concentration. Six strains of Lact. acidophilus and one strain of Lact. casei subsp. rhamnosus inhibited H. pylori growth where as Bifidobacterium bifidus, Ped. pentosaceus and Lact. bulgaricus did not. Concentrations of lactic acid produced by these strains ranged from 50 to 156 mmol 1⁻¹ and correlated with H. pylori inhibition. The role of probiotic organisms and their metabolic by-products in the eradication of H. pylori in vivo remains to be determined.     

Improvement of ethanol production of themophilic Clostridium sp. by Mutation

Three thermophilic Clostridium strains were isolated from soil as cellulose-fermenting bacteria wich produced ethanol, lactic acid, and acetic acid from cellulose at 60°C. To increase ethanol productivity, strains no. 187 was mutated with N-methyl-N′-nitro-N-nitrosguanidine. The resultant mutant, no 187-102-27, was superior to the original strain in ethanol production, and produced less lactic and acetic acid. The activities of some enzymes involved in the biosynthesis of the lactic and acetic acid of mutant no. 187-102-27 were lower than those of the original strain. These results are highly consistent with the acid production of the mutant strain being low.     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

Quantitative gas chromatography of Bacteroides species under different growth conditions

From 56 strains of strictly anaerobic gram-negative rods isolated from stool and purulent lesions the fermentation products in the presence and absence of hemin were determined by quantitative gas-solid chromatography, using a simple and more rapid chromatographic procedure. With hemin the fermentation products were propionic, acetic, lactic and succinic acid. Without hemin no or little succinic acid was formed and the main products were lactic and acetic acid. In both groups the distribution of subspecies was determined and the production of fatty acids measured quantitatively.Fourteen strains of the lesion group showed a higher metabolic activity, resulting in an increased total acid production caused by an excessive production of acetic and lactic acid. This characteristic is probably a virulence factor in these strains.All strains were protoporphyrin- and oxgall-dependent. It is postulated that these substances are used for the production of cytochromes which permits the formation of succinic acid by a fumarate reductase resulting in an increased growth rate and growth yield.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Thin layer chromatographic determination of organic acids for rapid identification of bifidobacteria at genus level

This study presents a simple and fast method for the identification of bifidobacteria using a thin layer chromatographic (TLC) analysis of the short chain fatty acids in a culture broth. When the chromatogram was sprayed with the indicator solution (methyl red-bromophenol blue in 70% ethanol), lactic acid exhibited two red spots, and acetic acid, propionic acid, and butyric acid all produced blue spots. Succinic acid and citric acid produced yellow and dark yellow spot, respectively. In addition, these organic acids showed different R(f) values. The total time taken to analyze the organic acids in the 10 bacterial culture broths using the proposed method was approximately 50 min. The proposed TLC method was used to analyze the organic acids in culture broths of the following strains, five Bifidobacterium species. (Bifidobacterium longum, B. breve, B. infantis, B. bifidum, and B. adolescentis) and five other lactic acid bacteria strains (Lactobacillus casei, L. bulgaricus, L. acidophilus, Streptococcus thermophilus, and S. lactis). Both spots of lactic acid and acetic acid were detected on all the TLC plates from the five bifidobacterial culture broths. The five other lactic acid bacterial culture broths, however, only exhibited lactic acid spots. Accordingly, the proposed TLC method would appear to be a useful tool for rapid identification of Bifidobacterium spp. at the genus level.     

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.     

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.     

Comparison of Ethanol Production by Different Zymomonas Strains

A comparison of the rates of growth and ethanol production by 11 different strains of Zymomonas revealed a wide range of characteristics, with some strains being more tolerant of high sugar or ethanol concentrations and high incubation temperatures than others. Some strains were unable to utilize sucrose; others produced large amounts of levan, and one strain grew well but produced no levan. One strain, CP4, was considerably better in all respects than most of the other strains and was chosen as a starting strain for genetic improvement of ethanol production.     

Performance of Rhizopus,Rhizomucor, and Mucor in ethanol production from glucose,xylose, and wood hydrolyzates

In searching for ethanol producing microorganisms also capable of fermenting pentoses, nine zygomycetes strains including three strains of Rhizopus oryzae, Mucor corticolous, M. hiemalis, M. indicus, Rhizomucor pusillus, R. miehei, and zygomycete IT were examined. Each strain was cultivated on glucose, xylose or dilute-acid hydrolyzate (DAH) as carbon sources, and the production of ethanol, lactic acid, glycerol, xylitol, and succinic acid were investigated. Great similarities but also conspicuous differences were seen between the species, to some extent linked to the genera. All strains were capable of growing on glucose or xylose as single carbon source. With the exception of the two Rhizomucor strains, all produced ethanol. All the strains produced glycerol as by-product, while Rhizopus and Rhizomucor but not Mucor produced lactic acid in significant amounts. All Mucor and Rhizopus strains and one strain of Rhizomucor produced xylitol in the xylose medium, but no xylitol was detected after growth on DAH. All Mucor and two R. oryzae strains were capable of growing on DAH. Two Mucor species, M. hiemalis and M. indicus showed greater ethanol production than the other strains. The ethanol yields by M. hiemalis on glucose, xylose, and DAH were 0.39, 0.18, and 0.44 g/g, respectively, whereas the corresponding results for M. indicus were 0.39, 0.22, and 0.44 g/g. The strains also rapidly consumed hydroxymethyl furfural present in DAH.     

Candida krusei produces ethanol without production of succinic acid; a potential advantage for ethanol recovery by pervaporation membrane separation

The development of fermentative yeasts secreting no organic acids is highly desirable for ethanol production coupled with membrane separation processes, because the acidic byproduct, succinic acid, significantly inhibits the membrane permeation of ethanol. Of the Pichia and Candida yeasts tested, Candida krusei IA-1 showed the highest ethanol productivity [55 g L(-1) day(-1) from 150 g L(-1) (w/v) of glucose], comparable to the strains of Saccharomyces cerevisiae, and produced much less of the acid (0.6 g L(-1) day(-1)) than the Saccharomyces strains (1.5-1.8 g L(-1) day(-1)) under semi-aerobic conditions. Interestingly, under aerobic conditions, strain IA-1 showed no production of the acid. Stain IA-1 exhibited a good assimilation of the acid, while S. cerevisiae NBRC 0216 showed no assimilation. The activity of succinate dehydrogenase (SDH) in strain IA-1 was 37.5 mU mg(-1), and 7.8-fold higher than that in S. cerevisiae strain NBRC 0216. More significantly, SDH1 was abundantly transcribed in strain IA-1, different from that in strain NBRC 0216, regardless of the culture conditions. From these results, C. krusei IA-1 efficiently takes up succinic acid and metabolizes it in the Krebs cycle, producing an extremely low level of byproducts in the culture medium. Therefore, C. krusei is not only a promising alternative to S. cerevisiae but also a suitable model for metabolic engineering of S. cerevisiae.     

Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds

Synthetic mixtures of predominant lignocellulosic hexose sugars were supplemented with separate aliquots of three inhibitory compounds (furfural, hydroxymethylfurfural (HMF), and acetic acid) in a series of concentrations and fermented by the spent sulfite liquor (SSL)-adapted yeast strain Tembec T1 and the natural isolate Saccharomyces cerevisiae (S. cerevisiae) Y-1528 to compare tolerance and assess fermentative efficacy. The performance of Y-1528 exceeded that of Tembec T1 by a significant margin, with faster hexose sugar consumption, higher ethanol productivity, and in the case of furfural and HMF, faster inhibitor consumption. Nevertheless, furfural had a dose-proportionate effect on sugar consumption rate and ethanol productivity in both strains, but did not substantially affect ethanol yield. HMF had a similar effect on sugar consumption rate and ethanol productivity, and also lowered ethanol yield. Surprisingly, acetic acid had the least impact on sugar consumption rate and ethanol productivity, and stimulated ethanol yield at moderate concentrations. Sequential iterations of softwood (SW) and hardwood (HW) SSL were subsequently inoculated with the two yeast strains in order to compare adaptation to, and performance in lignocellulosic substrates in a cell recycle batch fermentation (CRBF) regime. Both strains were severely affected by the HW SSL, which was attributed to specific syringyl lignin-derived degradation products and synergistic interactions between inhibitors. Though ethanologenic capacity was preserved, a net loss of performance was evident from both strains, indicating the absence of adaptation to the substrates, regardless of the sequence in which the SSL types were employed.     

Strain variation in Hymenolepis diminuta: enzyme profiles

In comparative study of respiratory metabolism, it was established that the relative proportions of respiratory end-products (succinic, acetic and lactic acids) differed consistently in two strains of Hymenolepis diminuta (Toronto and ANU). The ANU strain produced more lactic acid and less succinic acid under aerobic and anaerobic conditions. In the shift from aerobic to anaerobic conditions both strains compensated by increasing their outputs of succinic acid. The ANU strain possessed significantly higher activities of hexokinase, pyruvate kinase, lactate dehydrogenase, cytosolic and mitochondrial malic enzyme and cytosolic α-glycerophosphate dehy drogenase. The Toronto strain had significantly higher activities of fumarase, succinate dehydrogenase, and fumarate reductase. There were no significant differences in the activities of phosphoenolpyruvate carboxykinase and malic dehydrogenase between strains. The fumarase activity in the Toronto strain was 16 times that of the ANU strain, its K_m (malate) was 0.8mM, as opposed to 2.5 mM, and it was less sensitive to inhibition by NAD or ATP. These observations are consistent with the patterns of end-product formation in the two strains. Ratios of end-products and calculations of approximate redox balance suggest that the Toronto strain may have a greater capacity for aerobic metabolism.     

Prevention of gut inflammation by Bifidobacterium in dextran sulfate-treated gnotobiotic mice associated with Bacteroides strains isolated from ulcerative colitis patients

Indigenous Bacteroides strains are closely associated with the occurrence and exacerbation of ulcerative colitis (UC). In this study, we aimed to clarify the effect of Bifidobacterium strains, another major member of colonic bacteria, on the development of gut inflammation using gnotobiotic mouse models associated with Bacteroides strains isolated from UC patients. Dextran sulfate (DSS) administration induced inflammation in the large intestine, in particular of the cecum, in the gnotobiotic mice associated with three strains of Bacteroides vulgatus, judging from the myeloperoxidase activity, occult blood score, and IgG leakage into the intestinal contents. However, the severity of the inflammation was greatly reduced in the gnotobiotic mice associated with both B. vulgatus and Bifidobacterium strains. The severity of the cecal inflammation was well correlated with the concentration of succinic acid in the cecum. Bacteriologically, the density of B. vulgatus strain A (BV-A) greatly decreased and the predominant strain changed from BV-A to BV-B on additional association with Bifidobacterium strains. Among gnotobiotic mice associated with a single B. vulgatus strain, the severity of cecal inflammation in BV-A-associated mice was greater than that in BV-B-associated mice. Each Bifidobacterium strain produced compound(s) more effectively inhibiting the growth of BV-A than BV-B in in vitro culture. Taken together, these results suggest that the severity of DSS-induced gut inflammation is closely associated with a particular B. vulgatus strain, and that Bifidobacterium strains may repress exacerbation of intestinal inflammation through growth inhibition of the B. vulgatus strain.     

High levels of malic acid production by the bioconversion of corn straw hydrolyte using an isolated Rhizopus delemar strain

The microbial fermentation of malic acid, which is one of the most important organic acid platforms used widely in food and chemical engineering, has attracted considerable interest. A malate production strain was isolated, a mutation was induced, and regulation of the metabolic network was then conducted. The identification results showed that the malic acid production strain, HF- 119, belonged to Rhizopus delemar. An analysis of the metabolic pathway showed that the malic acid flux of this strain occurred through three main pathways, and many byproducts, such as succinic acid, fumaric acid and ethanol, were produced. Although corn straw hydrolyte was used, the metabolism of xylose was not as rapid as that of glucose. Subsequently, breeding of the strains and regulation of the metabolic network resulted in an increase in malate yield, and the strain HF-121 produced more than 120 g/L malic acid within 60 h. The ability to produce malic acid from biomass hydrolyte highlights the industrial development potential of this strain.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.