Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

Syntrophic Association by Cocultures of the Methanol- and CO(2)-H(2)-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H(2)-CO(2), and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO(2), and H(2) from pectin. The coculture produced one-third more acetate, and butyrate and CO(2) were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

Syntrophic Association by Cocultures of the Methanol- and CO2-H2-Utilizing Species Eubacterium limosum and Pectin-Fermenting Lachnospira multiparus During Growth in a Pectin Medium

Lachnospira multiparus grew very well in an anaerobic 0.2% pectin medium, whereas Eubacterium limosum, which utilizes methanol, H₂-CO₂, and lactate, did not. Cocultures of the two species grew at a somewhat more rapid growth rate than did L. multiparus alone and almost doubled the amount of growth as measured by optical density. In model experiments with cultures transferred once a day with a 2-day retention time, L. multiparus produced mainly acetate, methanol, ethanol, formate, lactate, CO₂, and H₂ from pectin. The coculture produced one-third more acetate, and butyrate and CO₂ were the only other significant end products. The results are discussed in relationship to microbial metabolic interactions and interspecies hydrogen transfer.     

Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium.

A defined medium with glucose as the carbon source was used to quantitatively determine the metabolic end products produced by Listeria monocytogenes under aerobic and anaerobic conditions. Of 10 strains tested, all produced acetoin under aerobic conditions but not anaerobic conditions. Percent carbon recoveries of end products, typified by strain F5069, were as follows: lactate, 28%; acetate, 23%; and acetoin, 26% for aerobic growth and lactate, 79%; acetate, 2%; formate, 5.4%; ethanol, 7.8%; and carbon dioxide, 2.3% for anaerobic growth. No attempt to determine carbon dioxide under aerobic growth conditions was made. The possibility of using acetoin production to assay for growth of L. monocytogenes under defined conditions should be considered.     

Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic,acetate-utilising,butyrate-producing bacterium from human faeces

Two strains of a previously undescribed Eubacterium-like bacterium were isolated from human faeces. The strains are Gram-variable, obligately anaerobic, catalase negative, asporogenous rod-shaped cells which produced acetate, butyrate and lactate as the end products of glucose metabolism. The two isolates displayed 99.9% 16S rRNA gene sequence similarity to each other and treeing analysis demonstrated the faecal isolates are far removed from Eubacterium sensu stricto and that they represent a new subline within the Clostridium coccoides group of organisms. Based on phenotypic and phylogenetic criteria, it is proposed that the two strains from faeces be classified as a new genus and species, Anaerostipes caccae. The type strain of Anaerostipes caccae is NCIMB 13811T (= DSM 14662T).     

Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5-7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO(2), and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO(2) represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields ( Y(X/ATP)), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1-7.2 g mol(-1). When this extra ATP was omitted, it was 11.9-13.1 g mol(-1). The comparison of these values with those of Y(X/ATP) in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici.     

Control of lactate production by Selenomonas ruminantium: homotropic activation of lactate dehydrogenase by pyruvate.

Selenomonas ruminantium produced one mole of D(-)-lactate per mole of glucose used at all dilution rates in ammonia-limited continuous culture. In contrast, lactate production varied according to the dilution rate when glucose was the limiting nutrient. At dilution rates of less than 0.2 h-1, acetate and propionate were the main fermentation products and lactate production was low. At dilution rates above 0.2 h-1, the pattern changed to one of high lactate production similar to that under ammonia limitation. Experiments with cell-free extracts of S. ruminantium showed that D(-)-lactate dehydrogenase had sigmoidal kinetics consistent with homotropic activation of the enzyme by its substrate, pyruvate. This feature allows S. ruminantium to amplify the effects of relatively small changes in the intracellular concentration of pyruvate to cause much larger changes in the rate of production of lactate. Some confirmation that this mechanism of control occurs under physiological conditions was obtained in glucose-limited culture, in which the sigmoidal increase in lactate production was accompanied by a linear increase in pyruvate excretion as the dilution rate increased.     

Growth stimulation of Treponema denticola by periodontal microorganisms

Previous experiments have indicated that enrichment of subgingival plaque in human serum can lead to the accumulation of Treponema denticola. T. denticola depends on bacterial interactions for its growth in serum. Aim of the present study was to identify specific microorganisms involved in the growth stimulation of T. denticola. To this end, strains isolated from previous plaque enrichment cultures were tested for growth stimulation in co-cultures with T. denticola. In addition, growth of T. denticola was tested in culture filtrates of the same strains, Bacteroides intermedius, Eubacterium nodatum, Veillonella parvula and Fusobacterium nucleatum were found to enhance growth of T. denticola in co-cultures. A continuous co-culture of T. denticola, F. nucleatum and B. intermedius in human serum gave very high levels of T. denticola, up to 3.10(9).ml-1. Mechanisms involved in growth stimulation may include the ability of B. intermedius and E. nodatum to cleave the protein-core of serum (glyco-)proteins, making these molecules accessible for degradation by T. denticola. In addition, E. nodatum was found to produce a low-molecular weight growth-factor for T. denticola, that was heat-stable and acid as well as alkaline resistant. V. parvula may provide peptidase activities complementary to those of T. denticola. The nature of the growth enhancing activity of F. nucleatum is yet unknown. The data support the dependency of T. denticola on other bacterial species for growth in the periodontal pocket.     

Influence of heme and vitamin B12 on growth and fermentations of Bacteroides species.

We examined the effects of heme on the growth and fermentations of Bacteroides species. Bacteroides fragilis ATCC 25285 required heme for growth and produced malate and lactate as major products of glucose fermentation when the concentration of heme was 1 ng/ml. With 1 microgram of heme per ml, malate was not formed, lactate production decreased, and succinate and acetate were the major fermentation products. B. eggerthii ATCC 27754 grew without heme, with the production of mainly malate and lactate from glucose. Its fermentation with 1 microgram of heme per ml was similar to that of B. fragilis grown with the same concentration of heme. B. splanchicus VPI 6842 grew without heme, with the production of mainly malate, acetate, and H2 from glucose. With 1 microgram of heme per ml, malate disappeared, H2 decreased significantly, and succinate, acetate, and butyrate were the major products. The addition of vitamin B12 to media containing 1 microgram of heme per ml caused all species to produce propionate at the expense of succinate and, with B. splanchnicus, also at the expense of butyrate. Thus, the concentration of heme and the presence of vitamin B12 significantly influenced the course of glucose fermentation by these bacteria.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Acetoin production in Leuconostoc mesenteroides NCDO 518

Abstract Cell suspensions of Leuconostoc mesenteroides NCDO 518 converted pyruvate to acetoin and a small amount of lactate and acetate. Acetoin was not produced from mixtures of pyruvate and glucose unless the ratio of pyruvate to glucose was greater than 2:1. In the presence of glucose, external pyruvate was first used as an electron acceptor, being reduced to lactate, and was converted to acetoin only after the exhaustion of glucose. Use of added pyruvate as an electron acceptor suppressed ethanol formation and the products of glucose fermentation were then lactate and acetate; 2 mol of pyruvate per mol of glucose were required to completely suppress ethanol formation. It is suggested that acetoin is produced by heterofermentative organisms when available pyruvate is in excess of that required for reoxidation of all NADH produced during glucose fermentation.     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Interaction of lipogenic substrates in porcine adipose tissue in vitro

• 1.1. Porcine adipose tissue was incubated with radiolabeled glucose, acetate or lactate. Saturation curves indicated that lactate > glucose > acetate in providing two-carbon units for fatty-acid synthesis.
• 2.2. Competition between individual substrates indicated that lactate was the best lipogenic substrate.
• 3.3. Incubation of all three substrates at concentrations observable in serum indicated that at 5.56mM, glucose was the preferred lipogenic substrate in the presence of 0.1 mM acetate and 1.0 mM lactate.
• 4.4. At elevated concentrations (18.52mM glucose, 1.0 mM acetate and 10.0 mM lactate), acetate and lactate were preferred to glucose as lipogenic substrates.

     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by 'in vitro' culture

Abstract By culturing Trypanosoma cruzi epimastigotes in modified Grace's medium with 10% foetal bovine serum, a significant quantity of metacyclic forms could be obtained. Transformation was observed after 8 days of culture, with metacyclic forms reaching 75%. Cultured Vero cells were infected with metacyclic forms and maintained until free-amastigote forms were obtained. Additionally, amastigote-like forms could be obtained by subjecting metacyclic cultures to heat shock. Parasites were grown with glucose as the major carbon source. The metabolites produced and excreted during culture were identified by difference proton nuclear magnetic resonance spectroscopy and quantified by enzymatic methods. The final products of glucose catabolism differed not only quantitatively but also qualitatively for the three major life-cycle stages of T. cruzi . The end products of metabolism produced by epimastigote forms were mainly acetate and pyruvate and, to a lesser extend, l-alanine and ethanol. Differences between epimastigotes and metacyclic forms were only quantitative. However, free amastigotes as well as amastigote-like forms, excreted acetate, glycerol, and pyruvate and to a lesser extent succinate, but no l-alanine or ethanol.     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

Isolation, Culture, and Fermentation Characteristics of Selenomonas ruminantium var. bryanti var. n. from the Rumen of Sheep

Large forms of Selenomonas sp. were isolated from the sheep rumen on a rumen fluid-glucose-agar medium by using a differential centrifugation technique to purify the inoculum. The cells from the six isolated strains were curved, gram-negative, strictly anaerobic crescents, and rapidly motile by flagella attached to the concave side of the cell. One or more of the volatile fatty acids were essential for growth. None of the strains produced indole or reduced nitrate. All strains grew on fructose, glucose, mannose, cellobiose, maltose, sucrose, and salicin. Fermentation end products from glucose were mainly lactate, acetate, propionate, and formate. Small amounts of succinate were formed. The final pH in a glucose medium ranged between 4.3 and 4.5. On the basis of the sugar fermentation characteristics and the capacity to form hydrogen sulfide from cysteine, it is suggested that one of the strains is a large form of Selenomonas ruminantium. The other five strains are designated S. ruminantium var. bryanti, var. n.     

The regulation of carbohydrate metabolism in Klebsiella aerogenes NCTC 418 organisms,growing in chemostat culture

Klebsiella aerogenes NCTC 418 was grown in chemostat cultures (D=0.17 hr^-1; pH 6.8; 35° C) that were, successively, carbon-, sulphate-, ammonia-, and phosphate-limited, and which contained as the sole carbon-substrate first glucose, then glycerol, mannitol and lactate. Quantitative analyses of carbon-substrate used and products formed allowed carbon balances to be constructed and direct comparisons to be made of the effciency of substrate utilization. With all sixteen cultures, carbon recoveries of better than 90% were obtained.Optimum utilization of the carbon substrate was invariably found with the carbon-limited cultures, the sole products being organisms and carbon dioxide. But the extent to which excess substrate was over-utilized varied markedly with both the nature of the growth-limitation and the identity of the carbon-substrate. In general, sulphate-, ammonia-, and phosphate-limited cultures utilized glycerol more efficiently than mannitol, mannitol better than lactate, and glucose least efficiently. Glucose-containing cultures also synthesized some extracellular polysaccharide.When the carbon source was in excess, a range of acidic compounds generally were excreted. Sulphate-limited cultures, growing on glucose, excreted much pyruvate and acetate, whereas similarly-limited cultures growing on glycerol, mannitol or lactate produced only acetate. Ammonialimited cultures invariably excreted 2-oxoglutarate and acetate, whereas phosphate-limited cultures produced gluconic acid, 2-ketogluconic acid and acetate, when growing on glucose, but only acetate when growing on mannitol or lactate.From the rates of substrate and oxygen consumption, and the rates of cell synthesis, yield values for both substrate and oxygen were calculated. These showed different trends, but were similar in being highest under carbon-limitation and substantially lower under all other limitations.The physiological significance of these findings, and the probable nature of the regulatory mechanisms underlying overflow metabolism are discussed.     

Paraliobacillus ryukyuensis gen. nov., sp. nov., a new Gram-positive,slightly halophilic,extremely halotolerant,facultative anaerobe isolated from a decomposing marine alga

A slightly halophilic, extremely halotolerant, alkaliphilic, and facultatively anaerobic rod bacterium was isolated from a decomposing marine alga collected in Okinawa, Japan. The isolate, designated O15-7(T), was Gram-positive, endospore-forming, catalase-positive, menaquinone-7-possessing bacterium that is motile by peritrichous flagella. The isolate was an inhabitant of marine environments; the optimum NaCl concentration for growth was 0.75-3.0% (w/v) with a range of 0-22.0%, and the optimum pH was 7.0-8.5 with a range of 5.5-9.5. Catalase was produced in aerobic cultivation but not in anaerobic cultivation. Carbohydrate, sugar alcohol or a related carbon compound was required for growth. In aerobic cultivation, the isolate produced pyruvate, acetate and CO(2) from glucose, and in anaerobic cultivation, it produced lactate, formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 for the last three products. No gas was produced anaerobically. Lactate yield per consumed glucose was markedly affected by the pH of the fermentation medium: 51% at pH 6.5 and 8% at pH 9.0. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Phylogenetically, the isolate occupied an independent lineage within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1 with the highest 16S rRNA gene sequence similarity of 95.2% to the genus Gracilibacillus. For this isolate, Paraliobacillus ryukyuensis gen. nov., sp. nov. was proposed. The type strain, O15-7(T) (G+C535.6 mol%), has been deposited in the DSMZ, IAM, NBRC, and NRIC (DSM 15140(T)=IAM 15001(T)=NBRC 10001(T)=NRIC 0520(T)).