Effects of various organic acid supplements on growth rates of Eubacterium limosum B2 on methanol

Summary During anaerobic growth on methanol, Eubacterium limosum B2 produces acetic and butyric acids as overflow metabolites, but can be induced to produce other organic acids. All organic acids (C₂–C₆) tested had a similar effect on growth, although the toxicity of each was different e.g. increasing inhibition by acids of increasing chain length. Inhibition was only observed above a threshold concentration related to the molecular size of the organic acids. At higher concentrations the degree of inhibition was a linear function of concentration. In a mathematical treatment of the data the inhibition constant (K _p) was shown to be proportionate to the threshold value (P _c) of each organic acid and accurately predicted the growth characteristics of Eubacterium limosum on methanol following the addition of organic acid supplements.     

Sequential demethoxylation reactions during methylotrophic growth of methoxylated aromatic substrates with Eubacterium limosum

The exploitation of methoxylated aromatic monomers by Eubacterium limosum was restricted to the cleavage and consumption of the methoxyl substituents: the phenolic residues were not further attacked. Growth characteristics were similar to those previously described for this organism on methanol. Degradation of aromatics containing more than one methoxyl group occurred in a sequential manner and transient accumulation of intermediates (particularly methyl-gallate) took place, though the enzymic mechanism for this phenomenon remains obscure. Degradation of 3,4,5-trimethoxybenzoate necessitated the initial attack of the para-methoxyl group before those groups in meta positions could be metabolised.     

Organic acid production during methylotrophic growth of Eubacterium limosum B2: displacement towards increased butyric acid yields by supplementing with acetate

Summary During anaerobic growth on methanol/CO₂ the fermentative bacterium Eubacterium limosum B2 produced mixtures of acetic and butyric acids as overflow metabolites. The proportion of each product was shown to vary according to the initial acetate concentration. At low concentrations, acetate provoked a displacement of the organic acid ratio culminating in homobutyric fermentations at 100 mM initial acetate. This metabolic shift was accompanied by a proportionate increase in the methanol dissimilated to CO₂, enabling a constant NAD(P)H₂/NAD(P) metabolite pool to be maintained. Higher initial acetate concentrations could not be balanced by further changes to the substrate stoichiometry and resulted in less rapid growth. The yield of butyric acid was enhanced further by some consumption of acetate. A mathematical model is presented relating initial acetate concentration to butyric acid production.     

Methanol metabolism byEubacterium limosum B2: Effects of pH and carbon dioxide on growth and organic acid production

Growth ofEubacterium limosum B2 on methanol-CO₂ was dependent upon the pH; optimum growth rates were obtained at pH 7.3–7.4. Carbon dioxide, a necessary cosubstrate for methylotrophic growth on methanol, was a determining factor for both growth and the nature of the organic acid produced. The coefficient of affinity for CO₂ was not affected by variations in pH if calculated relative to the concentration of hydrogen carbonate (16 mM). Similarly the conversion rate for methanol into acetic acid and butyric acid remained constant regardless of the level of pH with a fixed concentration of hydrogen carbonate. The metabolism ofE. limosum B2 grown on methanol-CO₂ was regulated by the HCO ₃ ^- concentration in the medium.     

Butyrate production in the acetogen Eubacterium limosum is dependent on the carbon and energy source

Eubacterium limosum KIST612 is one of the few acetogenic bacteria that has the genes encoding for butyrate synthesis from acetyl-CoA, and indeed, E. limosum KIST612 is known to produce butyrate from CO but not from H₂ + CO₂. Butyrate production from CO was only seen in bioreactors with cell recycling or in batch cultures with addition of acetate. Here, we present detailed study on growth of E. limosum KIST612 on different carbon and energy sources with the goal, to find other substrates that lead to butyrate formation. Batch fermentations in serum bottles revealed that acetate was the major product under all conditions investigated. Butyrate formation from the C1 compounds carbon dioxide and hydrogen, carbon monoxide or formate was not observed. However, growth on glucose led to butyrate formation, but only in the stationary growth phase. A maximum of 4.3 mM butyrate was observed, corresponding to a butyrate:glucose ratio of 0.21:1 and a butyrate:acetate ratio of 0.14:1. Interestingly, growth on the C1 substrate methanol also led to butyrate formation in the stationary growth phase with a butyrate:methanol ratio of 0.17:1 and a butyrate:acetate ratio of 0.33:1. Since methanol can be produced chemically from carbon dioxide, this offers the possibility for a combined chemical-biochemical production of butyrate from H₂ + CO₂ using this acetogenic biocatalyst. With the advent of genetic methods in acetogens, butanol production from methanol maybe possible as well.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum . increased the extent of microcrystalline cellulose degradation by 10·12% and 7·96%, respectively. Biomass yield in co-cultures was increased by 89·9% and 59·4% for M. elsdenii and E. limosum . Y_cellulose for fungus alone was 52·29 g dry matter mol^-1 glucose. These values were 64·93 and 55·92 g mol^-1 glucose unit in co-culture with M. elsdenii and E. limosum , respectively.     

Influence of produced acetic acid on growth of sulfate reducing bacteria

Summary A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under gás (H₂S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated acetic acid concentrations of approximately 54 mg/L.     

Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum

Aims: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. Methods and Results: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0·5%, 1%, 1·5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose‐dependent fashion, whereas butyric acid production showed a significant linear increase. Conclusions: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro‐organisms selected for the study. Significance and Impact of the Study: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.     

The effect of L-alpha-amino-n-butyric acid on growth and production of extracellular isoleucine and valine by Eubacterium ruminantium and a related rumen isolate

Two anaerobic rumen bacteria, Eubacterium ruminantium and a closely related isolate, were studied to determine the effect of the valine antimetabolite alpha-aminobutyric acid on growth and production of extracellular isoleucine and valine in an amino acid free medium. In the absence of alpha-aminobutyrate, these organisms actively excreted valine during growth (90-195 microgram/mL) but only accumulated limited concentrations of isoleucine (3-7 microgram/mL) in the culture broth. Growth of both organisms was reduced in the presence of 0.5-1.5% alpha-aminobutyrate but this inhibition was largely overcome by the use of preadapted inoculum. Metabolism of alpha-aminobutyrate was also increased using preadapted inoculum. During growth in the presence of 0.5-1.5% alpha-aminobutyrate, both organisms accumulated high concentrations of isoleucine (100-225 microgram/mL) while the normal accumulation of valine was unaffected. alpha-Ketobutyrate, a product of alpha-aminobutyrate metabolism, also stimulated isoleucine excretion by these organisms. The results are discussed in relation to the regulation of the biosynthetic pathways of isoleucine and valine in these rumen anaerobes and the potential significance of this amino acid excretion in ruminant nutrition.     

Formation of N,N-Dimethylglycine, Acetic Acid, and Butyric Acid from Betaine by Eubacterium limosum

Two bacterial strains that grow anaerobically on betaine were isolated from enrichment cultures and identified as strains of Eubacterium limosum. In a mineral medium supplemented with yeast extract and Casitone, the doubling time of E. limosum strain 11A on betaine was 6 h at 37°C. The molar growth yield amounted to 9 g of dry cell mass per mol. Betaine was fermented in accordance with the following equation: 7 betaine + 2 CO₂ → 7 N,N-dimethylglycine + 1.5 acetate + 1.5 butyrate. E. limosum also grew on methanol and choline. The former was converted to acetate and butyrate, and the latter was converted to N,N-dimethylethanolamine, acetate, and butyrate. The conditions for the quantitative determination of N,N-dimethylglycine by capillary tube isotachophoresis have been determined.     

Influence of antimicrobial agents on contamination and chlortetracycline production

The possibility of shortening the thermal sterilization time for cultivating media was demonstrated in chlortetracycline fermentation with an industrial strain ofStreptomyces aureofaciens. The medium was artificially contaminated with a mixture of eight strains of G + and G-bacteria isolated from contaminated industrial fermentors, and the following chemical agents, either alone or in combination, were added: formaldehyde, phenol, dimethylformamide,p-aminosalicylic acid and nitrofurazone. Dimethylformamide was inhibitory even at 0.08%. formaldehyde concentrations higher than 0.05%, Nitrofurazone stimulated chlortetracycline production, The best combination was 0.01% formaldehyde added before, and 2.10^-3% nitrofurazone added after short sterilization at 120 °C.     

Use of an industrial grade medium and medium enhancing effects on high cell density CO fermentation by Eubacterium limosum KIST612

Studies were made on the composition of the growth medium to increase the cell concentration in a cell-recycled continuous culture (Eubacterium limosum KIST612) with carbon monoxide as a sole energy source using phosphate-buffered basal medium (PBBM) and modified PBBM. One of major limiting factors in PBBM might be nitrogen during the high cell density culture. This limitation could be overcome by increasing of inorganic nitrogen or yeast extract concentration in the medium. Anaerobic digester fluid, which could replace the organic nitrogen in PBBM, was used to develop an industrial grade medium for conversion of CO to multi-carbon compound.     

Energy Conservation Model Based on Genomic and Experimental Analyses of a Carbon Monoxide-Utilizing,Butyrate-Forming Acetogen,Eubacterium limosum KIST612

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na⁺ dependent. Consistent with the finding of a Na⁺-dependent Rnf complex is the presence of a conserved Na⁺-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H₂ + CO₂. The energetic benefit may be one reason that butyrate is formed only from CO but not from H₂ + CO₂.     

Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species

Eubacterium limosum was isolated as the most numerous methanol-utilizing bacterium in the rumen fluid of sheep fed a diet in which molasses was a major component (mean most probable number of 6.3 X 10(8) viable cells per ml). It was also isolated from sewage sludge at 9.5 X 10(4) cells per ml. It was not detected in the rumen fluid of a steer on a normal hay-grain diet, although Methanosarcina, as expected, was found at 9.5 X 10(5) cells per ml. The doubling time of E. limosum in basal medium (5% rumen fluid) with methanol as the energy source (37 degree C) was 7 h. Acetate, cysteine, carbon dioxide, and the vitamins biotin, calcium-D-pantothenate, and lipoic acid were required for growth on a chemically defined methanol medium. Acetate, butyrate, and caproate were produced from methanol. Ammonia or each of several amino acids served as the main nitrogen source. Other energy sources included adonitol, arabitol, erythritol, fructose, glucose, isoleucine, lactate, mannitol, ribose, valine, and H2-CO2. The doubling time for growth on H2-CO2 (5% rumen fluid, 37 degree C) was 14 h as compared with 5.2 h for isoleucine and 3.5 h for glucose. The vitamin requirements for growth on H2-CO2 were the same as those for methanol; however, acetate was not required for growth on H2-CO2, although it was necessary for growth on valine, isoleucine, and lactate and was stimulatory to growth on glucose. Acetate and butyrate were formed during growth on H2-CO2, whereas branched-chain fatty acids and ammonia were fermentation products from the amino acids. Heat tolerance was detected, but spores were not observed. The type strain of E. limosum (ATCC 8486) and strain L34, which was isolated from the rumen of a young calf, grew on methanol, H2-CO2, valine, and isoleucine and showed the same requirements for acetate as the freshly isolated strains.