Robert E. Hungate: pioneer of anaerobic microbial ecology.
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Journal Article↗
Biomedical subjects
M P Bryant
Publications and source records attributed to M P Bryant.
Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses.
After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.
Bacteria, Anaerobic↗
Proposal of Quinella ovalis gen. nov., sp. nov., based on phylogenetic analysis.
Quin's oval is a relatively large bacterium often seen in the rumens of sheep fed diets containing some readily fermented carbohydrates. It has not been obtained in axenic cultures, but a number of its features have been determined by various methods, such as studying cell suspensions purified from rumen fluid by differential centrifugation. We obtained similarly purified suspensions from a sheep fed a diet containing a large amount of molasses. Nearly complete 16S rRNA sequence analysis of these cells as well as cells as Selenomonas ruminantium subsp. ruminantium GA192 (ATCC 12561; type strain) and S. ruminantium subsp. lactilytica HD4 (ATCC 27209) was done. These sequences were compared with those of other bacteria. Evolutionary distance estimates indicated that Quin's oval was most closely related to the Selenomonas-Megasphaera-Sporomusa group in the gram-positive phylum but that it belongs in a new genus. We propose the name Quinella ovalis gen. nov., sp. nov., with its description based on previously known features.
Animals↗
Lipid metabolism in anaerobic ecosystems.
In anaerobic ecosystems, acyl lipids are initially hydrolyzed by microbial lipases with the release of free fatty acids. Glycerol, galactose, choline, and other non-fatty acid components released during hydrolysis are fermented to volatile fatty acids by the fermentative bacteria. Fatty acids are not degraded further in the rumen or other parts of the digestive tract but are subjected to extensive biohydrogenation especially in the rumen. However, in environments such as sediments and waste digestors, which have long retention times, both long and short chain fatty acids are beta-oxidized to acetate by a special group of bacteria, the H2-producing syntrophs. Long chain fatty acids can also be degraded by alpha-oxidation. Biotransformation of bile acids, cholesterol, and steroids by intestinal microorganisms is extensive. Many rumen bacteria have specific growth requirements for fatty acids such as n-valeric, iso-valeric, 2-methylbutyric, and iso-butyric acids. Some species have requirements for C13 to C18 straight-chain saturated or monoenoic fatty acids for growth.
Anaerobiosis↗
Assignment of Clostridium bryantii to Syntrophospora bryantii gen. nov., comb. nov. on the basis of a 16S rRNA sequence analysis of its crotonate-grown pure culture.
The Clostridium bryantii-Methanospirillum hungatei syntrophic coculture, grown on caproate, was adapted to grow on crotonate. Then, C. bryantii was isolated in pure culture from crotonate bottle plates. A 16S rRNA sequence analysis of the pure subculture revealed that, as a member of the gram-positive phylum, it was not closely related to any of the Clostridium species with which it was compared or to any of the other clusters in the gram-positive phylum with which it was compared. However, it was closely related to another syntrophic fatty acid-degrading bacterium, Syntrophomonas wolfei. On the basis of its phylogeny, physiology, and cell wall ultrastructure, we propose assignment of C. bryantii to Syntrophospora bryantii gen. nov., nov. comb.
Bacteriological Techniques↗
Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste.
The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.
Ammonia↗
Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans.
Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-C] pyruvate, and chemically synthesized O-[methyl-C]vanillate were used to further investigate the catabolism of one-carbon (C(1)) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO(2) was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO(2) readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO(2) and pyruvate predominantly labeled the carboxyl group of acetate. No CO(2) was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.
Journal Article↗
[O-demethylation and metabolism of the methoxyl group of vanillic acid, monomer model of lignin, by the rumen bacterium Syntrophococcus sucromutans].
The O-demethylation of a lignin monomer by washed cells of the rumen bacterium S sucromutans was studied using chemically synthesized O-[methyl-14C]vanillate. During cometabolism of pyruvate and vanillate, the label from the methoxyl group was incorporated into acetate, the sole product, which was singly labeled in the methyl group.
Animals↗
Lipid growth requirement and influence of lipid supplement on fatty acid and aldehyde composition of Syntrophococcus sucromutans.
Results concerning the ruminal fluid growth requirement of the ruminal acetogen, Syntrophococcus sucromutans, indicate that octadecenoic acid isomers satisfy this essential requirement. Complex lipids, such as triglycerides and phospholipids, can also support growth. The cellular fatty acid and aldehyde composition closely reflects that of the lipid supplement provided to the cells. Up to 98% of the fatty acids and 80% of the fatty aldehydes are identical in chain length and degree of unsaturation to the octadecenoic acid supplement provided in the medium. S. sucromutans shows a tendency to have a greater proportion of the aldehyde form among its 18 carbon chains than it does with the shorter-chain simple lipids, which may be interpreted as a strategy to maintain membrane fluidity. 14C labeling showed that most of the oleic acid taken up from the medium was incorporated into the membrane fraction of the cells.
Aldehydes↗
Growth of the syntrophic anaerobic acetogen, strain PA-1, with glucose or succinate as energy source.
Strain PA-1 (S. Barik, W.J. Brulla, and M.P. Bryant, Appl. Environ. Microbiol. 50:304-310, 1985) is an anaerobic, gram-negative rod that in pure culture decarboxylates succinate to propionate and that grows syntrophically as an acetogen with the H2 utilizer Methanospirillum hungatei if glucose, pyruvate, aspartate, or fumarate is provided. In pure culture, strain PA-1 grows optimally in a medium containing 5% ruminal fluid, 0.1% yeast extract, a 4:1 N2-CO2 gas phase, and 20 mM succinate. With the PA-1 plus M. hungatei coculture, good growth was obtained with 7.5 mM glucose and tryptophan could replace the yeast extract. Strain PA-1 in pure culture grew quite well in glucose medium if the large headspace was flushed intermittently with N2. Flushing with H2 inhibited this growth.
Culture Media↗
Characterization of the pyrogallol-phloroglucinol isomerase of Eubacterium oxidoreducens.
Cell extracts of Eubacterium oxidoreducens, in the presence of dimethyl sulfoxide, catalyzed the conversion of pyrogallol to phloroglucinol with methyl sulfide as a product. The isomerization reaction also proceeded when 1,2,3,5-benzenetetrol was present rather than dimethyl sulfoxide. An assay to quantitate this activity was developed. The assay followed the disappearance of 1,2,4-benzenetriol as determined colorimetrically after incubation with sodium molybdate at neutral pH. The products of this reaction were resorcinol and 2,6-dihydroxyquinone. The enzyme(s) catalyzing this reaction was purified fivefold from cells grown on gallate plus H2. The purification procedure involved treatment with 40% acetone, precipitation with ammonium sulfate, DEAE-cellulose chromatography, concentration by ultrafiltration (molecular weight cutoff, greater than 100,000), and hydroxylapatite chromatography. This preparation had a specific activity of 14.7 mumol/min per mg of protein and a pH optimum of about 7.3. It was strongly inhibited by p-chloromercuribenzoate. The mechanism of the reaction involved oxidation of the pyrogallol followed by introduction of water. The benzenetetrol intermediate was then reduced and dehydrated to phloroglucinol.
Colorimetry↗
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)
Animals↗
Quin's oval and other microbiota in the rumens of molasses-fed sheep.
Two rumen-cannulated wether sheep were fed a diet containing 1 kg of a liquid-molasses mixture, 80 g of soybean oil meal, and 100 g of chopped wheat straw once a day. In 6 weeks and thereafter, the microbiota adapted such that Quin's oval, a very large bacterium, was present in huge numbers (11.3 X 10(10) and 1.3 X 10(10) ml-1 after 73 days). Direct microscopic counts were also done on small bacteria, moderate-sized Selenomonas spp., and small Entodinium spp., which were the only protozoa seen. After the necessary dilution of rumen contents to make the microbial cells visible, Quin's ovals were seen to be much smaller in sheep 1 than in sheep 2. Most-probable-number estimates indicated that Methanobrevibacter spp. were present at 10(7) ml-1, Methanosarcina spp. were present at 10(3) ml-1, and Eubacterium limosum-like bacteria were present at 10(5) to 10(6) ml-1. In the adapted sheep, the dry portion of the diet was rapidly consumed, but the molasses mixture was consumed over a 9- to 10-h period. Volatile fatty acids in the rumen were present in very low amounts just prior to feeding and were much higher during the consumption of the diet, with about a 1:1 molar ratio of propionate to acetate between 1 and 9 h after feeding. Data were obtained on hourly feed consumption, levels of volatile fatty acids, and pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Animal Feed↗
Metabolism of gallate and phloroglucinol in Eubacterium oxidoreducens via 3-hydroxy-5-oxohexanoate.
The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems. Cells grown with gallate and crotonate, but with no formate or H2, excreted pyrogallol and phloroglucinol into the medium. Gallate was decarboxylated by crude cell extracts, with pyrogallol as the only detectable product. Whole cells converted pyrogallol to phloroglucinol. A phloroglucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol when NADPH was used as the source of electrons. Both formate dehydrogenase (EC 1.2.1.43) and hydrogenase (EC 1.18.99.1) were present in cell extracts of gallate-formate-grown cells. These two enzymes were both NADP linked. Since either H2 or formate is required for cell growth with gallate or phloroglucinol, these results suggest that the oxidation of the reduced substrate may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinol hydrolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product then presumably can be broken down by a series of reactions similar to beta-oxidation. These reactions cleaved the six-carbon acid to 3-hydroxybutyryl-coenzyme A yielding acetate and butyrate as end products. A number of key enzymes involved in beta-oxidation and substrate-level phosphorylation were demonstrated in cell extracts.
Caproates↗
Characteristics of methanogens isolated from bovine rumen.
Six strains of methanogens were isolated from 10(-8) and 10(-9) ml of bovine rumen contents. All strains had the morphologic and physiologic characteristics of Methanobrevibacter spp. Four strains required coenzyme M; two did not. Growth of all strains either depended on or was stimulated by a mixture of isobutyric, isovaleric, 2-methylbutyric, and valeric acids. None of the strains reacted with antiserum against the type strain of Methanobrevibacter ruminantium.
Animals↗
A rumen bacterium degrading quercetin and trihydroxybenzenoids with concurrent use of formate or H2.
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Animals↗
PA-1, a Versatile Anaerobe Obtained in Pure Culture, Catabolizes Benzenoids and Other Compounds in Syntrophy with Hydrogenotrophs, and P-2 plus Wolinella sp. Degrades Benzenoids.
Methanogenic enrichments catabolizing 13 mM phenylacetate or 4 mM phenol were established at 37 degrees C, using a 10% inoculum from a municipal anaerobic digester. By using agar roll tubes of the basal medium plus 0.1% yeast extract-25 mM fumarate, a hydrogenotrophic lawn of Wolinella succinogenes and phenol or phenylacetate, strains P-2 and PA-1, respectively, were isolated in coculture with W. succinogenes. With the lawn deleted, PA-1 was isolated in pure culture. Strain P-2 is apparently a new species of anaerobic, motile, gram-negative, spindle-shaped, small rod that as yet has been grown only in coculture with W. succinogenes. It used phenol, hydrocinnamate, benzoate, and phenylacetate as energy sources. Product recovery by the coculture, per mole of phenol and 4.4 mol of fumarate used, included 2.03, 0.12, 0.08, and 3.23 mol, respectively, of acetate, propionate, butyrate, and succinate. Carbon recovery was 75% and H recovery was 80%, although CO(2) and a few other possible products were not determined. That P-2 is an obligate proton-reducing acetogen and possible pathways for its degradation of phenol are discussed. Strain PA-1 is apparently a new species of anaerobic, motile, relatively small, gram-negative rod. It utilized compounds such as phenylacetate, hydrocinnamate, benzoate, phenol, resorcinol, gallate, 4-aminophenol, 2-aminobenzoate, pyruvate, Casamino Acids, and aspartate as energy sources in coculture with W. succinogenes. Per mole of phenylacetate and 1.44 mol of fumarate used, 1.04, 0.53, and 0.78 mol of acetate, propionate, and succinate, respectively, were recovered from the coculture. Only about 50% of the carbon and H were recovered. In coculture with Methanospirillum hungatei, 0.96 mol of acetate and 0.25 mol of methane were recovered per mol of pyruvate used; 0.90 mol of acetate and 0.33 mol of methane, per mol of fumarate used; 0.93 mol of acetate and 0.54 mol of methane, per mol of aspartate used; and 1.71 mol of acetate and 0.57 mol of methane, per mol of glucose used. Carbon and H recoveries, assuming CO(2) and ammonia were produced in stoichiometric amounts, were 97 and 98% for pyruvate, 72.5 and 82% for fumarate, 96.5 and 98% for aspartate, and 61.8 and 76% for glucose. No explanation such as contamination could be found for the fact that the coculture PA-1 plus Wolinella sp. did not use glucose; after growth with M. hungatei on pyruvate, however, the latter coculture used glucose. The PA-1 pure culture produced 0.86 mol of propionate per mol of succinate used during growth. PA-1 produced a small amount of H(2). Strain PA-1 is the most versatile anaerobic bacterium yet known that catabolizes monobenzenoids in the absence of electron acceptors such as sulfate or nitrate.
Journal Article↗
Peptostreptococcus productus strain that grows rapidly with CO as the energy source.
Anaerobic bacteria were enriched with a sewage digestor sludge inoculum and a mineral medium supplemented with B-vitamins and 0.05% yeast extract and with a 50% CO-30% N2-20% CO2 (2 atm [202 kPa]) gas phase. Microscopic observation revealed an abundance of gram-positive cocci, 1.0 by 1.4 micron, which occurred in pairs or chains. The coccus, strain U-1, was isolated by using roll tubes with CO as the energy source. Based on morphology, sugars fermented, fermentation products from glucose (H2, acetate, lactate, and succinate), and other features, strain U-1 was identified as Peptostreptococcus productus IIb (similar to the type strain). The doubling time with up to 50% CO was 1.5 h; acetate and CO2 were the major products. In addition, no significant change in the doubling time was observed with 90% CO. Some stock strains were also able to use CO, although not as well. Strain U-1 produced acetate during growth with H2-CO2. Other C1 compounds did not support growth. Most probable numbers of CO utilizers morphologically identical with strain U-1 were 7.5 X 10(6) and 1.1 X 10(5) cells per g for anaerobic digestor sludge and human feces, respectively.
Acetates↗