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Previous work has shown that the divergently transcribed Mycobacterium tuberculosis genes acr ( hspX , Rv2031c) and acg (Rv2032) are induced under conditions of shallow standing culture and low oxygen and intracellularly within macrophages. We used a combination of computational and experimental methods to identify promoters for eight additional genes that are regulated in a similar manner and that comprise an acr -coregulated promoter (ACP) family. Transcriptional regulation of these ACP family members was evaluated by using a plasmid-based promoter-green fluorescent protein fusion system and flow cytometry. All promoters showed increased expression in shallow standing versus shaking cultures, in low- versus high-oxygen conditions, and intracellularly within macrophages versus extracellularly in tissue culture medium. However, there were quantitative differences in expression among promoters and among conditions for each promoter. A conserved 18-bp palindromic sequence motif was identified in all ACPs by Gibbs sampling-based computational analyses. Two such motifs overlap regions in the acr and acg promoters that were previously shown to be required for their expression. In addition, we found that 5% carbon dioxide was required for growth of Mycobacterium bovis BCG under microaerophilic (1.3% O 2 ) culture conditions and fully prevented the growth cessation typically associated with rapid removal of oxygen. These findings are likely to be relevant to the in vivo environment and will contribute to our understanding of the pathogenesis of tuberculosis infection.

The prokaryote generic name Bryantella Wolin et al. 2004(VP) is illegitimate because it is a later homonym of Bryantella Chickering, 1946 (Animalia, Arthropoda, Arachnida, Araneae, Salticidae, Dendryphantinae, Dendryphantini) and a later homonym of Bryantella Britton, 1957 (Animalia, Arthropoda, Scarabaeoidea, Scarabaeidae, Melolonthinae) [Principle 2, Rule 51b(4) of the Bacteriological Code (1990 Revision)]. Bryantella represents a genus of jumping spiders within the family Salticidae and a genus of melolonthine scarab beetles within the family Scarabaeidae. Therefore, a new genus name, Marvinbryantia gen. nov., is proposed for this taxon. As a result, a new combination, Marvinbryantia formatexigens comb. nov., is required for the type species to replace the illegitimate combination Bryantella formatexigens Wolin et al. 2004(VP).

We characterized the effects of ciprofloxacin and rifampin alone and in combination on Staphylococcus aureus in vitro. The effects of drug combinations (e.g., indifferent, antagonistic, or additive interactions) on growth inhibition were compared by disk approximation studies and by determining the fractional inhibitory concentrations. Bactericidal effects in log-phase bacteria and in nongrowing isolates were characterized by time-kill methods. The effect of drug combinations was dependent upon whether or not cells were growing and whether killing or growth inhibition was the endpoint used to measure drug interaction. Despite bactericidal antagonism in time-kill experiments, our in vitro studies suggest several possible explanations for the observed benefits in patients treated with a combination of ciprofloxacin and rifampin for deep-seated staphylococcal infections. Notably, when growth inhibition rather than killing was used to characterize drug interaction, indifference rather than antagonism was observed. An additive bactericidal effect was observed in nongrowing bacteria suspended in phosphate-buffered saline. While rifampin antagonized the bactericidal effects of ciprofloxacin, ciprofloxacin did not antagonize the bactericidal effects of rifampin. Each antimicrobial prevented the emergence of subpopulations that were resistant to the other.

The pathways of short-chain fatty acid (SCFA; acetate, propionate, and butyrate) formation from glucose were determined for the human fecal microbial communities of two subjects. The pathways were identified by radioisotope analysis of the SCFA and CO2 obtained after incubation of fecal suspensions with glucose under 20% CO2 with [1-14C]glucose, [3,4-14C]glucose, or 14CO2. Acetate was chemically degraded to learn the labeling of the methyl and carboxyl carbons. The labeling of CO2 and acetate showed that the major route of glucose catabolism was the Embden-Meyerhof-Parnas pathway, with production of CO2 from pyruvate carboxyl carbon. Labeling of the methyl and carboxyl carbons of acetate by 14CO2 or [3,4-14C]glucose proved that acetate was formed from CO2 by the Wood-Ljungdahl pathway. CO2 reduction accounted for about one-third of the acetate formed by suspensions from subject 1 and about one-fourth of the acetate formed by suspensions from subject 2. Propionate was formed by a CO2 fixation pathway, and butyrate was formed by classical routes of acetyl-S coenzyme A condensation. The amount of CO2 formed from [1-14C] glucose and acetate labeling patterns obtained with the other 14C precursors indicated that the Entner-Doudoroff, transketolase-transaldolase, and heterolactic pathways were not significant. Fermentation of cabbage cellulose by subject 1 followed the same pathways as were used for glucose. The results with suspensions from subject 2 suggested that some radioactive acetate was formed from the C-3 of glucose by the Bifidobacterium pathway.

We used dilutions of fecal suspensions from a human volunteer to enrich cultures for bacteria that reduce CO2 to acetate in the colon. The soluble enrichment substrates used were glucose, methanol, formate, and vanillate, which were used with a gas phase that contained 80% N2 and 20% CO2. The gaseous enrichment substrates used were 80% H2-20% CO2 and 50% CO-50% CO2. We isolated three different strains that produced acetate from CO2. One strain produced acetate from methanol, vanillate, H2-CO2, glucose, and other sugars. The other two strains did not form acetate from methanol or vanillate. Both of the latter strains formed acetate from glucose and other sugars, but only one of these strains formed acetate from H2-CO2. Both of these strains cometabolized formate. However, none of the enrichment cultures or pure cultures used CO or formate as a substrate for growth. The two strains that produced acetate from H2 and CO2 grew slowly when the gases alone were used as substrates, but they rapidly cometabolized H2 and CO2 when they were grown with organic substrates. The ability of all of the strains to produce acetate from CO2 and/or other one-carbon precursors was verified by determining the radioactivity of the methyl and carboxyl groups of the acetate formed after growth with 14CO2 or other radioactively labeled one-carbon precursors.

Fermentations of cornstarch and a cabbage- fiber preparation by human fecal suspensions were studied. The molar percent of butyrate oftotal short-chain fatty acid products was significantly higher when cornstarch was the substrate. Higher molar percents of butyrate were also produced from cornstarch as compared with endogenous substrate when rat fecal suspensions were used. A range ofcornstarch fermentation rates was found with suspensions from 20 human subjects. Rapid fermentation was associated with the absence of methane pro- duction. Methane-negative rat fecal suspensions also fermented cornstarch more rapidly than did methane-positive suspensions. High butyrate production may be important because butyrate provides energy to colonocytes and it regulates differentiation ofcultured cells. Am J C/in Nutr i992;55:70-7.

We wished to characterize the carbohydrate fermentation by intestinal flora in formula-fed infants and in breast-fed infants. We also wished to compare the carbohydrate fermentation process in the two groups to determine whether differences that existed between groups could help explain the observation that breast-fed infants usually have milder forms of acute gastroenteritis. We performed in vitro incubations of fecal samples from nine formula-fed and 14 breast-fed infants and examined the samples for fermentation characteristics, the effect of acid pH on bacterial fermentation, and changes in carbohydrate fermentation in relation to the age of the infant. Fecal samples were incubated, with and without the addition of lactose, at a pH of 6.8 and at a pH of 5.5. Volatile fatty acids and carbohydrates were determined in the incubates. The addition of lactose to the incubate at pH 6.8 resulted in significantly increased volatile fatty acid production and larger amounts of lactose, glucose, and galactose compared with the values observed in 1-h incubates to which no lactose was added. At pH 5.5, volatile fatty acid production was significantly lower in both groups compared with that at pH 6.8, and the accumulation of monosaccharides in the incubate of feces of formula-fed infants increased significantly (p < 0.05). In contrast, in stools from breast-fed infants, incubation at pH 5.5 resulted in a greater proportion of saccharides as lactose, i.e. a decrease in the amount of lactose hydrolyzed (p < 0.01), accompanied by a nonsignificant increase in the amount of hexose. The decrease in lactose hydrolysis in breast-fed infants results in a lower luminal osmolar load, which may provide a partial explanation for the fact that acute gastroenteritis is milder in this population.

Formate stimulates growth of a new bacterium from human feces. With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway. The original isolate fermented vegetable cellulose and carboxymethylcellulose, but it lost this ability after storage at −76°C. 16S rRNA gene sequencing identifies it as a distinct line within the Clostridium coccoides supra-generic rRNA grouping. We propose naming it Bryantella formatexigens gen. nov., sp. nov.

Previous work has shown that the divergently transcribed Mycobacterium tuberculosis genes acr (hspX, Rv2031c) and acg (Rv2032) are induced under conditions of shallow standing culture and low oxygen and intracellularly within macrophages. We used a combination of computational and experimental methods to identify promoters for eight additional genes that are regulated in a similar manner and that comprise an acr-coregulated promoter (ACP) family. Transcriptional regulation of these ACP family members was evaluated by using a plasmid-based promoter-green fluorescent protein fusion system and flow cytometry. All promoters showed increased expression in shallow standing versus shaking cultures, in low- versus high-oxygen conditions, and intracellularly within macrophages versus extracellularly in tissue culture medium. However, there were quantitative differences in expression among promoters and among conditions for each promoter. A conserved 18-bp palindromic sequence motif was identified in all ACPs by Gibbs sampling-based computational analyses. Two such motifs overlap regions in the acr and acg promoters that were previously shown to be required for their expression. In addition, we found that 5% carbon dioxide was required for growth of Mycobacterium bovis BCG under microaerophilic (1.3% O(2)) culture conditions and fully prevented the growth cessation typically associated with rapid removal of oxygen. These findings are likely to be relevant to the in vivo environment and will contribute to our understanding of the pathogenesis of tuberculosis infection.

A system is described that combines the fermentation of cellulose to acetate, CH4, and CO2 by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH4 and CO2 by Methanosarcina barkeri to convert cellulose to CH4 and CO2. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H2-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH4 and CO2 was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5-3.8 days. Rates of acetate utilization were 9.5-17.3 mmol l(-1) day(-1) and increased with decreasing RT. The Ks for acetate utilization was 6-8 mM. The two-stage system can be used as a model system for studying biological and physical parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using phase of the overall fermentation.

Microbes in the adult human colon ferment dietary substrates chiefly to acetic, propionic and butyric acids and CO 2 , H 2 and CH 4 . How this fermentation evolves after microbial colonization of the neonate is unknown. We examined the fermentation of glucose by fecal suspensions of a breast-fed infant from d 16 to 158 and found that the fermentation changed with age. Acetate, ethanol, succinate, lactate, formate and H 2 were formed up to 117 d of age. Production of succinate, lactate, formate and H 2 ceased after 117 d and acetate production increased. Butyrate and propionate were minor products up to 117 d. Afterwards, there was a slight increase in propionate production with no change in butyrate formation. Acetate was always the major product of glucose fermentation by the fecal suspensions. Approximately the same amounts of ethanol were formed throughout the study period. The fermentations were similar to fermentations of Escherichia coli and streptococci through 117 d. Nuclear magnetic resonance (NMR) analysis of the acetate formed from 1- 13 C- and 3- 13 C-glucose showed that the dominant fermentation pathway used by the colonic microbes switched from the Embden-Meyerhof-Parnas pathway at 16 d of age to the Bifidobacterium pathway at 158 d of age. An increase in the contribution of the Bifidobacterium fermentation to the overall colonic fermentation after 117 d would account for the increase in the formation of acetate from glucose. Chemical and NMR analyses of products of fecal fermentations from two other breast-fed infants

The gastrointestinal tracts of breast-fed infants are colonized more easily with bifidobacteria than are those of formula-fed infants. Colonization is thought to reduce infant diarrhea. Amendments to formulas that improve colonization by bifidobacteria are being actively investigated. Colonization studies almost invariably require measurements of the concentration of the bifidobacteria in feces to assess their importance in the colon. We investigated the use of nuclear magnetic resonance (NMR) analysis of products of fermentations of 1- and 3- 13 C-glucose to evaluate the importance of bifidobacteria in the colonic ecosystem. Bifidobacteria use a unique pathway of hexose catabolism to produce primarily acetate and lactate. The fermentation yields 3 mol of acetate from 2 mol of glucose. Two of the acetates are formed from C1 and C2 of glucose and the third is formed entirely from C3 of glucose. We first employed high resolution NMR to verify the pathway used by a pure culture of Bifidobacterium bifidum. The major products of fermentation of 1- and 3- 13 C-glucose were acetate and lactate. Most of the 13 C from 3- 13 C-glucose was in 13 CH 3 13 COOH with equal enrichment in the methyl and carboxyl groups. The 13 C-acetate from 1- 13 C-glucose was almost entirely enriched in the methyl of acetate and no 13 CH 3 13 COOH was produced. NMR analysis of glucose fermentation by the colonic flora of a 158-d-old strictly breast-fed infant showed production of 13 CH 3 13 COOH from 3- 13 C-glucose. The amount of 13 CH 3 13 COOH formed established that the Bifidobacterium pathway was the major pathway used for glucose fermentation by this infant's colonic microbes.

Earlier studies suggest that butyrate has colonic differentiating and nutritional effects and that acarbose increases butyrate production. To determine the effects of acarbose on colonic fermentation, subjects were given 50-200 mg acarbose or placebo (cornstarch), three times per day, with meals in a double-blind crossover study. Fecal concentrations of starch and starch-fermenting bacteria were measured and fecal fermentation products determined after incubation of fecal suspensions with and without added substrate for 6 and 24 h. Substrate additions were cornstarch, cornstarch plus acarbose and potato starch. Dietary starch consumption was similar during acarbose and placebo treatment periods, but fecal starch concentrations were found to be significantly greater with acarbose treatment. Ratios of starch-fermenting to total anaerobic bacteria were also significantly greater with acarbose treatment. Butyrate in feces, measured either as concentration or as percentage of total short-chain fatty acids, was significantly greater with acarbose treatment than with placebo treatment. Butyrate ranged from 22.3 to 27.5 mol/100 mol for the 50-200 mg, three times per day doses of acarbose compared with 18.3-19.3 mol/100 mol for the comparable placebo periods. The propionate in fecal total short-chain fatty acids was significantly less with acarbose treatment (10.7-12.1 mol/100 mol) than with placebo treatment (13.7-14.2 mol/100 mol). Butyrate production was significantly greater in fermentations in samples collected during acarbose treatment, whereas production of acetate and propionate was significantly less. Fermentation decreased when acarbose was added directly to cornstarch fermentations. Acarbose effectively augmented colonic butyrate production by several mechanisms; it reduced starch absorption, expanded concentrations of starch-fermenting and butyrate-producing bacteria and inhibited starch use by acetate- and propionate-producing bacteria.

Figure 13.1 shows the general features of the colonic fermentation. The human diet contains high concentrations of plant polysaccharides (cellulose, hemicellulose, pectin, and starch). Except for starch, they are not digested by host enzymes and pass to the colon where they are fermented by the cooperative metabolism of a large number of different bacterial species (Cummings, 1985; Wolin and Miller, 1983). Significant amounts of starch escape host digestion and are fermented in the colon (Stephen et al., 1983; Thornton et al., 1987). The fermentation of plant polysaccharides produces acetate, propionate, and butyrate and the gases H2 and CO2 (Cummings, 1985; McNeil, 1984). CH4 is a major product of the colonie fermentation of some humans (Bond et al., 1971; Levitt, 1980). They harbor large concentrations of methane-forming Archae that use H2 to reduce CO2 to CH4 (Miller and Wolin, 1982; Weaver et al., 1986).

The hydrolysis and fermentation of cellulose (Avicel) by continuous cultures of Ruminococcus albus strain 7 and Methanobrevibacter smithii strain PS were studied. Cellulose destruction ranged from ca. 22% to 71% for 0.25 to 2.27 days solids retention time, respectively. The cellulose hydrolysis rate constant (k) was 1.3 days−1. Concentrations of soluble reducing sugars were low, showing that cellulose hydrolysis was the rate-limiting step of cellulose fermentation. The estimated methane-based molar growth yield for M. smithii was 2.8 g mol−1. Its maximum specific growth rate was ca. 4 days−1. The dissolved H2 half-saturation constant (K s ) for methanogenesis was ca. 1 μM. The final products of the co-culture were primarily acetate, CH4 and CO2 and low levels of ethanol and H2. The co-culture produced more H2 (used for reduction of CO2 to CH4) and acetate than a monoculture of R. albus. These differences coulb be accounted for by the lower production of ethanol, confirming to the theory of interspecies H2 transfer.

The nutritional requirements of Selenomonas ruminantium HD4 for growth on glucose, glycerol, or lactate were investigated to clarify the results of previous studies and to relate the nutrition of the organism to its physiology. The organism required l -aspartate, CO 2 , p -aminobenzoic acid, and biotin for growth on a lactate-salts medium that contained small amounts of dithiothreitol. Aspartate could be replaced by l -malate or fumarate but not by succinate or l -asparagine. Requirements for growth with glycerol as an energy source were similar, except that aspartate was not required. With glucose as the energy source, neither aspartate nor p -aminobenzoic acid was required, but a requirement for volatile fatty acids, which could be met by n -valerate, was observed. CO 2 was required for growth on lactate or glycerol but not on glucose on complex media containing Trypticase and yeast extract. Sulfide could be used as the sole source of sulfur.

The activities of populations in complex anaerobic microbial communities that perform complete bioconversion of organic matter to CH4 and CO2 are reviewed. Species of eubacteria produce acetate, H2, and CO2 from organic substrates, and methanogenic species of archaebacteria transform the acetate, H2, and CO2 to CH4. The characteristics and activities of the methanogenic bacteria are described. The impact of the use of H2 by methanogens on the fermentations that produce acetate, H2, and CO2 and the importance of syntrophy in complete bioconversion are discussed.

Summary Methanogenic bacteria were isolated from feces of horse, cow. sheep, pig, rat, goose, chicken and turkey. All isolates were obtained from either direct platings or enrichments of feces with H 2 and CO 2 as the major substrates for production of CH 4 . Morphologic and physiologic characterization and mol % G + C of DNA indicate the predominant methanogens in the large intestines of horse, sheep, goose, cow, pig and rat are members of the genus Methanobrevibacter. Characterization of the chicken and turkey fecal isolates indicate Methanogenium may be a major component in the large intestine of these fowl.

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g −1 , and the ethanol yield was 0.41 g g −1 . The product yield was constant, indicating that product formation was growth linked.

A plating medium containing cephalothin and clindamycin was developed for enumeration and isolation of methanogens in human feces. Specimens from nine CH4-producing subjects had total anaerobe counts of 1-8 x 1011/g dry weight. Methanogen counts on the antibiotic medium ranged from 0.001 - 12.6% of the total anaerobe count. There was no correlation between age, sex or percent dry fecal weight and the ratio of methanogens to total counts. Specimens from eight non-CH4-producing individuals contained bacteria that yielded nonmethanogenic colonies on the antibiotic medium. The means +/- SD of the logarithm of the total counts per gram dry weight were 11.4 /+- 0.29 and 11.38 +/- 0.44 for the positive and negative groups respectively. Values for the antibiotic-resistant flora were 8.8 /+- 1.13 and 7.78 /+- 1.08 respectively. Methanogens were isolated from the most dilute inoculum of each specimen from CH4-producing subjects. All isolates were morphologically, physiologically, and immunologically identical to Methanobrevibacter smithii. Growth of methanogens in media that were essentially extracts of CH4-negative feces suggested that no nutrients were lacking or inhibitors present in intestinal contents that prevent the growth of methanogens in these individuals.

Summary Methanogens are members of microbial communities that inhabit the large intestine of man and animals and the specialized forestomachs of some herbivores (e. g., ruminants). Nonmethanogenic populations of these communities ferment substrates to short chain volatile fatty acids, H2 and CO2. Methanogens use the H2 to reduce CO2 to CH4. Methanol and methylamines derived from dietary sources are used in lesser amounts for the production of CH4. Some humans excrete only a few methanogens per g dry weight (gdw) of feces and some excrete 1010 per gdw. About 1/3 of the population has 108 to 1010 methanogens per gdw. The predominant methanogen in all subjects is Methanobrevibacter smithii. A coccus that uses H2 to reduce methanol to CH4 is often present at lower concentrations. This coccus, Methanosphaera stadtmaniae, belongs to the Methanobacteriaceae. The major methanogen in Wistar rats belongs to the same genus as Mb. smithii, but DNA hybridization studies indicate that it is a different species. A coccus resembling Ms. stadtmaniae is present in lower concentrations. Methanogens have been isolated from feces of the horse, sheep, cow, pig, goose, turkey and chicken. The predominant methanogen in all except the chicken and turkey is a Methanobrevibacter species. The chicken and the turkey harbor species of Methanogenium. Methanogens have been isolated from feces of a variety of wild animals and insects and most are Methanobrevibacter species which are also the most common methanogens in the forestomach of the bovine rumen.

Methanosphaera stadtmaniae is a non-motile, Gram-positive spherical-shaped organism that obtains energy for growth by using hydrogen to reduce methanol to methane. It does not produce methane from hydrogen and carbon dioxide, formate, acetate or methylamines and cannot grow with hydrogen and carbon monoxide, nitrate, fumarate, sulfate or choline. Its pH optimum is 6.5 to 6.9 and its temperature optimum is 36 degrees to 40 degrees C. It is not inhibited by bile salts, inhibitors of the synthesis of folic acid coenzymes, cephalothin or clindamycin but is inhibited by metronidazole, bacitracin, monensin, lasalocid, or bromoethanesulfonate. It requires acetate, carbon dioxide, isoleucine, ammonium, and thiamin for growth and biotin is stimulatory. It does not contain cytochromes and the mol % G + C of its DNA is 25.8. The composition of its cell wall and 16 S rRNA and its immunological fingerprint are consistent with characterization of the organism as a member of a new genus of the family Methanobacteriaceae. The habitat of the type strain is the human large intestine.

Many cultures of the Streptococcus mitis group have been reported to oxidize butyric acid with the accumulation of hydrogen peroxide in both growing cultures and resting cell suspensions (Niven, Evans, and White, 1945). An investigation of this reaction was undertaken in this laboratory with the ultimate aim of obtaining information concerning the mechanism of the oxidation. The mechanism of butyric acid oxidation by enzymatic preparations of Clostridium kluyveri has been extensively investigated (Stadtman and Barker, 1949; Kennedy and Barker, 1951a). However, since these workers were studying the oxidation with preparations from an obligate anaerobic organism having a rather unusual energy metabolism, it seemed of interest to pursue similar studies with an organism having more orthodox metabolic patterns. This report will be confined to an account of the isolation of a strain of S. mitis that oxidizes butyric acid, and to some of the characteristics of the butyrate-oxidizing system in resting suspensions of this organism.

Fermentation of food by the microbial community of the rumen is essential for the maintenance and growth of ruminants. The microbial ecosystem and its interaction with the host are described, along with recent attempts to manipulate the composition and activity of the microbial community by adding antibiotics and other chemicals to ruminant diets. A similar microbial community and fermentation occur in the large intestine or cecum of most nonruminant animals including the large intestine of humans. The microbial ecosystems of the rumen and human large intestine are compared.

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant ( k ) of 1.18 day −1 . The true microbial growth yield ( Y ) was 0.11 g g −1 , the maintenance coefficient ( m ) was 0.10 g g −1 h −1 , and the maximum Y ATP was 7.7 g of biomass (dry weight) mol of ATP −1 .

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.

Total anaerobic bacteria and Methanobrevibacter smithii populations were enumerated in fecal specimens from two individuals over 10- and 13-month periods. The ratio of M. smithii to total anaerobic count varied between the individuals, but it was a relatively constant proportion of the large-bowel microbial flora within each individual. Neither a barium enema examination of one subject nor a radical change in the diet of the other had any long-term effect on the methanogen populations.

The antigenic map of 17 methanogenic bacteria representing the entire range of available species was determined by multiple assay with antibody probes. Four major clusters of antigenically related strains coincide with the females proposed on the basis of 16S ribosomal RNA analysis. Immunological mapping uncovered relationships not yet shown by other methods and allowed identification and classification of two new bacterial isolates.

H2 production from glucose by Ruminococcus albus was almost completely inhibited by 10(-5) M molybdate only when sulfide was present in the growth medium. Inhibition was accompanied by a significant increase in the production of formate. Extracts of molybdate-sulfide-grown cells did not contain hydrogenase activity. Active enzyme in extracts of uninhibited cells was not inhibited by the molybdate-sulfide-containing growth medium. The results indicate that a complex formed from molybdate and sulfide prevents the formation of active hydrogenase and electrons otherwise used to form H2 are used to reduce CO2 to formate. Growth was significantly inhibited when molybdate was increased to 10(-4) M. Reversal of growth inhibition but not inhibition of H2 production occurred between 10(-4) and 10(-3) M molybdate. H2 production by R. bromei but not by R. flavefaciens, Butyrivibrio fibrisolvens, Veillonella alcalescens, Klebsiella pneumoniae and Escherichia coli was inhibited by molybdate and sulfide.

During enterohepatic circulation conjugated steroids are hydrolyzed by intestinal bacteria, further metabolized mainly by anaerobic bacteria, reabsorbed, reconjugated and delivered to the blood for renal excretion. 21-Dehydroxlylation of tetrahydrodeoxycorticosterone (THCOD) to pregnanolone is performed exclusively by Eubacterium lentum, a normal inhabitant of the intestinal flora. The concentration of E. lentum in feces can be determined from the highest dilution of fresh voided stool capable of 21-dehydroxylating THDOC. Such studies revealed that the concentration increases from 106/g wet feces during the first year of life to 108/g feces in the 7th and 8th decade. Colonization appears to be independent of diet since the organisms are present in babies whether breast-fed or on formula, in subjects on a “Western diet” and in Africans consuming a diet low in meat and fat.

It is thought that monensin increases the efficiency of feed utilization by cattle by altering the rumen fermentation. We studied the effect of monensin and the related ionophore antibiotic lasalocid-sodium (Hoffman-LaRoche) on the growth of methanogenic and rumen saccharolytic bacteria in a complex medium containing rumen fluid. Ruminococcus albus, Ruminococcus flavefaciens , and Butyrivibrio fibrisolvens were inhibited by 2.5 μg of monensin or lasalocid per ml. Growth of Bacteroides succinogenes and Bacteroides ruminicola was delayed by 2.5 μg of monensin or lasalocid per ml. Populations of B. succinogenes and B. ruminicola that were resistant to 20 μg of either drug per ml were rapidly selected by growth in the presence of each drug at 5.0 μg/ml. Selenomonas ruminantium was insensitive to 40 μg of monensin or lasalocid per ml. Either antibiotic (10 μg/ml) inhibited Methanobacterium MOH, Methanobacterium formicicum , and Methanosarcina barkeri MS. Methanobacterium ruminantium PS was insensitive to 40 μg of monensin or 20 μg of lasalocid per ml. The methanogenic strain 442 was insensitive to 40 μg of monensin but sensitive to 10 μg of lasalocid per ml. The results suggest that monensin or lasalocid acts in the rumen by selecting for succinate-forming Bacteroides and for S. ruminantium , a propionate producer that decarboxylates succinate to propionate. The selection could lead to an increase in rumen propionate formation. Selection against H 2 and formate producers, e.g. R. albus, R. flavefaciens , and B. fibrisolvens , could lead to a depression of methane production in the rumen.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.

A ruminant can be thought of as a fermentation factory (Fig. 1). The animal ingests plant polymers (in grasses, hay, corn, silage, etc.) which are the raw material for its fermentation. Preliminary processing occurs in the oral cavity and consists mainly of comminution of food by mastication. The plant material is then swallowed and transported to the ruminant’s complex stomach. The stomach, called the rumen reticulum or, more simply, rumen, is the site of fermentation. A massive community of microorganisms, bacteria and protozoa, ferments the plant material to short-chain volatile fatty acids, methane, and carbon dioxide. The acids are removed from the rumen by absorption into the bloodstream and are subsequently used as the animal’s primary sources of energy and carbon. Gases are waste products and are removed by belching. The fermentation provides nutrients and energy for the growth and division of the microbial populations participating in the fermentation. Microorganisms and undigested food are semicontinuously removed from the rumen by passage to the lower part of the ruminant’s digestive tract.

Methanogens are present in the colons of our local Wistar rat colony. We studied the changes in concentrations of their fecal methanogenic and nonmethanogenic bacteria with age as a model of the development of these communities in humans. We found that the predominant methanogen in the rats is a Methanobrevibacter species. The log of the concentration of total anaerobes increased from 9.8/g (dry weight) at 3.0 weeks of age (shortly after weaning) to 10.7/g (dry weight) at 96 weeks (shortly before the end of the life span). In contrast, the log concentration of methanogens increased from 5.5 to 9/g (dry weight) during the same time period. Therefore, methanogens increased as a percentage of the total anaerobes from 0.005% at 3.0 weeks to 2.0% at 96 weeks. About 12 doublings of the methanogenic population and 3.3 doublings of the nonmethanogenic population took place from weaning until death. The slow increase in the ratio of methanogens to total anaerobes with age followed the same pattern in cecal contents as found in feces. There were no relationships between animal weights or fecal outputs and the increase in total anaerobe and methanogen concentrations in feces. A possible explanation for the slow increase in the Methanobrevibacter species in Wistar rats with age is a gradual shifting of the use of electrons from the reduction of CO2 to acetate by acetogens to the reduction of CO2 to CH4. The results provide the first evidence for an age-related change in the nonmethanogenic bacteria of the colon and supporting microbiological evidence for physiological studies that have shown age-related increases in colonic methane production in humans.

Specific antisera were raised in rabbits to 19 methanogenic bacteria representing the species available in pure culture at the present time. The antisera were characterized, labeled, and organized in a bank to serve as a source of material for preparation of antibody probes and thus provide standardized reagents for immunological analysis of methanogens. An indirect immunofluorescence procedure was standardized for optimal staining of homologous and heterologous bacterial strains. Two immunoenzymatic assays were developed: (i) a simple slide assay, useful for rapid antibody detection in small samples, antibody titrations, and disclosure of cross-reactions among methanogens, and (ii) a quantitative method. The latter is useful for quantification of antigenic relatedness. Procedural details were developed to obtain optimal bacterial preparations for use as immunogens to raise antibodies in vivo, and as antigens for antibody assay in vitro.

Preparation of new antisera has permitted more comprehensive immunological analyses of the two families of Methanomicrobiales. Methanomicrobiaceae and Methanosarcinaceae, and the species Methanobrevibacter arboriphilus. Immunological analysis was carried out with antibody probes against 23 strains, including almost all genera and species of methanogens. The absence of cross-reactions between families of methanogens was confirmed. Methanomicrobium and Methanogenium were found to be immunologically related. Extensive cross-reactions occurred among six strains of Methanosarcinaceae, but none occurred among three strains of M. arboriphilus when tested with the S probe, i.e., the last antiserum dilution of the titration curve's plateau.

The oxidation of pyruvate by resting-cell suspensions of a coagulasepositive, enterotoxin-producing strain of Staphylococcus aureus has been studied. With moderate substrate levels (10–20 μM per Warburg cup) pyruvate was oxidized almost exclusively to acetate, carbon dioxide, and water. The respiratory quotient of this oxidation varied from 1.4 to 1.9 depending on the cell preparation used. The results indicate that some of the substrate was oxidized beyond the acetate level (R.Q., 2.0). Methylene blue or oxygen exerted an inhibitory effect upon pyruvate oxidation, and, under certain conditions, thiamine and magnesium overcame this inhibition. Evidence is presented to support the hypothesis that this organism possesses two separate systems for pyruvate oxidation. Washed-cell suspensions oxidize pyruvate predominantly by a system that is sensitive to methylene blue and oxygen. When this system is inhibited the competing system becomes dominant. However, the washed cells are deficient in thiamine and magnesium for optimum functioning of this latter system. A by-product of this second system is acetoin, indicating that there is an intermediate compound that may be either oxidized to acetate or condensed to form acetoin. If the washed cells are provided with a high substrate level or are supplemented with additional thiamine and magnesium, some of the pyruvate is metabolized by the second pathway even in the absence of an inhibitor for the first system.

IN VITRO UTILIZATION OF LACTOSE BY THE FECAL FLORA DIFFERS IN BREAST-(BR) AND BOTTLE-FED (BO) INFANTS