Meyer J. Wolin

Methanosphaera stadtmaniae gen. nov., sp. nov.: a species that forms methane by reducing methanol with hydrogen

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° to 40° 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.

Methanogens in human and animal intestinal Tracts

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.

Enumeration of Methanobrevibacter smithii in human feces

A platin 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 1–8×1011/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 thay 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.

The Rumen Fermentation: A Model for Microbial Interactions in Anaerobic Ecosystems

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.

Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii

SummaryThe 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 (Ks) 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.

Isolation and characterization of methanogens from animal feces

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.

Bioconversion of organic carbon to CH4 and CO2

Abstract 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.

Formate-Dependent Growth and Homoacetogenic Fermentation by a Bacterium from Human Feces: Description of Bryantella formatexigens gen. nov., sp. nov

ABSTRACT 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.

Molybdate and sulfide inhibit H2 and increase formate production from glucose by Ruminococcus albus.

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.

Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri.

Abstract 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.