Comparative study of bacterial groups within the human cecal and fecal microbiota.

The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides, and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces.

A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products.

This paper introduces a new type of system to simulate conditions in the large intestine. This system combines removal of metabolites and water with peristaltic mixing to obtain and handle physiological concentrations of microorganisms, dry matter and microbial metabolites. The system has been designed to be complementary to the dynamic multi-compartmental system that simulates conditions in the stomach and small intestine described by Minekus et al. [Minekus M, Marteau P, Havenaar R, Huis in't Veld JHJ (1995) ATLA 23:197-209]. High densities of microorganisms, comparable to those found in the colon in vivo, were achieved by absorption of water and dialysis of metabolites through hollow-fibre membranes inside the reactor compartments. The dense chyme was mixed and transported by peristaltic movements. The potential of the system as a tool to study fermentation was demonstrated in experiments with pectin, fructo-oligosaccharide, lactulose and lactitol as substrates. Parameters such as total acid production and short-chain fatty acid (SCFA) patterns were determined with time to characterize the fermentation. The stability of the microflora in the system was tested after inoculation with fresh fecal samples and after inoculation with a microflora that was maintained in a fermenter. Both approaches resulted in total anaerobic bacterial counts higher than 10(10) colony-forming units/ml with physiological levels of Bifidobacterium, Lactobacillus, Enterobacteriaceae and Clostridium. The dry matter content was approximately 10%, while the total SCFA concentration was maintained at physiological concentrations with similar molar ratios for acetic acid, propionic acid and butyric acid as measured in vivo.

Effect of yeast extract on growth and metabolism of H2-utilizing acetogenic bacteria from the human colon.

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1-2 g. L-1 in the medium did not enhance utilization of H2/CO2. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species.

Fermentation of green alga sea-lettuce (Ulva sp) and metabolism of its sulphate by human colonic microbiota in a semi-continuous culture system.

The green alga, sea-lettuce (Ulva sp), could be considered as a new source of dietary fibre. Ulva, however, contains high levels of sulphate, part of which is chemically bound in soluble polymers (ulvan). The purpose of this study was to assess the fermentation characteristics and sulphate metabolism of Ulva and ulvan by human faecal bacteria fermentation system using a semi-continuous fermenter. Ulva and ulvan were poorly fermented, even after adaptation of the microbiota. Only 16.6% and 8.9% of Ulva and ulvan organic matter, respectively, were recovered as short chain fatty acids. Nevertheless, 40% of the sulphate in Ulva was dissimilated to sulphide by sulphate-reducing bacteria. Supplementation of Ulva with more fermentable polysaccharides, such as algal xylan and resistant starch, though decreasing the ammonia production originating from Ulva protein degradation, did not significantly reduce the sulphide levels. It is postulated that unless crude Ulva is desulphated, its daily consumption at a level of 20 g of dry product could stimulate colonic microbial sulphate reduction, which may have detrimental effects for the host.

13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon.

Glucose fermentation by four acetogenic species (two Clostridium strains, one Streptococcus strain and Ruminococcus hydrogenotrophicus) isolated from the human colon was of a mixed-acid type, whereas pyruvate metabolism was characterised by homoacetogenesis. Acetate formation from [1-13C] and [2-13C]glucose was consistent with the formation of acetyl-SCoA from pyruvate generated by the Embden-Meyerhof-Parnas pathway. Labelling of lactate and ethanol demonstrated that these metabolites were formed by reduction of pyruvate and acetyl-SCoA, respectively. In contrast, the reductive pathway of acetate formation was the preferential means of re-oxidising cofactors formed during [1-13C]pyruvate catabolism.

Utilization of algal polysaccharides by human colonic bacteria, in axenic culture or in association with hydrogenotrophic microorganisms.

The ability of different hydrolytic bacteria from the human colon to grow on various algal polymers (carrageenans, Palmaria palmata xylan, ulvan, desulphated ulvan and laminaran) was investigated and the interactions between Bacteroides thetaiotaomicron and H2-utilizing microorganisms (one methanogenic archaea and an acetogenic bacterium) were studied during laminaran degradation. None of the algal polysaccharides supported the growth of any of the hydrolytic species tested, except for laminaran, which allowed substantial growth of B thetaiotaomicron. This suggested that bacterial consortia were involved in algal polymer breakdown rather than one specific bacterial species. The presence of H2-utilizing microorganisms did not increase the extent of laminaran degradation by B thetaiotaomicron. Whereas the decrease in formate and H2 concentrations attested to their utilization by both hydrogenotrophic microorganisms, the large increase in acetate production observed in the coculture with acetogenic bacteria was mainly due to acetogenic fermentation of sugars released during laminaran hydrolysis.

H2/CO2 metabolism in acetogenic bacteria isolated from the human colon.

The present work reports on autotrophic metabolism in four H2/CO2-utilizing acetogenic bacteria isolated from the human colon (two Clostridium species, one Streptococcus species, and Ruminococcus hydrogenotrophicus). H2/CO2-utilization by these human acetogenic strains occurred during both exponential and stationary phases of growth. Acetate was the major metabolite produced by all isolates following the stoichiometric equation of reductive acetogenesis. Furthermore, the ability of these acetogenic bacteria to incorporate 13CO2 into acetate in the presence of H2 in the gas phase demonstrated the utilization of the reductive pathway of acetate formation from a one-carbon compound. Energy conservation during the autotrophic metabolism in colonic acetogens might involve sodium- or proton-chemiosmotic mechanisms. A sodium-dependent ATP generation was only demonstrated in one Clostridium species, whereas sodium could be replaced by potassium in other strains. The minimal thresholds of hydrogen uptake were determined and varied from 1100 to 3680 ppm depending on the acetogenic strain. These values appeared higher than those measured for the colonic methanogen,Methanobrevibacter smithii.

Ruminococcus hydrogenotrophicus sp. nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces.

A new H2/CO2-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject. The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli. The isolates grew autotrophically by metabolizing H2/CO2 to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds. The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate. The G+C content of DNA of strain S5a33 was 45.2 mol%. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed. The type strain of R. hydrogenotrophicus is S5a33 (DSM 10507). Furthermore, H2/CO2 acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus).

Diversity of H2/CO2-utilizing acetogenic bacteria from feces of non-methane-producing humans.

The purpose of this work was to study H2/CO2-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H2-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H2/CO2 as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H2/CO2 to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H2/CO2.

Inhibition of the cellulolytic activity of Neocallimastix frontalis by Ruminococcus flavefaciens.

A study was made of the antagonistic effect of Ruminococcus flavefaciens on the cellulolytic activity of Neocallimastix frontalis. An extracellular factor inhibiting the cellulolytic activity of the fungus was detected in the bacterial supernatant. The antagonistic factor, which precipitated with ammonium sulphate at 40% saturation, was temperature-sensitive and was destroyed at temperatures above 60 degrees C. After separation by anion-exchange chromatography, sequential precipitation, dialysis and SDS-PAGE, two protein species of 100 and 24 kDa were identified as being involved in this antagonistic effect. It is known whether the proteins are two subunits of a single protein or represent two different proteins. The inhibitory factor, which is not a bacterial cellulase, did not affect fungal growth, but it inhibited the activity of the fungal cellulases.

Effect of Eubacterium limosum, a ruminal hydrogenotrophic bacterium, on the degradation and fermentation of cellulose by 3 species of rumen anaerobic fungi.

The degradation and fermentation of cellulose filter paper were studied in axenic cultures of 3 species of rumen anaerobic fungi, Neocallimastix frontalis, Piromyces communis and Caecomyces communis, and in cocultures containing 1 of these fungal strains and Eubacterium limosum, a hydrogenotrophic rumen bacterial species. When E limosum was introduced into fungal cultures a slight decrease in fungal cellulolytic activity was observed. The end products of the fermentation of cellulose found in the cocultures were different from those found in the fungal monocultures. E limosum used formate and part of the hydrogen produced by the fungi and probably created a shift in the metabolism of the fungi resulting in a reduction of lactate and ethanol production.

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose.

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.

Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus.

A new species of strictly anaerobic fungus was isolated from the cow rumen. It is characterized by a polycentric thallus, a polynuclear rhizomycelium, mucronate zoosporangia and uniflagellated zoospores. It is also singular in that the sporocysts do not react to the specific lectins of L-fucose, N-acetyl-D-galactosamine and diacetyl chitobiose. These characteristics justify the creation of a new genus.

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii.

A new strain of strictly anaerobic fungi was isolated from the rumen of sheep. This strain is characterized by a polycentric thallus, an extensive and polynuclear rhizomycelium, polyflagellated zoospores with gamma particle-like bodies. We propose to assign this strain in a new species: Neocallimastix joyonii.