Polysaccharide breakdown by mixed populations of human faecal bacteria

Measurements of polysaccharide-degrading activity in different fractions of human faeces showed that bacterial polysaccharidases and glycosidases were primarily associated with the washed bacterial fractions. Amylase, pectinase and xylanase were the major polysaccharide-hydrolysing enzymes detected, whilst α-L-arabinofuranosidase, β-D-xylosidase, β-D-galactosidase and β-D-glucosidase were the most active glycosidases. Starch and 3 non-starch polysaccharides (NSP; pectin, xylan and arabinogalactan) were fermented by mixed populations of human faecal bacteria in batch culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly degraded substrates and that arabinogalactan and the relatively insoluble polysaccharide xylan were broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the batch culture fermentations showed that the arabinose side chains of pectin, xylan and arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but extracellular polysaccharidase activity increased as the fermentations progressed. Molar ratios of acetate, propionate and butyrate produced in these experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate, propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were 50:22:29, 50:42:8, 82:15:3, and 84:14:2, respectively. The presence of starch did not inhibit the breakdown of arabinogalactan, xylan or pectin by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided with mixed polysaccharide substrates.     

Metabolism of unsaturated bile acids and androstanes by human faecal bacteria

The metabolism of unsaturated bile acids and androstanes by mixed human faecal cultures has been studied. The reactions observed were mainly reductive. Unsaturated 4-ene-3-oxo and 1,4-diene-3-oxo bile acids were reduced in Ring A. 5 beta-3-Oxo bile acids were reduced to 5 beta-3-hydroxy bile acids. 4-Ene, 1,4-diene and 4,6-diene-3,17-dioxo-androstanes were reduced in Ring A with concomitant reduction of oxo groups to hydroxyl groups. The Gram-negative facultative anaerobic faecal bacteria are implicated in the reductive process, whilst the genus Clostridium does not appear to be important. Inclusion of menadione, a synthetic form of vitamin K, retards the reductive process.     

Comparison of populations of human faecal bacteria before and after in vitro incubation with plant cell wall substrates

Human faecal slurries were incubated anaerobically with larchwood xylan, oat spelt xylan, wheat bran, apple cell walls or sugar beet pulp as sole carbon sources. The populations which developed during incubation were different from the inoculum, the most marked changes being an increase in the number of Bacteroides species and a decrease in the number of Fusobacterium species for all carbon sources tested. With a water-soluble preparation of larchwood xylan the population was dominated by species able to ferment this substrate, in contrast to the population which developed with the insoluble substrates. The ability to use one plant cell wall substrate appeared to be related to the ability to use others. Strains capable of using plant cell wall substrates included Bacteroides spp., Clostridium clostridiiforme, Bifidobacterium longum, Fusobacterium spp. and Escherichia coli. When incubation with two contrasting substrates (bran and larchwood xylan) was replicated, the populations which developed were reproducibly different from the inoculum and from each other.     

Comparison of populations of human faecal bacteria before and after in vitro incubation with plant cell wall substrates

Human faecal slurries were incubated anaerobically with larchwood xylan, oat spelt xylan, wheat bran, apple cell walls or sugar beet pulp as sole carbon sources. The populations which developed during incubation were different from the inoculum, the most marked changes being an increase in the number of Bacteroides species and a decrease in the number of Fusobacterium species for all carbon sources tested. With a water-soluble preparation of larchwood xylan the population was dominated by species able to ferment this substrate, in contrast to the population which developed with the insoluble substrates. The ability to use one plant cell wall substrate appeared to be related to the ability to use others. Strains capable of using plant cell wall substrates included Bacteroides spp., Clostridium clostridiiforme, Bifidobacterium longum, Fusobacterium spp. and Escherichia coli. When incubation with two contrasting substrates (bran and larchwood xylan) was replicated, the populations which developed were reproducibly different from the inoculum and from each other.     

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased.     

Hydrolysis of iodothyronine glucuronides by obligately anaerobic bacteria isolated from human faecal flora

Abstract 35 bacterial strains isolated from the human faecal flora were screened for hydrolysis of the glucuronides of 3,3',5-triiodothyronine and 3,3'-diiodothyronine. Two Gram-positive obligately anaerobic strains possessed glucuronidase activity. These strains probably belong to the genus Eubacterium , but ethanol was produced in high concentrations during glucose fermentation, which makes final classification difficult. Considering the number of bacteria in the intestinal flora (> 10⁸/ml) and the biliary excretion of iodothyronine conjugates, the strains must be able to hydrolyse a major part of the total daily intestinal supply of these iodothyronine metabolites. The study extends previous observations with faecal suspensions of human and rat origin [24]. The relevance of bacterial β-glucuronidase activity for a possible enterohepatic circulation of iodothyronines is discussed.     

Effect of nitrate on methane production and fermentation by slurries of human faecal bacteria

Most probable number counts showed that denitrifying species were the numerically predominant NO3- reducing bacteria in the faeces of five methanogenic individuals [about 10(10) bacteria (g dry wt faeces)-1]. In faecal slurries, however, denitrification was a relatively minor route of NO3- dissimilation, since only about 3% of the NO3- was converted to gaseous products, with NO3- being mainly reduced to NO2- and NH4+. When KNO2 was added to the slurries, denitrification became quantitatively more significant with approximately 23% of the NO2- being lost as gaseous products. The addition of KNO3 (10 mM) to slurries containing either starch or casein significantly decreased H2 and CH4 production. The effect of NO3- on methanogenesis was twofold: firstly, H2 accumulation decreased due to diversion of electrons towards NO3-/NO2- reduction, and as a result of H2 being used as an electron donor for NO3- reduction, resulting in the removal of the methanogenic substrate; secondly, there was direct inhibition of methane-producing bacteria by NO3- and NO2-. In starch-containing slurries, acetate: butyrate molar ratios were increased when NO3- was added but this effect was not observed when casein replaced starch. These results show that the ability of NO3-/NO2- to act as an electron sink can significantly influence the major products of the human colonic fermentation.     

Response of populations of human faecal bacteria to viscosity in vitro

The effects of viscosity on cultures of human faecal bacteria in vitro were studied. The composition of the microbial populations which developed during in vitro incubation were affected by viscosity. The proportion of Bacteroides species was decreased at high viscosities and it is concluded that the 'viscotolerance' of a bacterial population contributes towards its composition. Viscosity alters the enzyme activity of cultures. Cell-associated total, and specific, beta-galactosidase activities of cultures of human faecal bacteria were decreased at high viscosity.     

Response of populations of human faecal bacteria to viscosity in vitro

The effects of viscosity on cultures of human faecal bacteria in vitro were studied. The composition of the microbial populations which developed during in vitro incubation were affected by viscosity. The proportion of Bacteroides species was decreased at high viscosities and it is concluded that the 'viscotolerance' of a bacterial population contributes towards its composition. Viscosity alters the enzyme activity of cultures. Cell-associated total, and specific, β-galactosidase activities of cultures of human faecal bacteria were decreased at high viscosity.     

Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen

AIMS: To investigate the mode of action of a blend of essential oil compounds on the colonization of starch-rich substrates by rumen bacteria. METHODS AND RESULTS: Starch-rich substrates were incubated, in nylon bags, in the rumen of sheep organized in a 4 x 4 latin square design and receiving a 60:40 silage : concentrate diet. The concentrate was either high or low in crude protein, and the diet was supplemented or not with a commercial blend of essential oil compounds (110 mg per day). The total genomic DNA was extracted from the residues in the bags. The total eubacterial DNA was quantified by real-time PCR and the proportion of Ruminobacter amylophilus, Streptococcus bovis and Prevotella bryantii was determined. Neither the supplementation with essential oil compounds nor the amount of crude protein affected the colonization of the substrates by the bacteria quantified. However, colonization was significantly affected by the substrate colonized. CONCLUSIONS: The effect of essential oils on the colonization of starch-rich substrates is not mediated through the selective inhibition of R. amylophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enhances our understanding of the colonization of starch-rich substrates, as well as of the mode of action of the essential oils as rumen manipulating agents.     

Endophytic colonization of plant roots by nitrogen-fixing bacteria

Plant and Soil - Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In...     

Adsorption of Thermomonospora curvata cellulases on insoluble substrates

Cellulosic substrates were tested for their ability to bind the cellulases of Thermomonospora curvata . Protein-extracted lucerne fibres had the highest adsorptive capacity while native cotton fibres had the least. Analysis of binding as a Langmuir adsorption isotherm yielded maximal binding capacities of 0.012 filter paper units and 0.51 endoglucanase units/mg lucerne fibre at enzyme saturation. This capacity was increased about 12-fold by fibre pre-treatment in hot NaOH solution.     

Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

BACKGROUND: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. AIM: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. METHODS: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). RESULTS: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. CONCLUSIONS: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).     

Effect of nematodes on rhizosphere colonization by seed-applied bacteria

There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of -10 and -40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.