pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon

Abstract

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.

FIG. 1.

Time course of SCFA and ammonia formation in continuous fermentor cultures maintained at pH 6.5 on high (0.6% peptide) and low (0.1%) peptide, following inoculation with a fecal sample from donor 2. Concentrations of products are indicated by symbols as follows: formate (▴), acetate (⋄), propionate (▪), butyrate (•), valerate (□), lactate (⧫), and succinate (×). Ammonium concentrations (▵) are shown on a 10-fold expanded scale, indicated in parentheses. Each value is the average of three independent analyses; error bars indicate the standard deviations.

FIG. 2.

Time course of SCFA formation in continuous fermentor cultures initiated at pH 5.5 and 0.6% peptide input after inoculation with a fecal sample from donor 1 (a) and donor 2 (b). The pH was shifted progressively to pH 6.5 within the interval indicated by the triangle. The outcome of parallel experiments (not shown) at 0.1% peptide input is summarized in Table 2. Concentrations of products are indicated as follows: acetate (⋄), propionate (▪), butyrate (•), and valerate (□). Concentrations of formate, lactate, succinate, and caproate (not shown) did not exceed 2 mM. Each value is the average of three independent analyses; error bars represent the standard deviations.

FIG. 3.

Effect of pH upon the abundance of major bacterial groups, expressed as a proportion of the total (Eub338) counts, in continuous fermentor cultures with high (0.6%) (a) and low (0.1%) (b) peptide input. Mean proportions are calculated from the FISH counts given in Table 3 and refer to all of the experiments involving the two donors that are summarized in Table 2 and illustrated in Fig. 1 and 2. Means are given for the initial 0.5-h time point (▪), after incubation at pH 5.5 (▧), and after incubation at pH 6.5 (□). Statistical significance for the effects of different pH and peptide regimes is indicated as follows: ✽, P = <0.05; ✽✽, P = <0.01; ✽✽✽, P = <0.001.

FIG. 4.

Comparison of growth rates (h⁻¹) for 14 representative pure strains of human fecal bacteria grown in batch culture on YCFAG medium at initial pH values of 6.7, 6.2, and 5.5 (see Materials and Methods). Where growth occurred, final pH values were up to 0.8 U lower than the initial pH (lowest value 5.1). ✽, Lack of growth at pH 5.5. The phylogenetic group or cluster to which each strain belongs (clostridial clusters XIVa, IV, IX, and XVI, Atopobium [Ato]; Bifidodobacterium [Bif]; and Bacteroides) and the probes that recognize them are shown above the histogram.