Several different approaches can be employed to understand and predict the selective influence of ND carbohydrates upon the gut microbiota. Analysis of carbohydrate active enzyme (CAZyme, Carbohydrate Active Enzymes database [16, 17], URL http://www.cazy.org/) complements from individual genomes [18] and growth tests on isolated species [19] may be indicative, but cannot predict how different organisms will compete and interact within the complex intestinal community. At the other extreme, in vivo feeding experiments cannot readily distinguish effects mediated via the gut environment, for example, changes in gut transit or pH, from the direct selective effects of the substrate. Furthermore, in vivo nutritional studies do not provide precise control over the substrates available to the microbiota, since endogenous substrates such as mucin and other food components will always be present. The approach that we have adopted here of using in vitro continuous flow fermentors to study the human intestinal microbiota as a microcosm has the considerable advantage of allowing precise control over both substrate supply and pH.

Detailed investigations into polysaccharide utilization have so far been mainly undertaken for Bacteroides spp. among predominant human gut anaerobes [20], although more recently studies have also been conducted in the Firmicutes representative Eubacterium rectale [21]. In Bacteroides spp., genes responsible for the degradation of particular polysaccharides are organized at polysaccharide utilization loci (PULs) that also encode transport functions and transcriptional regulators. Individual Bacteroides genomes typically possess large numbers of PULs (over 80) that are concerned with degradation of different host- and diet-derived polysaccharides [22, 23]. Variations in PUL distribution between species have been demonstrated to correlate with the ability to utilize particular polysaccharides in vitro and in gnotobiotic animal models [23–25]. Our results show that all of the eight most abundant Bacteroides OTUs present in the initial faecal microbiota gave a strong differential response in the mixed fermentor communities, being stimulated either by inulin (in two cases) or by pectin (in six cases). Similar selectivity was evident for many Firmicutes. Among the top 38 OTUs (each representing 0.5 % or more of the total sequences), five Firmicutes OTUs increased in abundance with inulin, while a further five Firmicutes OTUs increased with pectin (Additional files 3 and 4: Tables S2 and S3).

Previous studies on cultured isolates indicated that pectin utilization is widespread among Bacteroides spp., but relatively uncommon among human colonic Firmicutes [26, 27]. Nevertheless, the strong and highly specific enrichment of the pectin-utilizing Firmicutes species E. eligens [26, 27] could provide the basis for an effective prebiotic strategy while enrichment of one F. prausnitzii OTU by pectin was also observed here. Inulin stimulated the Firmicutes species F. prausnitzii and A. hadrus (strains related to the isolate SS2/1) as has also been reported in an in vivo human study [10, 15]. Both species are butyrate-producing bacteria that offer potential benefits to health, via anti-inflammatory action in the case of F. prausnitzii [28], and via conversion of D-lactate to butyrate in the case of A. hadrus [29, 30]. In addition to promoting particular Bacteroidetes and Firmicutes OTUs, inulin also promoted some Proteobacteria, including Sutterella wadsworthensis. This may warrant further investigation as S. wadsworthensis has been isolated not only from healthy individuals but also from gastrointestinal disease states [31].

Many studies have shown that inulin has a bifidogenic effect in vivo [9, 32–34] and there is also a report suggesting that pectin can be bifidogenic [35]. Interestingly, we found no evidence in these experiments for an overall stimulation of bifidobacteria by inulin or pectin either from sequence analysis, even though B. pseudocatenulatum was detected as one of the 10 most abundant OTUs, or from qPCR monitoring of the Bifidobacterium genus. Since the representation of bifidobacteria was greatest at pH 5.5 in the inulin fermentors (Fig. 7), it therefore seems likely that the promotion of bifidobacteria by inulin that is observed in vivo depends critically on acidification in the colonic lumen that results from SCFA production [12]. At pH values closer to neutrality our evidence suggests that other bacteria will tend to out-compete bifidobacteria for inulin. It is also known that the chain length of inulin is critical in determining its utilization by isolated gut anaerobes [19], which can complicate the comparison of different studies. Many isolated bifidobacteria cannot utilize long chain inulin, although they are able to benefit from fructo-oligosaccharide breakdown products via cross-feeding in co-cultures [36]. In general, metabolic cross-feeding involving partial degradation products, fermentation products and growth factors, but also inhibitory interactions, can be expected to have played a role in the community changes occurring in these studies [37, 38].

The overall decrease in microbiota diversity in the fermentor community relative to the faecal inoculum (seen from Fig. 6) is assumed to result mainly from the supply of a single substrate as energy source. By contrast, typical human diets supply a very wide variety of types of fibre and a wide array of host-derived glycans and proteins (especially mucins). Another important factor is likely to be the relative stability of conditions within the fermentor compared with the fluctuations that occur in vivo as a result of periodic meals.

Our experiments also show that pH exerts a strong influence on competition between bacteria from different phyla or families that share the ability to utilize the same polysaccharide. This effect was particularly clear for the inulin fermentors, where the proportion of Bacteroidetes 16S rRNA gene copies fell from around 60 % at pH 6.9 to around 30 % at pH 5.5. This suppression of Bacteroides spp. at slightly acidic pH appears to reflect growth inhibition by SCFAs at pH values below 6 [11, 13]. Interestingly, this trend was not evident for the pectin upshift fermentors where E. eligens was apparently able to compete with pectin-utilizing Bacteroides spp. across the pH range. The correlation between Bacteroides representation in the community and the proportion of propionate that was seen here has also been noted for faecal samples from an in vivo study [6]. This is assumed to reflect the dominant role of the succinate pathway, which is found mainly in the Bacteroidetes among human colonic bacteria, in the formation of propionate from carbohydrates [39]. Thus, there was considerable functional redundancy with respect to propionate formation, as substrate-driven changes in the dominant species of Bacteroidetes appeared to have little influence. On the other hand, the species level changes seen here could potentially have many other effects on the host, including those via immune signalling and metabolite transformation and which will warrant investigation in future studies.

We observed some asymmetry in the response of the microbial community to low pH in parallel downshift and upshift fermentors that received the same inoculum. A possible explanation for this lies in the initial reduction in community diversity in the fermentors, discussed above. Theoretical modelling suggests that diversity will also have decreased at the strain level as strains that have optimal characteristics for pH tolerance and substrate utilization are selected [40]. Thus, the community at the start of the pH 5.5 phase in the downshift fermentors was considerably less diverse than the inoculum that initiated the pH 5.5 phase (P <0.005) in the upshift fermentors. This lower diversity may have led to the absence of Bacteroides strains tolerant of pH 5.5 at the end of the downshift runs, thus allowing the growth of more low-pH tolerant competitors such as F. prausnitzii [27, 40] and perhaps explaining the observed ‘blooms’ in F. prausnitzii that were seen in several fermentor runs (Fig. 5). Interestingly, sudden shifts in the ratio of Bacteroides to F. prausnitzii have also been reported in vivo in human subjects [41].

In summary, we have shown that two ND carbohydrates, inulin and pectin, promote very different community profiles when supplied as sole energy sources to human colonic microbial communities under conditions of controlled pH and turnover. Notably, these differences were found to lie at the species level rather than at the phylum or family level. We found very little overlap between the species stimulated by the two substrates, implying that evolution in the highly competitive environment of the large intestinal microbiota has favoured a high degree of nutritional niche specialization at the species level. At the same time, it is evident that a number of phylogenetically distant organisms belonging to different phyla have evolved convergently to gain the ability to utilize the same substrates. With a simple homopolymer (inulin) as a substrate, a single species was found to dominate the community at a given pH, but several species were apparently able to co-exist on pectin at the same pH. It is likely that the extreme chemical complexity of pectin [42] creates multiple nutritional niches which can explain the greater microbiota diversity seen in the pectin-fed relative to the inulin-fed communities. Our experiments also show that the species that become dominant with a given substrate can vary for individual microbial communities, and are likely to depend on the precise mix of competing strains within each microbiota.