Chemostat enrichments of human feces with resistant starch are selective for adherent butyrate-producing clostridia at high dilution rates

Resistant starch (RS) enrichments were made using chemostats inoculated wit h human feces from two individuals at two dilution rates (D = 0.03 h(-1) an d D = 0.30 h(-1)) to select for slow- and fast-growing amylolytic communiti es. The fermentations were studied by analysis of short-chain fatty acids, amylase and ol-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide pr obes. Considerable butyrate was produced at D = 0.30 h(-1), which correspon ded with reduced branched-chain fatty acid formation. At both dilution rate s, high levels of extracellular amylase activity were produced, while or-gl ucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia be came more important at D = 0.30 h(-1). Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominate d in RS enrichments at D = 0.30 h(-1). This organism attached apically to R S granules and formed rosette-like structures which, with glycocalyx format ion, agglomerated to form biofilm networks in the planktonic phase. Attempt s to isolate this bacterium in pure culture were repeatedly unsuccessful, a lthough a single colony was eventually obtained. On the basis of its 16S rD NA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I Clostridium sp, A 16S rRNA-targe ted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at D = 0.03 and 0.30 h(-1), respecti vely. This study indicates that bacterial populations with significant meta bolic potential can be overlooked using culture-based methodologies. This m ay provide a paradigm for explaining the discrepancy between the low number s of butyrate-producing bacteria that are isolated from fecal samples and t he actual production of butyrate.