Molecular ecology of plant cell wall hydrolysis by mixed cultures of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85

Abstract

A total of 6 specific oligonucleotide probes were designed for the Ruminococci, 3 targeted to sites on 16S rRNA of Ruminococcus albus and 3 targeted to sites on 16S rRNA of Ruminococcus flavefaciens. A subspecies probe for Fibrobacter succinogenes S85 (Stahl et al., 1988) and a eubacterial kingdom probe (Stahl and Amann, 1991) also were used. Bacteria were grown as monocultures, dicultures and in triculture on cellobiose, cellulose and alkaline hydrogen peroxide-treated wheat straw to study competition among the three bacterial species on these substrates. The cellubiose-grown cultures were incubated at 39$\sp\circ$C for 10 hours and sampled hourly. The cellulose and alkaline hydrogen peroxide-treated wheat straw-grown cultures were incubated at 39$\sp\circ$C for 70 hours and sampled every 8 hours. Ribosomal RNA was extracted from all the samples, blotted on nylon membranes and hybridized with labelled $\sp{32}$P probes. The membranes the were exposed to films. Results showed that R. albus 8 inhibited the growth of R. flavefaciens FD-1 when grown as a diculture on cellubiose or cellulose. However, R. albus 8 was outnumbered by F. succinogenes S85 on cellobiose but not on cellulose. R. flavefaciens FD-1 outnumbered F. succinogenes S85 on both cellobiose and cellulose. In the triculture, R. flavefaciens FD-1 was inhibited, R. albus 8 appeared to dominate during early incubation (12-48 hours) on cellulose, while F. succinogenes S85 became predominant during the late incubation phase (60-70 hours). On alkaline hydrogen peroxide-treated wheat straw, F. succinogenes S85 grew better than either R. albus 8 or R. flavefaciens FD-1. However, R. flavefaciens FD-1 was present in low numbers throughout the incubation period unlike on cellobiose and cellulose. The results on cellobiose and cellulose suggest that R. albus 8 produced a substance that inhibited the growth of R. flavefaciens FD-1.