| Three experiments were performed using 16S rRNA-targeted oligonucleotide probes to study ruminal cellulolytic populations in the presence of condensed tannins. Initially, tannin-binding buffers were developed to remove tannins prior to bacterial RNA extraction. Cultures of Ruminococcus albus 8 were exposed to 8 g/l tannic acid or to 1 g/l condensed tannins from Acacia angustissima, Desmodium ovalifolium, Inga edulis, banana, grape skins or no tannin. Cultures without added tanin served as the negative control. Tris buffer pH 7 containing either 8% polyethylene glycol (PEG) or 6% polyvinylpyrrolidone (PVP) was used. The inclusion of PEG or PVP in the buffer yielded similar amounts of RNA.;A set of two experiments estimated microbial populations using either nitrogen determinations or 16 S rRNA-targeted probes. Using pure cultures, there were strong relationships (r = 0.97) among optical density, protein (Lowry), RNA extracted, and equivalents of RNA estimated by the purine assay. RNA hybridization with a universal probe tended to underestimate the amounts of RNA from mixtures of pure cultures while the R. albus-specific probe yielded signals proportional to the R. albus cell numbers. Both the Lowry and the purine methods produced highly variable estimates of microbial population probably due to the high plant dry matter content of the samples. The universal probe yielded satisfactory estimations of RNA. The signal from R. albus-specific probe estimated a 3% population, although coefficients of variation varied from 40% to 60%. Apparently plant material contaminants were co-extracted with RNA causing nonspecific probe binding. Microbial populations were more accurately estimated using the molecular probes compared with estimates using the nitrogen methods tested.;Using gas production, volatile fatty acids, neutral detergent fiber disappearance, and RNA hybridization to universal and species-specific probes we monitored cocultures of R. albus 8 and Fibrobacter succinogenes S85 digesting Cynodon plectostachyus in the presence of 0, 100, 150 or 250 mug D. ovalifolium condensed tannin/ml. Increasing tannin concentrations increased (P < 0.05) the lag phase. Increased production of succinic acid was found in samples with the strongest signal from the F. succinogenes probe. Because there was wide variation within replicates and among samples, the hybridization results were difficult to interpret. |
