This study investigated the effect of saponins gypenoside (gynosaponins) on methane production and microbe numbers in a co-culture of a fungus, Piromyces sp. F1, and a methanogen, Methanobrevibacter sp.. Two co-culture systems were used: with methanogen (co-culture I) and without methanogen (co-culture II; methanogen growth inhibited by chloramphenicol). Each co-culture system was treated with 0, 50, 100 or 200 mg gynosaponins/l culture medium. Gas production, methane concentration and volatile fatty acid concentration (VFA) were measured for each treatment group. The numbers of anaerobic fungi and methanogen were quantified by real time PCR. The results showed that, compared with the control, gynosaponin significantly reduced the gas production, methane concentration, methane to TVFA ratio (total volatile fatty acid), TVFA concentration, number of fungi (except for 50 mg dose of gynosaponin in co-culture I) and number of methanogens. Methane was not detected in co-culture II. The individual VFAs proportion of TVFA were not affected by gynosaponins in either of the co-cultures. The pH was higher in both co-cultures than that of the control (P<0.01). These data suggest that gynosaponins has the potential for being used as feed additive to modulate the ruminal fermentation, inhibit the methanogen growth and reduce methane production.

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and α-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and α-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

BackgroundThe novel archaea belonging to Rumen Cluster C (RCC), which may play an important role in methane production in the rumen have received increased attention. However, the present information on RCC in the rumen is limited by the unsuccessful isolation of axenic pure RCC from the rumen. In the present study, RCC grown in anaerobic fungal subcultures was identified by the molecular and culture methods.ResultsA novel RCC species existing in the fungal subcultures was identified and demonstrated by the 16S rRNA gene clone library. Interestingly, the novel RCC species survived in the fungal cultures over all the subculture transferring, even in the 62nd subculture, in contrast to the other methanogens, which disappeared during subcultures. Further work showed that subculture transfer frequency significantly affected the relative abundance of the novel RCC species in the fungal subcultures. The five-day and seven-day transfer frequencies increased the relative abundance of the RCC species (P<0.05). In addition, quantitative real-time PCR revealed that high concentrate diets did not affect the abundance of archaea, but numerically reduced the abundance of the novel RCC species in the rumen. In addition, the relative abundance of the RCC species was numerically higher in the rumen liquid fraction than in the rumen epithelium and solid fractions. Finally, a purified fungal culture containing the RCC species was successfully obtained. PCR and sequencing analysis showed that the novel RCC species contained a mcrA gene, which is known to play a crucial role in methanogenesis, and thus could be identified as a methanogen.ConclusionIn this study, a novel RCC species was identified as a methanogen and closely associated with anaerobic fungi. This novel approach by using co-culture with anaerobic fungi may provide a feasible way to culture and investigate not yet identified methanogens.

An intercomparison of methodology and of dating results, from α-counting and from the more recent γ-spectrometry method, has been made for marine sediment cores from several locations. The former method involves considerable chemical manipulation, whereas the latter is more straightforward and enables direct interpretation of the results. Similar precisions (∼ 12%) were found for both methods. With the constant flux model, the methods gave calculated sedimentation rates between 4.3 and 7.8 cm a−1 for replicate cores near a ferry pier in Hong Kong Harbor. The γ-spectrometry method is thus a reliable, more rapid method for routine monitoring and investigation of the harbor dynamics and history of pollution.