| The runmen of herbivores is a comlpex ecosystem with potential for kinds of inter-microbial which includes anaerobic fungi, archaea and protozoa. Anaerobic fungi play important roles in the digestion of plant material and subsequent conversion for energy requirements to the host. It is well-known that some metabolites of anaerobic fungi such as formate and H2/CO2 are suitable substrates for methanogens, and the hydrogen transfer-species makes anaerobic fungi and methanogens interact closely. As one of the end-products, Methane could not only cuase the waste of animals, but also make contributions to the greenhouse. Therefore, the mechanism of the formation of methane has attracted great attetions. Although several simple and stable natural cultures of anaerobic fungus and its specific indigenously associated methanogen have been obtained, but the metabolic mechanism of the anaerobic fungi and methanogens need further investigation. Therefore, this study was conducted to investigate metabolism relationship between anaerobic fungi and methanogens in herbivorous by the approach of in vitro enrichment with the procedure for isolating anaerobic fungus and methanogen using Hungate roll-tube technique and dynamic change of mono anaerobic fungi and co-cultures of anaerobic fungi and methanogens of 96 hours fermentation on glucose, combined with HPLC and GC-MS method we have conducted, which aimed to further investigate the metabolic profile of anaerobic fungi and the effect with associated methanogens.1. Isolation and identification of cellulolytic anaerobic fungi and their associated methanogens from Holstein cowWe studied the microbial interaction between anaerobic fungi and methanogens in the rumen. Co-cultures of anaerobic fungus with indigenously associated methanogen were isolated by Hungate roll-tube technique. The anaerobic fungi were identified by morphology and 4’,6 diamidino-2-phylindole nucleus staining and the methanogens were identified by 16S rRNA gene sequencing. A total of 28 co-cultures of anaerobic fungus with indigenously associated methanogen were obtained. The anaerobic fungi in the co-cultures were identified as monocentric genera Piromyces, Neocallimastix and Caeomyces. The indigenously associated methanogens were Methanobrevibacter olleyae like strains and Methanobrevibacter thaueri like strains. Four different phylotypes of fungus-methanogen co-cultures were obtained, which were PiromyceslMethanobrevibacter olleyae like strains, Neocallimastix/Methanobrevibacter olleyae like strains, Neocallimastix/Methanobrevibacter thaueri ke strains and Caecomyces / Methanobrevibacter olleyae like strains, respectively. Our study isolated and identified 28 co-cultures of anaerobic fungus and associated methanogens, which provided new materials for further study the mechanism of methane emission in the rumen.2. The determination of main metabolic profile of mono anaerobic fungi and co-cultures of anaerobic fungi and methanogens by gas chromatography-mass spectrometerThe co-culutures of Piromyces sp. F1 and Methanobrevibacter thaueri like strain was used in this study and the mono anaerobic fungi was obtained by the addition of chloromycetin. To evaluate the effects of co-exist methanogens on metabolism of anaerobic fungi, we compared the intracellular and extracellular metabolites of mono anaerobic fungi and co-culutres by gas chromatography-mass spectrometer (GC-MS). The result of PLS-DA revealed the separation of metabolite profiles of the fungal mono-culture and co-cultures in both suspensions and cell-free extracts. The main different metabolites in suspensions were malic acid, pyruvic acid, citric acid and lactate. The mean values of lactate, citric acid and malic acid in fungal monoculture suspensions were significant higher than those in co-cultures (P< 0.05), while with a significant lower lactate concentration compared to co-cultures (P< 0.05). When comes to the cell-free extracts, the main different metabolites in cell-free extracts were citric acid, glucose, malic acid and pyruvic acid. The mean values of citric acid in monoculture were significant lower than those in co-cultures, while with a significant higher glucose concentration compared to co-cultures (P< 0.05). What is more, the mean values of malic acid and pyruvic acid were lower than those in co-cultures with no significant differences. These result indicated that anaerobic fungal metabolism was shifted by associated methanogens through hydrogenosome.3. Determination of orgnic acid in mono-anaerobic fungi and co-cultures anaerobic fungi with methanogens by high performance liquid chromatographyA high performance liquid chromatography method was developed for simultaneous determining formate, lactate, acetate, succinic acid, citric acid and a-ketoglutarate in mono and co-cultures anaerobic fungi and methanogens. Agilent SB-Aq column (2.6×250 mm×5 um) was used at 25 ℃. Mobile phase was 20 mM Na2HPO4 (pH 2.4) with stable flow rate which controlled by program. Detection UV wave length was at 214 nm. The method could fast and effectively separate and detect the organic acids in suspensions and cell-free extracts of anaerobic fungi. There coveries were 92.17%-101.61% and the relative standard deviations were 1.07%-8.30%. All the correlation co efficients were no less than 0.9992, with a proper precision and accuracy. The results showed that compared with the co-cultures, the formate concentration was significant higher in monoculture in both suspensions and cell-free extracts (P< 0.05), while significant lower acetate concentration was observed in monoculture than in co-cultures (P< 0.05). The concentration of lactate in suspensions was significant higher than in co-cultures (P< 0.05). The ethanol concentration of cell-free extracts in co-cultures was significant lower than in monoculture (P< 0.05). However, there were no significant differences in succinic acid, citric acid and a-ketoglutarate between two cultures. In all, this method could apply for determining the main orgnic acid in mono-anaerobic fungi and co-cultures anaerobic fungi with methanogens, the associated methanogens could shift the metabolism of anaerobic fungi.4. The dynamic state of mono anaerobic fungi and co-cultures of anaerobic fungi with methanogens on the fermentation of glucoseTo further investigate the metabolism profile of anaerobic fungi and the effect with associated methanogens, we researched the dynamic change of mono anaerobic fungi and co-cultures of anaerobic fungi and methanogens of 96 hours fermentation on glucose, combined with the HPLC method we have conducted. The results showed that total gas of co-cultures was significantly higher than monoculture (P< 0.05). The concentration of formate in both two groups were similar within 42 h, whereas the concentration of formate were sharp decreased after 48 h in co-cultures, with the accumulation of formate in monoculture, the concentration of lactate and ethanol were higher in monoculture than those in co-cultures all the time, especially at 60 h and 30 h, respectively (P< 0.05), while the concentration of acetate was higher in co-cultures than in monoculture. Besides, the citric acid, succinic acid and α-ketoglutarate had a fluctuant and increasing trend in both two cultures, with a higher α-ketoglutarate concentration in monoculture, the concentration of citric acid was lower in monoculture, especially at 60 h to 72 h (P< 0.05). These results indicated the associated methanogens shift the metabolism of anaerobic fungi significantly. |
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