| Previous study of our laboratory found that the anaerobic fungi and methanogens co-cultures enriched from the goat rumen showed robust growth of anaerobic fungi and methanogens maintained over 62 transfers lasting for more than 200 days.Also,members of our laboratory obtained numerous natural co-cultures of anaerobic fungi and their indigenously associated methanogens,and found that the degradation of lignocellulosic substrates by anaerobic fungi was significantly enhanced by the presence of methanogens.However,the species-specificity within enriched ruminal cultures of anaerobic fungi and methanogens and the mechanism that the associated methanogens enhanced the utilization of a series of substrates by anaerobic fungi remain unclear.At first,the enriched ruminal cultures of anaerobic fungi and methanogens were used to dissect the degradation of the lignocellulosic substrates and the species-specificity of co-occurrence between anaerobic fungi and methanogens.After that,the present study focused on the simple natural co-culture to investigate the metabolism of anaerobic fungi in the presence of the associated methanogens and the potential mechanism that the associated methanogens enhanced the utilization of substrates by anaerobic fungi.1 Characteristics of Enriched Ruminal Cultures of Anaerobic Fungi with Methanogens and Bacteria with Methanogens on the In Vitro Degradation and Metabolism of Lignocellulosic MaterialsThe two specific microbial groups,ruminal anaerobic fungi with methanogens and bacteria with methanogens,were selected by the addition of streptomycin-penicillin or cycloheximide respectively,to ruminal content and the degradation and metabolism of rice straw and wheat straw by these two treatments and ruminal content were evaluated in vitro.Bottles were incubated at 39? for 70 h.Gas production and methane production were measured at intervals.The pH was determined immediately upon removing crimp-seals and stoppers.Samples were collected for analysis of in vitro digestibility,water-soluble end-products and microbial community.The order of in vitro digestibility of rice straw was as follows:ruminal content(RC)>streptomycin-penicillin treatment(FM group)>cycloheximide treatment(BM group).The order of in vitro digestibility of wheat straw was as follows:FM group>RC>BM group.The pH of FM group was significantly lower than that of RC and BM group(P<0.05).Total gas production of FM group in rice straw or wheat straw was similar to that of RC,while it was significantly higher than that of BM group(P<0.05).The methane production of FM group was significantly higher than that of RC and BM group(P<0.05).Though the concentration of total VFA of FM group was the lowest among the three groups,the concentration of acetate and formate in FM group was significantly higher than that in RC and BM group(P<0.05).However,there were no substantial propionate,valerate,isobutyrate and isovalerate produced in FM group.The concentration of ammonia in FM group was significantly lower than that in RC and BM group(P<0.05).The copy number of anaerobic fungi in FM group was significantly higher than that in RC and BM group(P<0.05),whereas the copy number of bacteria in FM group was significantly lower than that in RC and BM group(P<0.05).The copy number of archaea in FM group was significantly higher than that in BM group(P<0.05).The copy number of anaerobic fungi in BM group was the lowest among the three groups,and was significantly lower than that in RC and FM group(P<0.05).However,The copy number of bacteria in BM group was the highest among the three groups,and was significantly higher than that in FM group(P<0.05).Results above indicated that anaerobic fungi-methanogens enriched culture was more efficient on lignocellulose degradation than bacteria-methanogens enriched culture.The anaerobic fungi-methanogens enriched culture had a potent ability in bioconversion of lignocellulosic materials to methane.2 Diversity of Anaerobic Fungi and Methanogens and Co-occurrence Patterns between Anaerobic Fungi and Methanogens in Enriched Ruminal CultureBased on the first chapter of in vitro fermentation,samples from the incubation of anaerobic fungi-methanogens system(FM system)and ruminal content system(RC system)with rice straw or wheat straw as substrate,were collected for DNA extraction.The obtained DNA was subsequently pyrosequenced to analyze the diversity of anaerobic fungi and methanogens,and network analysis was used to explore co-occurrence patterns between anaerobic fungi and methanogens.The results indicated that there was no significant??difference in diversity of anaerobic fungi and methanogens between rice straw and wheat straw used as substrate in FM system or RC system(P>0.05).The archaea community was dominated by Methanobrevibacter in both FM and RC system.The anaerobic fungi community was dominated by Caecomyces in both FM and RC system.The diversity of anaerobic fungi and methanogens in FM system was decreased compared with that in RC system.The abundance of Methanobrevibacter and Methanosphaera in FM system was significantly higher than that in RC system(P<0.05).However,The abundance of Group9,Group 10 and Group8 in FM system was significantly lower than that in RC system.Regarding to anaerobic fungi,The abundance of Caecomyces in FM system was significantly increased compared to that in RC system(P<0.05),whereas the abundance of Piromyces,Orpinomyces and Neocallimastix in FM system was significantly lower than that in RC system(P<0.05).In the FM system,the interaction between Methanobrevibacter OTUs and anaerobic fungi OTUs was enhanced compared to that in RC system,whereas the interaction between OTUs belonging to Methanomassiliicoccales and anaerobic fungi OTUs was reduced compared to that in RC system.Results above indicated that there was interdependence between methanogens and anaerobic fungi.And,Methanobrevibacter was the most frequent genus to co-occur with anaerobic fungi.3 Effect of the Associated Methanogens on the Response of Anaerobic Fungi to NitrovinFor in vitro fermentation,mono-culture of anaerobic fungi Piromyces sp.and co-culture of Piromyces sp.and Methanobrevibacter thaueri were respectively inoculated into fresh media in which rice straw was used as substrate.Nitrovin hydrochloride was added to the final concentrations of 0,5,10 and 25 mg/L-nitrovin.Bottles were incubated at 39? for 96 h without shaking.Gas production and methane production were measured at intervals.The pH was determined immediately upon removing crimp-seals and stoppers.Samples were collected for analysis of in vitro digestibility and water-soluble end-products of formate,lactate and acetate.In the mono-culture,nitrovin(5,10 and 25 mg/L)significantly reduced the fermentation of rice straw by anaerobic fungi(P<0.05).In the co-culture,no significant impact was observed when adding 5 mg/L nitrovin(P>0.05).However,the fermentation activity was significantly depressed at the concentrations of 10 and 25 mg/L.At the concentration of 5 and 10 mg/L nitrovin,the activity of fermentation by the co-culture was significantly higher than that by the mono-culture(P<0.05).Our results showed that there was a mutualism between anaerobic fungi and methanogens,where anaerobic fungi provided substrates for methanogens to grow and methanogens,in turn,relieved the catabolic repression of anaerobic fungi,leading to the enhanced degradation of rice straw by anaerobic fungi.4 Effect of the Associated Methanogens on the Metabolism of Anaerobic Fungi with Monosaccharide as SubstrateTen milliliters of 3 days inocula were inoculated into 90 ml of prewarmed fresh medium containing glucose or xylose as substrate,and incubated at 39? for 96 h without shaking.The cumulative gas.hydrogen and methane production was determined at intervals.Parallel anaerobic cultures were incubated.At regular intervals(0,24,48,72,and 96 h),six bottles were sacrificed to collect the samples for measurement of pH,concentration of the substrate,cell dry weight(CDW)and water-soluble metabolites.The results were presented with two parts(part ? with glucose as substrate,part ? with xylose as substrate).In part ?,glucose fermentation by the anaerobic fungal monoculture resulted in mixed fermentation end products,mainly including hydrogen,formate,acetate,lactate,and ethanol.The dominant end-products produced by the co-culture of anaerobic fungi and methanogens were methane,acetate,lactate,and ethanol.The presence of methanogens shortened the growth lag time of anaerobic fungi.The occurrence of the maximum cell dry weight and the disappearance of most of glucose were observed at 24 h for the co-culture and 48 h for the fungal mono-culture.In the co-culture,hydrogen was detected at a very low level during fermentation,and formate transitorily accumulated at 24 h and disappeared at 48 h.resulting in an increase of pH.At 24 h,malate and lactate in the co-culture were higher than those in the monoculture(P<0.05).However,malate and lactate in the co-culture were lower than those in the monoculture over 48-96 h(P<0.05).Acetate in the co-culture was higher than that in the monoculture during the fermentation(P<0.05).In part ?,xylose fermentation by the monoculture led to end products mainly including hydrogen,formate,acetate,lactate,and ethanol.The dominant end-products produced by the co-culture of anaerobic fungi and methanogens were methane,acetate,lactate,and ethanol.Before 48 h,there was no significant difference in gas production,pH and end-products(except formate)between the two cultures(P>0.05).After 48 h,accumulated formate was remarkably consumed by co-cultured methanogens,accompanied by significantly increased acetate,CO2 and pH,and decreased lactate and malate.Xylose utilization,in both cultures,was similar during fermentation.However,the relative flux of carbohydrate in hydrogenosomes in the co-culture was higher than that in the monoculture.Results above indicated that both monoculture and co-culture can grow on both glucose and xylose,and respectively produce a relatively similar spectrum of fermentation products.When grown on medium containing glucose,the co-cultured methanogens not only can facilitate the metabolism in hydrogenosomes,but they can also facilitate the uptake of glucose into cells.However,the co-culture grown on xylose only showed the increased metabolism in hydrogenosomes.5 Study on the Mechanism of the Associated Methanogens Enhancing the Metabolism of Glucose by Anaerobic FungiTen milliliters of 3 days inocula were inoculated into 90 ml of prewarmed fresh medium containing glucose as substrate,and incubated at 39? without shaking.The cumulative gas,hydrogen and methane production was determined at intervals.At regular intervals(36 h,co-culture mid log stage;51 h,monoculture mid log stage;66 h,co-culture late log stage;80 h,monoculture late log stage),a batch of fermentation was stopped.Supernatant was collected for determination of pH,concentration of glucose,ammonia,protein and water-soluble end-products.Thallus were collected for RNA extraction and subsequent RNA-Seq analysis.Results showed that there was no significant difference in gas production between co-culture and monoculture at the mid log stage(P>0.05).However,the gas production in co-culture was significantly higher than that in monoculture at the late log stage(P<0.05).Co-culture utilized glucose at a faster rate compared with monoculture.There was no significant difference in glucose utilization between co-culture and monoculture at the mid log stage and at the late log stage,respectively(P>0.05).Concentration of formate and lactate in co-culture was lower than that in monoculture at the mid log stage(P<0.05).At the mid log stage,acetate in co-culture tended to be higher than that in monoculture(P=0.054).Formate in co-culture was at a undectable level at the late log stage.Lactate in co-culture was significantly lower than that in monoculture at the late log stage(P<0.05),whereas acetate in co-culture was significantly higher than that in monoculture at the late log stage(P<0.05).Examination of transcriptional patterns indicated that strain F1 constitutively transcribes a high level of transcripts of enzymes for cellulose and hemicellulose saccharification on glucose.Many transcripts of enzymes for cellulose and hemicellulose saccharification was significantly upregulated at the late log stage compared to that at the mid log stage,whereas most of the transcripts belonging to enzymatic activities required for intracellular carbohydrate degradation were significantly downregulated at the late log stage.The transcripts of genes of sugar transporter and hexokinase in co-culture were significantly upregulated compared to that in monoculture at the mid log stage.Results above indicated that the associated methanogens may enhance the metabolism of anaerobic fungi by facilitating transportion of glucose,phosphorylation of the intracellular glucose and metabolism in hydrogenosomes.In conclusion,the anaerobic fungi-methanogens system owned a potent ability in degradation of lignocellulose and bioconversion of lignocellulose to methane.Methanobrevibacter was the most frequent genus to co-occur with anaerobic fungi within this system.There was a mutualism between anaerobic fungi and methanogens,where anaerobic fungi provided substrates for methanogens to grow and methanogens,in turn,relieved the catabolic repression of anaerobic fungi and facilitated the metabolism in hydrogenosomes of anaerobic fungi,leading to the enhanced degradation and utilization of substrate by anaerobic fungi. |
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