本实验室前期利用连续传代的方法获得了来自瘤胃的厌氧真菌和甲烷菌的富集培养物，并发现厌氧真菌和甲烷菌可以在体外长期稳定共存；本实验室前期还从不同草食动物肠道及粪便分离得到了多个厌氧真菌和甲烷菌的共培养，并发现共培养降解一系列粗纤维的能力显著高于厌氧真菌纯培养。然而，在厌氧真菌和甲烷菌富集培养物中，厌氧真菌和甲烷菌之间的紧密关系是否存在种属特异性，目前并不是很清楚。此外，甲烷菌共存时厌氧真菌的代谢以及甲烷菌促进厌氧真菌降解和利用一系列底物的机制并不明了。因此，本论文首先以厌氧真菌和甲烷菌富集培养物为研究对象，揭示其代谢粗纤维的特性以及厌氧真菌和甲烷菌共发生的种属特异性；接着选取典型的厌氧真菌和甲烷菌共培养菌株，探究甲烷菌共存时厌氧真菌的代谢以及甲烷菌促进厌氧真菌利用底物的机制。 1 瘤胃厌氧真菌与甲烷菌富集培养物和瘤胃细菌与甲烷菌富集培养物发酵秸秆特性的研究 以稻秸或麦秸为底物，将瘤胃内容物分别接种于含青链霉素（FM组，厌氧真菌和甲烷菌富集培养物）、含放线菌酮（BM组，细菌和甲烷菌富集培养物）以及不含抗生素（RC组）的培养基中，于39 ºC体外发酵70 h。测定发酵不同时间点的产气量和甲烷产量；发酵结束后测定pH值、体外降解率、发酵代谢产物以及微生物的数量。各处理组降解稻秸存在如下顺序：RC组 > FM组 > BM组。各处理组降解麦秸存在如下顺序：FM组 > RC组 > BM组。以稻秸及麦秸为底物进行发酵时，FM组的pH值显著低于RC组和BM组（P < 0.05）；FM组总产气量与RC组均无显著差异（P > 0.05），且显著高于BM组（P < 0.05）；FM组甲烷产量显著高于RC组和BM组（P < 0.05）；FM组总VFA浓度在三个处理组中最低，然而乙酸浓度显著高于RC组和BM组（P < 0.05），无丙酸、戊酸、异丁酸和异戊酸的产生；FM组甲酸的浓度显著高于其它两组（P < 0.05），然而氨氮的浓度显著低于其它两组（P < 0.05）。以稻秸或麦秸为底物进行发酵，FM组厌氧真菌的数量显著高于RC组和BM组（P < 0.05），然而其细菌的数量则显著低于其它两组（P < 0.05）；FM组古菌的数量最高，显著高于BM组（P < 0.05）；BM组厌氧真菌的数量最低，显著低于RC组和FM组（P < 0.05），然而其细菌数量则最高，显著高于FM组（P < 0.05）。结果表明：厌氧真菌-甲烷菌富集培养物具有很强的粗纤维降解能力，其降解稻秸和麦秸的能力强于细菌-甲烷菌富集培养物；厌氧真菌-甲烷菌富集培养物具有很好的降解粗纤维产甲烷的能力。 2 厌氧真菌和甲烷菌富集培养物中甲烷菌和厌氧真菌多样性以及两者共发生的研究 本试验以第一章为基础，采集了甲烷菌和厌氧真菌富集培养物（FM）以及瘤胃内容物体系（RC）发酵稻秸和麦秸后的样品，提取DNA，对甲烷菌和厌氧真菌进行高通量测序，利用网络分析的方法研究甲烷菌和厌氧真菌共发生的种属特异性。结果显示，两种不同底物（稻秸和麦秸）对两体系中甲烷菌和厌氧真菌的多样性无显著影响（P > 0.05）。Methanobrevibacter是两体系中的优势甲烷菌属；Caecomyces则是优势厌氧真菌属。相比于RC组，FM组中甲烷菌和厌氧真菌的多样性降低。FM组中Methanobrevibacter和Methanosphaera的相对丰度显著高于RC组（P < 0.05），而Group9、Group10和Group8的相对丰度则显著低于RC组（P < 0.05）。FM组中Caecomyces的相对丰度显著高于RC组（P < 0.05），而Piromyces、Orpinomyces和Neocallimastix的相对丰度则显著低于RC组（P < 0.05）。相比于RC组，FM组中Methanobrevibacter与厌氧真菌的共发生关系得到加强，而Methanomassiliicoccales与厌氧真菌的共发生关系则减弱。上述结果表明，甲烷菌和厌氧真菌之间存在着紧密的互作关系，而Methanobrevibacter是与厌氧真菌关系最为密切的甲烷菌属。 3 共存甲烷菌影响厌氧真菌对真菌抑制剂硝呋烯腙响应的研究 以稻秸为底物，厌氧真菌纯培养和厌氧真菌-甲烷菌共培养为接种物进行体外发酵。两培养物中各添加不同浓度的硝呋烯腙（终浓度为0，5，10，25 mg/L），于39 ºC培养箱中静置培养96 h。在特定时间点测定产气量和甲烷含量；发酵结束后立即测定pH；剩余底物用于测定体外降解率；上清液用于代谢产物浓度的测定。相比于未添加硝呋烯腙纯培养组，5，10，25 mg/L硝呋烯腙纯培养组发酵稻秸的活性被显著抑制（P < 0.05）。相比于未添加硝呋烯腙共培养组，5 mg/L硝呋烯腙共培养组发酵稻秸的活性并未显著降低：两者pH、产气量、甲烷产量、底物降解率以及乙酸浓度皆无显著差异（P < 0.05）。然而10和25 mg/L硝呋烯腙共培养组发酵稻秸的活性则显著降低（P < 0.05）。5，10 mg/L硝呋烯腙共培养组发酵稻秸的活性分别显著高于5，10 mg/L硝呋烯腙纯培养组（P < 0.05）。当厌氧真菌被抑制后甲烷菌的生长也受到影响。上述结果表明，厌氧真菌和甲烷菌之间存在着互利共生的关系，厌氧真菌为甲烷菌提供底物，维持甲烷菌的生长；甲烷菌的存在则可以促进厌氧真菌对底物的降解。 4 以单糖为碳源研究甲烷菌共存对厌氧真菌代谢的影响 接种10 mL已生长三天的培养物悬浮液于预热至39 ºC的90 mL培养基中（以葡萄糖或木糖为碳源），39 ºC静置培养96 h。于发酵特定时间点测定产气量、氢气和甲烷产量。在0、24、48、72及96 h五个时间点终止一批发酵，测定pH值、底物浓度、菌体重量以及水溶性代谢产物。试验结果以葡萄糖和木糖两部分呈现。当以葡萄糖为底物进行发酵时得到如下结果：纯培养发酵葡萄糖的代谢产物主要为H2、甲酸、乙酸、乳酸和乙醇；共培养发酵葡萄糖的代谢产物主要为CH4、乙酸、乳酸和乙醇；甲烷菌共存缩短了厌氧真菌的生长延滞期；共培养组的葡萄糖最大消失率和最大细胞干重出现在发酵24 h，而纯培养组则出现在48 h；在共培养中，氢气在整个发酵期间都处于一个较低水平，甲酸虽然在24 h存在大量积累，然而在48 h则没有甲酸积累，pH则较24 h升高；共培养组24 h的苹果酸和乳酸浓度显著高于纯培养组（P < 0.05），在48、72及96 h则显著低于纯培养组（P < 0.05）；共培养组乙酸的浓度在整个发酵期间显著高于纯培养组（P < 0.05）。当以木糖为底物进行发酵时得到如下结果：纯培养发酵木糖的代谢产物主要为H2、甲酸、乙酸、乳酸和乙醇；共培养发酵木糖的代谢产物主要为CH4、乙酸、乳酸和乙醇；在48 h前，共培养和纯培养的产气量、pH和代谢产物（除甲酸）无显著差异（P > 0.05），在48 h后，共培养中积累的甲酸被甲烷菌大量利用，这导致共培养中的乙酸、CO2和pH显著增加（P < 0.05），乳酸和苹果酸则显著降低（P < 0.05）；共培养和纯培养对木糖的利用没有显著差异（P > 0.05），然而共培养的氢化酶体代谢的碳源比例显著高于纯培养（P < 0.05）。综上所述，厌氧真菌利用葡萄糖和木糖具有类似的代谢产物谱，厌氧真菌和甲烷菌共培养利用葡萄糖和木糖也具有类似的代谢产物谱。当以葡萄糖为底物进行发酵时，共存甲烷菌对厌氧真菌的促进作用主要为1）促进厌氧真菌对葡萄糖的吸收或转运2）增强厌氧真菌氢化酶体的能量代谢；当以木糖为底物进行发酵时，共存甲烷菌对厌氧真菌的促进作用主要表现在增强厌氧真菌氢化酶体的能量代谢。 5 共存甲烷菌影响厌氧真菌代谢葡萄糖分子机制的研究 以葡萄糖为底物，接种10 mL已生长三天的培养物悬浮液于预热至39 ºC的90 mL培养基中，39 ºC静置培养。于发酵特定时间点测定产气量、氢气和甲烷。在纯培养发酵中点（51 h）和终点（80 h），共培养发酵中点（36 h）和终点（66 h），四个时间点各终止一批发酵，取上清液测定其pH、葡萄糖、氨氮、蛋白浓度和水溶性代谢产物，菌体用于微生物定量以及转录组测序。结果显示，共培养发酵中点与纯培养发酵中点的产气量无显著差异（P > 0.05），共培养发酵终点的产气量显著高于纯培养发酵终点的产气量（P < 0.05）。共培养利用葡萄糖的速率高于纯培养：共培养发酵36 h和66 h利用的葡萄糖分别与纯培养发酵51 h和80 h利用的葡萄糖无显著差异（P > 0.05）。共培养中点组（C中点）的甲酸和乳酸显著低于纯培养中点组（M中点）（P < 0.05），C中点组乙酸有高于M中点组的趋势（P = 0.054）。共培养终点组（C终点）中未检测到甲酸，C终点组的乳酸显著低于纯培养终点组（M终点）（P < 0.05），而C终点组乙酸则显著高于M终点组（P < 0.05）。转录组测序发现，厌氧真菌F1在以葡萄糖为底物进行生长时表达大量粗纤维降解酶基因的转录本。在纯培养和共培养组中，相比于发酵中期，发酵末期许多与粗纤维降解酶相关的基因显著上调；大部分与胞内碳水化合物代谢相关的转录本则显著下调。比较C中点和M中点的转录本发现，甲烷菌共存显著上调了厌氧真菌糖转运体基因和己糖激酶基因的表达。以上结果表明，甲烷菌共存可能通过促进葡萄糖的转运，胞内葡萄糖的磷酸化以及氢体的代谢来促进厌氧真菌对葡萄糖的利用。 综上所述，厌氧真菌和甲烷菌富集培养物具有很好的降解粗纤维产甲烷的能力，在这个系统中甲烷短杆菌是与厌氧真菌互作关系最为紧密的甲烷菌属。厌氧真菌和甲烷菌存在互利共生的关系：厌氧真菌为甲烷菌提供底物；甲烷菌通过利用厌氧真菌的代谢产物，消除产物抑制作用，增强厌氧真菌氢化酶体能量的代谢，进而促进厌氧真菌对底物的降解和利用。

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 Materials The 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 ºC 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 Culture Based 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, Group10 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 Nitrovin For 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 ºC 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 Substrate Ten milliliters of 3 days inocula were inoculated into 90 ml of prewarmed fresh medium containing glucose or xylose as substrate, and incubated at 39 ºC 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 Ⅰ with glucose as substrate, part Ⅱ with xylose as substrate). In part Ⅰ, 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 Ⅱ, 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 Fungi Ten milliliters of 3 days inocula were inoculated into 90 ml of prewarmed fresh medium containing glucose as substrate, and incubated at 39 ºC 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.