Diversity Of Fungi And Methanogen Co-cultures From The Rumen Of Yaks And Their Fibrolytic Characteristics

The yaks graze on the Qinghai-Tibetan Plateau at an elevation higher than 3000 m throughout the year, and mainly live on wild grass to thrive in a harsh environment. The microbes that live in the yak rumen act synergistically to degrade wild pasture grass and withered grass in cold weather to provide nutrients for the yaks, which makes the rumen of yaks a residence for highly active lignocellulose hydrolyases. Anaerobic fungi are important fiber-degrading microorganisms in the rumen. Most previous studies have noted that the methanogen can improve fiber-degrading ability of anaerobic fungi mono-culture. fungus-methanogen co-cultures can offer a highly effective combination in developing fermentative system for the bioconversion of lignocellulose to CH4, acetate and high activity fiber-degrading enzyme. Thus far, The study on fungus-methanogens natural co-cultures is still very sparse. no literature on the interaction between anaerobic fungi and indigenous methanogens in the rumen of yaks is available. There are still a blank about fungus-methanogen co-cultures from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and their degradation ability for lignocellulose. In order to develop and utilize superior fiber-degrading fungi resources under extreme environment in China, for the first time, the present study isolated, identified the fungus-methanogen co-cultures from the rumen of Tianzhu yaks grazing on the Qinghai-Tibetan Plateau, revealed their possible symbiosis relationships in the rumen, selected the high effectively fungus-methanogen co-cultures for fiber-degrading by in vitro studies with wheat straw as substrate and investigated their ability to degrade wheat straw, corn stalk and rice straw and fermentation end-products. The main conclusions are as follow:1.Diversity of anaerobic fungi and methanogens co-cultures from the rumen of yaks:Twenty natural fungus-methanogen co-cultures were obtained from the rumen of Tianzhu yaks grazing on the Qinghai-Tibetan Plateau by Hungate roll tube technique.The fungi were identify according to the morphological characteristics and兰州大学博士学位论文牦牛瘤胃厌氧真菌及甲烷菌共培养物多样性及其纤维降解特性研究internal transcribed spacer 1(ITS1)sequences analysis.The co-cultures were confirmed as the one fungus-one methanogen pattern by the PCR-DGGE assay.All the methanogens were identified as Methanobrevibacter sp.by 16sr RNA gene sequencing.There were 4 types among 20 co-cultures:Neocallimastix frontalis with Methanobrevibacter ruminantium,Orpinomyces joyonii with Methanobrevibacter ruminantium,Orpinomyces joyonii with Methanobrevibacter millerae and Piromyces with Methanobrevibacter ruminantium.2. Screening for high effectively fungus-methanogens co-cultures: In vitro studies with wheat straw as substrate showed that 8 N. frontalis with M. ruminantium co-cultures and 3 Piromyces with M. ruminantium co-cultures grew more stably and presented stronger lignocellulose degradation ability including higher gas yield, xylanase, filter paper cellulase(FPase), ferulic acid esterase(FAE) and acetyl esterase(AE) activities, in vitro dry matter digestibility(IVDMD), ferulic acid and p-coumaric acid releases, CH4 and acetate yield than 9 Orpinomyces joyonii with Methanobrevibacter. Among 20 co-cultures, the high effectively fungus-methanogens co-cultures of 3 genus of fungi with methanogen were: O. joyonii Yak7+M. ruminantium, N. frontalis Yak16+M. ruminantium and Piromyces Yak18+M. ruminantium.3. The straw degradation potential of the high effectively fungus-methanogens co-cultures and fungus mono-cultures: Through enlargeing anaerobic culture volume, under wheat straw, corn stalk and rice straw as the substrates, during the 7 days incubation, the N. frontalis Yak16+M. ruminantium co-culture exhibited strongest lignocellulose degradation among the 3 high effective fungus-methanogen co-cultures. The methanogen improved biodegrading capacity of fungi mono-cultures, especially in the fungus mono-culture N. frontalis Yak16. Not only the fungus mono-culture N. frontalis Yak16 showed high IVDMD, but also the N. frontalis Yak16+M. ruminantium co-culture showed the strongest biodegration among 20 co-cultures. The N. frontalis Yak16+M. ruminantium co-culture reached the highest values were 12500 m U on wheat straw, FPase 430.3 m U, FAE 11.0 m U, AE 199.3 m U, CAE 5.0 m U, neural detergent fiber digestibility(NDFD) 61.7%, acid detergent fiber digestibility(ADFD) 55.7%, ferulic acid release 24.1 μg/m L and p-coumaric acid release 50.3 μg/m L on corn stalk, and IVDMD 71.9% on rice straw, respectively. 4. The fermentation end-products of the high effective fungus-methanogens co-cultures and fungi mono-cultures: Through enlargeing anaerobic culture volume, under wheat straw, corn stalk and rice straw as the substrates, during the 7 days incubation, acetate, lactate, ethanol and H2 yield produced by 3 strains of fungus mono-cultures: N. frontalis Yak16 ﹥ Piromyces Yak18 ﹥ O. joyonii Yak7. Interestingly, the fungus mono-culture N. frontalis Yak16 produced much lactate and ethonal which was well above those recorded in the previously reported anaerobic fungi. Acetate, lactate and CH4 yiled produced by three high effective fungus-methanogen co-cultures: N. frontalis Yak16+M. ruminantium ﹥Piromyces Yak18+M. ruminantium ﹥ O. joyonii Yak7+M. ruminantium. Especially, the N. frontalis with M. ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, much CH4 and acetate due to the methanogen changed the metabolism pathway of the fungi mono-cultures. Our data suggest that the N. frontalis Yak16+M. ruminantium co-culture and the N. frontalis Yak16 mono-culture both have great potentials for different industrial use.The present study revealed the symbiotic relationship between fungi and methanogens in the rumen of yaks grazing on the Qinghai-Tibetan Plateau, investigated the ability of the fungus-methanogen co-cultures from yaks to degrade wheat straw, corn stalk and rice straw and their metabolism end-products and provide the experimental basis and scientific evidence in order to develop and utilize superior fiber-degrading microbial resources under extreme environment in the Qinghai-Tibetan Plateau.