Screen Of Facultative Anaerobic Cellulolytic Microbial Consortia From The Rumen Of Holstein Cows And Yaks And Their Application In Silage

Ensiling is the main means to preserve green feed and an effective biological pretreatment for lignocellulosic biomass.Lignocellulosic biomass is difficult to ensile due to its high content of crude fiber.The biological additives can effectively improve the fermentation quality of silage.Cellulolytic microbial consortia were composed of many different bacterial communities,which secreted large numbers of various enzymes.The cellulolytic microbial consortium can effectively degrade plant cell wall into water-soluble carbohydrates for lactic acid bacteria to improve the fermentation quality of raw materials.The rumen of ruminants is a perfect natural digestion and fermentation system and micro-ecological system in nature,and it is hailed as a rich resource bank of lignocellulose degrading bacteria.Therefore,this study aimed to screen facultative anaerobic microbial consortia with high cellulose-degrading ability from the rumen of Holstein cows and yaks,and further evaluated their effects on rice straw silages and sweet sorghum silages,which provided a theoretical basis for the application of ruminal facultative anaerobic cellulolytic microbial consortium in silage production and the development of the new type of silage additives.1.Screening of facultative anaerobic cellulolytic microbial consortium from the rumen of Holstein cows and its effects on the fermentation quality of rice straw silageThis study aimed to screen facultative anaerobic cellulolytic microbial consortium from the rumen of Holstein cows and further evaluate its effect on the fermentation quality and structural carbohydrates degradation of rice straw silage.The facultative anaerobic cellulolytic microbial consortium M6 was screened from the fresh rumen contents of Holstein cows by continuous enrichment cultivation,Congo red staining,acid-resistant induction,secondary screening through filter paper degradation,and enzyme activity assay.The dominant functional bacteria in consortium M6 were Escherichia,Lactococcus,and Enterococcus through molecular biological identification.Rice straw was treated with following:（1）WCK,natural fermentation of non-irradiated rice straw;（2）WIRR,irradiated rice straw ensiled directly;（3）CLAB,irradiated rice straw ensiled with combined lactic acid bacteria CLAB（Lactobacillus plantarum,Pediococcus pentosaceus,and L.buchneri）;（4）M6,irradiated rice straw ensiled with M6.Each treatment was sampled randomly after 3,6,15,45,60,and 90 days of ensiling for laboratory analysis.The results showed that throughout the ensiling period,the p H of M6 silages was lower than that of other silages and reached the minimum（4.62）on the 60-day of ensiling.After 45 days of ensiling,the lactic acid content of M6 silages was significantly（P<0.05）higher than that of other silages and reached the maximum(23.90 g?kg^-1 DM)on the 90-day of ensiling.After 15 days of ensiling,the contents of NDF,ADF,and cellulose in M6 silages were significantly（P<0.05）lower than that of other silages,and represented the lowest on the 90-day of ensiling.After 60 days of ensiling,the WIRR and M6 silages maintained higher level of water-soluble carbohydrate contents,followed by the CLAB silages.In summary,the facultative anaerobic cellulolytic microbial consortium M6 has effects on degrading cellulose and promoting lactic acid fermentation of rice straw silage.The addition of ruminal cellulolytic microbial consortium could effectively improve the fermentation quality of rice straw and provided theoretical basis for the development and application of silage additives.2.Screening and identification of facultative anaerobic cellulolytic microbial consortium from the rumen of yak（Bos grunniens）This study aimed to screen facultative anaerobic cellulolytic microbial consortium with acid resistance from the rumen of yaks,to provide microbial resources for the development of biological additives in silage production.Two facultative anaerobic cellulolytic microbial consortia CF and PY were screened from the fresh rumen contents of Tibetan yak by continuous enrichment cultivation in rice straw or CMC as substrates,acid-resistant induction,Congo red staining,secondary screening through filter paper degradation,and enzyme activity assay.The dominant functional bacteria in consortium CF and PY were Enterococcus,Klebsiella,and Escherichia through molecular biological identification.It was found that consortium CF and PY had abundant enzyme systems through CAZymes annotation and had obvious advantages in degrading lignocellulose.The consortium CF and PY exhibited the maximal cellulase activity under natural cellulose as carbon source at p H4.5～5.5.In summary,the facultative anaerobic cellulolytic microbial consortium CF and PY had acid tolerating and cellulose-degrading capacities,and they would have great potential to be applied in silage production.3.Effects of facultative anaerobic cellulolytic microbial consortium from yak on the fermentation quality and microbial community structure of rice straw silageThis study aimed to evaluate the effects of facultative anaerobic cellulolytic microbial consortium CF and PY on the fermentation quality,carbohydrates composition,microbial community structure,and enzymatic hydrolysis of irradiated rice straw silage.Rice straw was treated with following:（1）WCK,natural fermentation of non-irradiated rice straw;（2）WIRR,irradiated rice straw ensiled directly;（3）CLAB,irradiated rice straw ensiled with combined lactic acid bacteria CLAB（L.plantarum,P.pentosaceus and L.buchneri）;（4）CF,irradiated rice straw ensiled with CF;（5）PY,irradiated rice straw ensiled with PY;（6）CF+CLAB,irradiated rice straw ensiled with CF and CLAB;（7）PY+CLAB,irradiated rice straw ensiled with PY and CLAB.Each treatment was sampled randomly after 3,6,15,45,60,and 90 days of ensiling for laboratory analysis.The results showed that the addition of facultative anaerobic cellulolytic microbial consortium with or without lactic acid bacteria significantly（P<0.05）decreased the p H and the contents of NDF,ADF,and cellulose,and significantly（P<0.05）increased the contents of lactic acid,sucrose,fructose,glucose,xylose,and cellobiose in rice straw silages,while the combined addition showed the best performance.On 3-day of ensiling,Lactobacillus was the predominant bacterial genera in microbial consortium inoculated silages（CF,PY,CF+CLAB,and PY+CLAB）,while on 90-day of ensiling,the abundance of Klebsiella and Bacillus with cellulolytic ability increased in various degrees.The higher water-soluble carbohydrates contents and lower p H value in the microbial consortium inoculated silages were associated with the large abundance of Lactobacillus,while the lower cellulose contents in the microbial consortium inoculated silages were associated with the large abundance of Bacillus.The results of enzymatic hydrolysis showed that the cellulolytic microbial consortium significantly（P<0.05）improved the yields of total reducing sugar,glucose,and xylose in rice straw silages.Therefore,the facultative anaerobic cellulolytic microbial consortium effectively improved the fermentation quality,cellulose conversion,and enzymatic hydrolysis efficiency of rice straw silage,and the combined addition with lactic acid bacteria performed better.4.Effects of facultative anaerobic cellulolytic microbial consortium from yak on the fermentation quality and carbohydrates composition of sweet sorghum silageThis study aimed to further evaluate the effects of facultative anaerobic cellulolytic microbial consortium CF and PY on the fermentation quality,carbohydrates composition,and enzymatic hydrolysis of sweet sorghum silage.Sweet sorghum was treated with following:（1）WCK（control）;（2）CLAB（L.plantarum,P.pentosaceus,and L.buchneri）;（3）CF;（4）PY;（5）CF+CLAB;（6）PY+CLAB.Each treatment was sampled randomly after3,7,14,30,and 60 days of ensiling for laboratory analysis.The results showed that the addition of facultative anaerobic cellulolytic microbial consortium with or without combined lactic acid bacteria decreased the p H and structural carbohydrates contents,and increased the contents of lactic acid,sucrose,fructose,glucose,xylose,and cellobiose of sweet sorghum silages in various degrees,and the combined addition showed the best performance.On the60-day of ensiling,the lactic acid content of PY+CLAB silages reached the maximum of85.91 g?kg^-1 DM and the p H value reached the minimum of 3.81.The results of enzymatic hydrolysis showed that the addition of cellulolytic microbial consortium improved the enzymatic hydrolysis efficiency of sweet sorghum silages.At 72-h of enzymatic hydrolysis,the enzymatic hydrolysis efficiency of PY+CLAB silages reached the maximum of 71.34%.Therefore,the facultative anaerobic cellulolytic microbial consortium effectively improved the fermentation quality,structural carbohydrates degradation,and enzymatic hydrolysis efficiency of sweet sorghum silage,and the combined addition with lactic acid bacteria performed better.