Isolation And Identification Of Cellulolytic Bacteria From Tibetan Yak Rumen And Their Application In Silage

Ensiling is a traditional conservation method of fresh forages and grains,which aimed at supplying year-round availability of nutritious and palatable feed for livestock.Furthermore,ensiling may function as a beneficial pretreatment for lignocellulosic materials before further processing to bioenergy production.Tropical grasses and crop stalks present difficulty to ensiling due to its low WSC and high lignocellulosic contents.Exogenous fibrolytic enzyme has been explored to induce direct conversion of structural carbohydrates into soluble sugars for LAB fermentation,but,high cost and instability of commercially enzymes limited their widespread application in silage.Microorganism action altogether presents a combined storage and pretreatment benefits which require less energy and affords easy handling.Therefore,screening and isolating fibrolytic microorganism lineages with high substrate specific activities and stability is very crucial.1.Isolation and identification of ruminal facultatively anaerobic cellulolytic bacteria from the Tibetan yaks (Bos grunniens)To isolate and identify facultatively anaerobic cellulolytic bacteria and provide the basis for studying the new type of silage additives.The fresh ruminal fluid was collected from Tibetan yak,highly cellulolytic facultatively anaerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media.The cellulolytic bacteria were identified based on morphological,physiological,and biochemical characteristics as well as 16S rRNA analysis.Examination of their growth characteristics and enzyme assay;the decomposition of rice straw of the strain was carried out by evaluating cellulose,hemicellulose and lignin contents.Strains were identified as Enterococcus casseliflavus JFL12?Enterococcus faecalis JF85?Enterococcus faecium Y83.Three strains grew well in a range of conditions:temperature 15?55?,pH 3.0?7.0 and NaCl 3.0% and 6.5%.They exhibited the maximal cellulase activity and enzyme stability under natural cellulose and organic nitrogen sources conditions at pH 5.0?5.5 and 50?.In simulated fermentation,compared to the control,adding of strains significantly (P <0.05) decreased lignocellulose contents (except ADL) of rice straw.The largest reduction in lignocellulose contents were observed in combined additives.Therefore,strains with highly cellulolytic abilities have acid and salty tolerating capacities,and they would have a great potential to be applied in the production of silage.2.Improvement of fermentation quality,carbohydrate compositions and enzymatic hydrolysis of Pennisetum sinese silage by inoculation of cellulolytic bacteriaThe first study was conducted to examine the effects of cellulolytic bacteria on the fermentation quality,carbohydrate compositions,and cellulose convertibility of P.sinese.Three strains were inoculated with or without Lactobacillus plantarum (Lp) to Pennisetum sinese silage for 90 days.All inoculants increased lactic acid content,decreased pH and lignocellulose contents compared with silage without additives (control).The lowest pH,highest lactic acid and largest reduction in lignocellulose contents were observed in JFL12+Lp,JF85+Lp,and Y83+Lp silages.Isolates alone or in combination with Lp significantly increased WSC,mono-and disaccharides contents as compared to the control.Combined addition efficiently improved enzymatic hydrolysis of P.sinese silage,indicated by higher glucose yield and cellulose convertibility.P.sinese ensiled with combined additives is a suitable storage and pretreatment method prior to sugars production from energy crop.The second experiment was conducted to evaluate the cellulolytic efficacy of E.faecium Y83 isolated from Tibetan yak on lignocellulosic materials.P.sinese was ensiled without additive,L.plantarum (Lp),Trichoderma reesei (Tr),fibrolytic enzymes (E),and E.faecium (Y83) for 90 days.Y83 silages had higher LA and lower AA and ammonia-N as compared to E and Tr silages.Tr and E had superior effects for degrading lignocellulose while Y83 had intermediate effects.The first-order exponential decay models(R2=0.928?0.998) predicted water soluble carbohydrates kinetics and demonstrated high WSC (g/kg DM) preservation potential in Y83 (21.40),followed by Tr (18.94) and E(16.74).Addition of Y83 improved the conversion efficiency of P.sinese silage than Tr and E,indicated by higher glucose and total reducing sugars yield (22.49 and 36.89 w/w % DM,respectively).In conclusion,Y83 can be exploited for the ensiling lignocellulosic biomass before grass processing.3.Improvement of fermentation quality,carbohydrate compositions and enzymatic hydrolysis of Napier grass silage by inoculation of cellulolytic bacteriaTo evaluate the effects of facultative anaerobic isolates with cellulolytic activities on the fermentation characteristics,carbohydrates components and cellulose convertibility of Napier grass silage.Three strains were inoculated with or without L.plantarum (Lp) to Napier grass silage for 60 days.Additives increased the lactic acid,while decreased pH,ammonia-N and all structural carbohydrate compositions except ADL as compared with control.Silages treated with JFL12+Lp,JF85+Lp and Y83+Lp decreased significantly (P <0.05) the contents of NDF,ADF,hemicellulose,and cellulose compared to Lp.Three strains with or without Lp significantly increased water soluble carbohydrates,glucose,fructose and sucrose contents,Y83+Lp>JF85+Lp>JFL12+Lp>Y83.Silages treated with Y83+Lp had highest glucose yield and cellulose convertibility after 60 days of ensiling.In conclusion,three isolates combined with Lp accelerated the silage fermentation and degradation of structural carbohydrates.The second experiment was conducted to examine the effects of biological inoculants on fermentation characteristics,structural carbohydrates degradation,and enzymatic conversion yields of Napier grass.Napier grass was ensiled without additive,L.plantarum(Lp),T.reesei (Tr),fibrolytic enzymes (E),and E.faecium (Y83) for 60 days.Additives increased significantly (P<0.05) the contents of LA content and the ratio of lactic acid to acidic acid,while decreased significantly (P<0.05) pH and ammonia N as compared with control.Y83 silages had higher LA and lower ammonia-N as compared to E and Tr silages.Tr and E had superior effects for degrading lignocellulose while Y83 had intermediate effects.The first-order exponential decay models predicted water soluble carbohydrates kinetics and demonstrated high WSC preservation potential in Y83,Tr,and E.Addition of Y83 improved the conversion efficiency of Napier grass silage than Tr and E,indicated by higher glucose,xylose,and total reducing sugars yield.In conclusion,the cellulolytic bacterium Y83 improved fermentation quality and enzymatic hydrolysis and could open a new approach for the development silage additive for lignocellulosic biomass.4.Effect of cellulolytic bacteria on fermentation quality,carbohydrate compositions,enzymatic hydrolysis and in vitro digestibility of rice straw silageTo evaluate the effects of facultative anaerobic isolates with cellulolytic activities on the fermentation characteristics,carbohydrates components,enzymatic hydrolysis and in vitro digestibility of rice straw silage.Rice straw was treated with no additive;JFL12;JF85;Y83;JFL12+JF85+Y83 (M).The silos were open after 3,5,7,14 and 30 days of ensiling for rice straw silage.Before the test,L.plantarum (1×106 cfu/g FW) was added to the five treatments to ensure the basic amount of lactic acid bacteria in the fermented material.All inoculants increased LA content and WSC,mono-and disaccharides contents,decreased pH,ammonia N and lignocellulose contents compared with control.The highest LA content was observed in M silages,the NDF and hemicellulose contents in Y83 and M were significantly lower than those in the control (P <0.05).The in vitro gas production and digestibility was higher in cellulolytic bacteria silages compared to control after 30 days of ensiling.Addition of cellulolytic bacteria improved the conversion efficiency of rice straw silage than control,indicated by higher enzymatic hydrolysis conversion efficiency.These results could provide insight and initiate the development of new silage additive from microbes.