Selection Of Excellent Inoculant Strains And Their Effects On Corn Stover Silages

Corn,as an important food and forage crop,was widely planted in north of China.After the corn was harvest,lots of stover was left.Ensilage is an effective technology with a long history of use in the preservation of forage crops for livestock.During the natural ensiling process,the epiphytic lactic acid bacteria(LAB)of plant surfaces play important roles in the acidification of silages under anaerobic conditions,resulting in inhibition the growth of undesirable microorganisms,reduction the risk of feedstock deterioration and keeping forage available throughout the year.Currently,corn stover silage is becoming attractive due to its high nutritional value and good palatability.To gugurantee the success of silage,additives were often applied.Therefore,it is necessary to seclect suitable silage inoculants for the environment in north of China.Homofermentative LAB species were frequently used to improve the silage quality by accelerating the initial phase of the ensiling process via the rapid fermentation of water soluble carbohydrate(WSC)into lactic acid.However,there were limited preserving effects that depend only on the pH value decreased by homofermentative LAB.Hence,there is a trend to using heterofermentative LAB species as silage inoculants which can produce more acetic acid.In addition,lignocellulosic degrading-enzymes were often used with LAB inoculants to improve the nutritional value and digestibility of silags.However,the knowledge about the association of lignocellulosic biomass-degrading enzymes and their producing microbes with LAB in ensilages is still limited.Therefore,the research content and main results are as follows:1.Dynamic changes of the core bacteria during corn stover ensilingLAB play important roles in ensilages.Monitoring the changes of their populations during corn stover ensiling are helpful for improving the silage quality.Here,Corn stover was harvested and ensiled in mini silos for a period of 45 days.Fermentation characteristics were assessed and bacterial communities were described along with the fermentation process.The ensiled corn stover exhibited chemical traits as low pH value(3.92 ± 0.02)and high levels of lactic acid(66.75± 1.97 g/kg dry matter)which were associated with well ensiled forages,as well as moderate concentrations of acetic acid(19.69 ± 1.51 g/kg dry matter)and small amounts of 1,2-propanediol(4.4 ± 0.11 g/kg dry matter).In the early stages of the ensiling process,a significant increase and then reduction of the abundance of species Lactococcus lactis,Leuconostoc pseudomesenteroides,Pediococcus pentosaceus and Weissella sp.were observed.The species Lactobacillus plantarum group and Lb.brevis grew vigorously,and then gradually instead by the species Lb.farciminis and Lb.parafarraginis along with the course of ensiling.The knowledges about the dynamic changes of the dominant LAB could lead to improved ensiling practices and the selection of corn stover silage inoculants.A total of 78 strains was isolated from the ensiled corn stover silages.They were identified and classified as 12 species,including Lb.plantarum,Lb.farciminis,Lb.acidophilus,Lb.amylovorus,Lb.fermentum,Lb.buchneri,Lb.brevis,Lb.parafarraginis,Lb.oris,P.acidilactici,P.pentosaceus and Bacillus coagulans.The growth characteristics of these bacteria were further studied,and those strains with excellent properties have great potential as inoculants.2.Effects of heterofermentative inoculants on the fermentation characteristics and microbial communities of corn stover silageTo improve silage quality of crop forages,bacterial inoculants are often employed.Nowadays,there is a trend to using heterofermentative LAB species as silage inoculants.In this study,Lb.brevis SDMCC050297 and Lb.parafarraginis SDMCC050300 were used as inoculants to corn stover in lab silos for ensiling.At the initial stage of ensiling,the pH value of the inoculated silages reduced more drastically,and the inoculated silages had higher lactic acid and acetic acid contents.After 20 days of ensiling,a reduction in lactic acid content coupled with an increase in acetic acid and 1,2-propanediol contents was observed in inoculated silages.Furthermore,both the amount of LAB and the abundance of order Lactobacillales in inoculated silages were higher than those of controls in the whole process.Meanwhile,high abundance of Lb.brevis and Lb.parafarraginis were detected in inoculated silages.Especially at day 10,the percentage of species Lb.brevis was 55.74%,compared to 5.58%of controls.The species Lb.parafarraginis increased dramatically after day 20.At day 45,it dominated the inoculated silages with the percentage of 61.63%.Except Lactobacillus,Enterobacter,Pseudomonas and Stenotrophomonas were predominant at the early stage of ensiling.Along with the fermentation,the abundance of Enterobacter and Pseudomonas decreased significantly,while the abundance of Stenotrophomonas only decreased a little.Some species of Salmonella,Klebsiella and Pseudomonas,considered as undesirable bacteria for silage,were also detected in the samples of this study.However,the abundance of these genera in inoculated silages is lower than that in controls.These results suggested that the Lb.brevis SDMCC050297 and Lb.parafarraginis SDMCC050300 had capability of improving the silage quality and affecting the microbial ecology over the course of the ensiling.3.The hydrolysis and secretion characteristics of feruloyl esterases produced by LactobacillusAlthough LAB play important roles in silage fermentation which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms,they are not able to degrade cellulose and hemicellulose.Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility.In the present study,we isolated four Lactobacillus strains(Lb.amylovorus CGMCC 11056,Lb.acidophilus CCTCC AB2010208,Lb.farciminis CCTCC AB2016237 and Lb.fermentum CCTCC AB2010204)with feruloyl esterase(FAE)activities from ensiled corn stover by a plate screening assay.These four FAEs could efficiently hydrolyze methyl ferulate,methyl caffeate,methyl p-coumarate and methyl sinapinate under the experimental conditions,suggesting that they belonged to the type C FAE family.Moreover,they were able to hydrolyze corn stover to release hydroxycinnamic acids.Furthermore,these strains could degrade hydroxycinnamic esters and hydroxycinnamic acids,and Lb.amylovorus CGMCC 11056 was the most efficient strain among these four isolates.These results provided a new target for the development of inoculants to improve silage quality and digestibility.A Lactobacillus/Escherichia coli shuttle vector was constructed to heterologously express FAE of Lb.amylovorus CGMCC 11056,which did not contain the signal peptide by the prediction of SignalP 4.0.The results showed that the FAE could be secreted to in the culture medium of Lb.plantarum CGMCC 6888 and E.coli DH5?.In addition,L.plantarum and E.coli cells producing inactive FAES130A were constructed,and it was found that the inactive enzyme was also could be secreted into the medium,suggesting the extracellular location did not cause by its activity.Furthermore,a truncated version of the FAE without the 20 amino acids of N-terminus was constructed and proved to locate in the cytoplasm.Moreover,fusion of heterologous proteins with N20 from FAE can carry the target proteins out of the cells.These results indicated the N-terminus of Lb.amylovorus FAE can be employed as carriers for extracellular production of recombinant proteins and thus has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.To our best knowledge,this is the first reported protein which can be secreted out of the cells of both Gram-positive and Gram-negative bacteria.4.Effects of the lignocellulosic biomass degrading-microbes on LAB inoculantsCorn stover(particular wilted corn stover)is mainly composed of cellulose,hemicellulose and lignin,but a small amount of WSC which limits the growth of LAB and the digestion and utilization by animals.LAB and lignocellulosic biomass-degrading microbes are naturally adhered to the plant surface.During the ensiling process,they co-exist in forage crops to carry out the fermentation.However,the knowledge about the association of lignocellulosic biomass-degrading microbes with LAB in ensilages is still limited.Paenibacillus strains are important microbes in sustainable agriculture.Here,P.panacisoli SDMCC050309 was isolated from ensiled corn stover and used as an example to investigate the effects on LAB.This strain produced at least 4 endoglucanases and 7 xylanases.By absorption on microcrystalline cellulose and zymogram results,two endoglucanases Cel5 and Cel9 were identified.They could degraded carboxymethyl cellulose into cellooligosaccharides.Silage inoculants Lb.plantarum CGMCC 6888 and Lb.farciminis CCTCC AB2016237 could utilize cellobiose and cellotriose present in the medium to produce more lactic acid.Furthermore,two xylanase Xyn10 and Xyn11 were also identified.They could degraded larch wood xylan and alkali-pretreated corn stover into xylooligosaccharides.Using the produced XOS to culture Lb.brevis SDMCC050297 and Lb.parafarraginis SDMCC050300,both of them grew well with high level of acetic acid production.These results provided insights into the effects of the lignocellulosic degrading-microbes on LAB during the process of ensiling.5.Application of the mixed inoculants in ensiling of corn stover and wilt corn stoverStrains Lb.plantarun CGMCC 6888,Lb.farciminis CCTCC AB2016237,Lb.brevis SDMCC050297,Lb.parafarraginis SDMCC050300 and P.panacisoli SDMCC050309were prepared as mixed inoculants to study the application effects on corn stover silages and compare with other commercial silage additives.The results showed that the inoculated sialges had lower pH value(control silages vs inoculated silages,3.75 ± 0.07 vs 3.64 ± 0.02)and higher LAB number(control silages vs inoculated silages,2.02 ±0.93×1010 vs 9.77 ± 6.81 ×1010 cfu/kg)and lactic acid content(control silages vs inoculated silages,42.31 ± 1.79 vs 53.27 ± 3.77 g/kg).Furthermore,comparing with other commercial additives,our inoculants had advantages in reducing the pH values,decreasing the loss of dry matter,inhibition the growth of undesirable microorganisms and increasing the contents of organic acids.These results indicate that our mixed inoculants can obviously improve the fermentation quality of silages and have the great application potentials.Furthermore,the mixed inoculants were applied to wilt corn stover which contains low contents of WSC(11.3 g/kg)and LAB(2.1 ×107 cfu/kg).The successful ensiling of wilt corn stover was depend on the inoculants,and high quality of silages was obtained when the inoculants were applied together with enzyme preparation.During the 14 days of fermentation,the amount of reducing sugar,LAB number and lactic acid content in the inoculated silages were 7.1 g/kg,3.3 × 1010 cfu/kg and 40.83 g/kg,respectively.Thus,the inoculants could compensate the deficiency of LAB in the original plant materials,and enzyme could decompose lignocellulose into WSC which promote the growth of LAB during the ensiling process.Moreover,the effects of different enzymes on the organic acids production in wilt corn stover silages were investigated.Results showed that adding cellulase and xylanase brought the ratio of lactic acid and acetic acid of 2.53± 0.16 and 1.49 ± 0.13,respectively.These results provided the theoretical and practical foundation for the promotion of the mixed inoculants in northern China.