| Chinese liquor has a long historical standing and it is one of the six major series of distillate spirits in the world.There are various kinds of Chinese liquor that can be classified as Luzhou-flavor,Maotai-flavor,rice-flavor,and Fen-flavor,of which Luzhou-flavor Chinese liquor is the mainstay in liquors by solid fermentation in China.Luzhou-flavor liquor such as Luzhou Laojiao and Wuliangye accounts for more than 70%of Chinese liquor production.Production of Luzhou-flavor liquor is based on pits where ample microbial communities are living especially bacteria,and interactions among all sorts of microbes in it make flavor components of Luzhou-flavor liquor very unique.The Luzhou-flavor liquor was obtained by distillation after the liquor fermentation,and the residue was distillers' grains(DG).As the main by-product of Chinese liquor industry,the annual production of wet DG had recently reached approximately 20 million tons.The major constituents of DG were sorghum and rice husk,which contained abundance of lignocelluloses.Chinese liquor industry has largle biomass resources,from which it not only contained the aromatic biomass that was influence to the liquor quality,but also had the lignocelluloses that was the main component in DG.Therefore,how to use of these biomass more efficiently had become a difficult issue which we were confronted.Based on the thoughts above,the 30-years and 300-years pit mud and distillers' grains were collected from Luzhou Laojiao,which were analysed the species composition and abundance of two pit muds through 16S rDNA high-throughput sequencing.Metagenomics and metaproteomics were used to analysis the metabolic mechanisms of flavor components in liquor.Understanding these mechanisms will help to improve the liquor quality with genetic technology or chemicrobiology.Furthermore,to utilization of biomass in DG sufficiently,analytic chemistry was also used to investigate the transition and utilization of lignocelluloses in DG.To studing on the microbial diversities of 30-and 300-years pit mud,16S rDNA sequences were sequenced on a high-throughput sequencer Miseq from Illumina.Sequencing data indicated that by 97%sequence similarity,there were 1062 and 1164 of operational taxonomic units(OTUs)in the 30-and 300-year pit muds,respectively.The most common microbe was firmicutes phylum in both samples in general and by genus,that was clostridium and lactobacillus,both of which,along with another common one methanobacterium,constituted important functional colonies in the pit muds serving to fermentation of Chinese liquor.The abundance of methanobacterium and clostridium were higher in PM300,whereas the abundance of lactobacillus was lower.With sequencing depth of 60,000,within-sample alpha diversity analysis revealed more plentiful diversities of microbial communities existing in the 300-than in the 30-year pit mud.We found plenty of microorganisms that were related to the quality of liquor through 16S rDNA analysis.Consequently,we carried out the research on metabolic and transition mechanism of aromatic biomass.To find the genes,which were related to the metabolism sufficiently,we studied on the abundance of species and functions of microorganisms in two pit muds and annotated through the metagenomic analysis.In addition,we also compared the interative pathways of the two samples.The result showed that there is a higher abundance of archea in 300-years pit mud(about 50%),especially the methanogens(about 30%).In the sample of 30 years pit mud,bacteria were the main microorganisms(about 80%),and in which the lactobacillus(about 50%)was shown a comparatively high amount compare to other bacterium.The annotation result of KEGG database shows that most genes in two samples were menbrance transport genes(12218 vs 8237).As the butyrate metabolism and the methane metabolism were very important during liquor brewing,we annotate the genes involved in these two metabolisms by KEGG database.We found that there were several genes were involved in the metabolisms in both samples,however,more genes were annotated in PM300.The annotation result of CAZy database and eggNOG database in two samples were the same.For CAZy database,there were 12000 genes annotated as glycoside hydrolases in PM300,whereas in PM30,8750 genes were annotated.For eggNOG database,21000 genes were annotated as function unknown in PM300,but there were only 7000 genes were annotated.Consequently,there were more genes were annotated in PM300 by these two databases.Interative pathway result shown that PM300 had more unique KEGG orthologous groups(KOs)than PM30,and for KOs in common,more KOs were annotated in PM300 than that in PM30.To find the proteins which were related to the metabolism sufficiently,we analyse the microbial proteins in pit mud through quantitative proteomics.To increasing the extracted protein content,four different protein extraction methods with minor modification were investigated in order to determine the optimal method for proteins extraction.A protein extraction method suitable for pit muds was established.iTRAQ-based proteomics was applied to investing the proteins of microbes in pit muds 135,930 proteins which were belong to 305 proteins were identified.To analyse the metabolic mechanism of aromatic biomass in liquor,we selected 59 proteins of functional microbes in 30-and 300-years pit muds.55 proteins were highly expressed in PM300.These functional proteins were found to be involved in methanogensis,formation of caproic acid and butyric acid.They were highly expressed in PM300 could explain the reason why the liquor quality from 300-year pit mud is better than the 30-year to some extent.To transition and utilization of another biomass in liquor industry,lignocelluloses,we conducted a study to analyse the nutrition composition of distillers' grains.We found that there were plenty of lignocelluloses in DG and could as a poteintial carbon source for cultivating microorganisms.Consequently,four different treatments,including sodium hydroxide(0.5-2%w/v.50-121 ?,1 h),calcium hydroxide(0.2-4%w/v,50-121 ?,1 h),dilute sulfuric acid(0.5-2%w/v,50-121 ?,1 h)and hot water(50-121 ?,1 h),were investigated in order to determine the optimal method for degrading the cellulose in DG for producing monomeric sugars.Enzymatic hydrolysis followed each treatment method.The results showed that the highest yield of monomeric sugars and cellulose degradation are obtained by using 2%dilute sulfuric acid for 1 h at 121 ?(313.50 g/kg DG)and this was still higher than the yields of monomeric sugars pretreated using the other three methods with the addition of enzymatic hydrolysis.These results suggested that one-step dilute sulfuric acid pretreatment method could be used as a cost-effective method to convert lignocelluloses in DG into monomeric sugars.16S rDNA analysis revealed that a lot of bacillus in cellars.Bacillus thuringiensis(Bt)was the most productive microbial insecticide in the world.Consequently,we were trying to isolate Bt from pit muds and distillers' grains,identification of it's molecular biological features and insecticidal acitivity,followed by cultivating Bt using distillers' grains extract.The results showed that one Bacillus thuringiensis strain was isolated from 168 distillers' grains and pit muds samples.PCR-RFLP analysis showed that the genotype of this strain was cry1Ac.The result of protein pattern was the same as PCR-RFLP,molecular weight of crystallin was 130 kD,which was encoded by crylA gene.Bioassay indicated this isolate was toxic to lepiteran pest diamondback moth,with mortality of 80%.However,it was non-toxic to dipteran pest Culex quinquefasciatus.Distillers' grains extract(2%H2SO4,121 ?,1 h)was selected for Bt fermentation,single factorial experiment showed that soybean meal,CaCO3,MnSO4 and K2HPO4 improved the production of Bt-based insecticide.Plackett-Burman design(PB),path of steepest ascent and response surface methodology(RSM)were employed to optimaize the medium components for fermentation of Bt.The optimal composition is as followed:52%DG extract,27.3%soybean meal,0.83 g/L CaCO3,0.4 g/L MnSO4,0.4 g/L K2HPO4,0.4 g/L Tween 100 and pH 7.2.25 times improvement in production of spores is achieved compare with original DG medium after optimization.The use of DG provided a carbon source that was effective not only for Bt production,but also for reducing industrial waste and environmental pollution. |
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