| Thermophilic phase is the main stage of composting,during which the organic macromolecules of composting material were destroyed,fractured and decomposed.It is also an important phase to ensure that the composting is harmless.However,due to the influence of high temperature,the microbial communities in the thermophilic phase are limited,which affects the degradation efficiency of macromolecules.Thermophiles are a group of microorganisms which can grow and propagate in thermophilic environment.For most thermophiles,the metabolism processes and reproductions are relatively faster,the growth activities are higher,and the extracellular enzymes are more thermostable.Inoculating thermophiles in composting can not only increase the number of microorganisms of the thermophilic phase,but also accelerate the decomposition and transformation of recalcitrant organic materials,which will result in improving composting efficiency.Thermophiles are mainly composed of bacteria and fungi.Among them,bacteria are the most nutritionally diverse group.They can secrete various kinds of enzymes which will be responsible for the decomposition of most organic matter and generate biological heat.The tolerable temperature for fungi is relatively lower in comparison to bacteria,but fungi perform better at degrading stubborn substrates,such as cellulose and hemicellulose.Although fungi almost stop growing when the temperature of composting heap exceeds 55℃,the cellulases and hemicellulases they secreted still exist,and most of the enzymes keep good activity at 70℃ for a long time.Strain A.fumigatus Z5 and G.stearothermophilus B5 were two efficient composting strains isolated from composting by our lab.Both of them are thermophiles and can survive in above 50℃ environment.In this paper,biochemistry and bioinformatics analyses were combined so as to explore their influence on the simulated and practical composting.Here,the degradation characteristics of Z5 strain on wheat straw were studied at 50℃by inoculating Z5 spores in simulated composting.Samples were taken at different times and related parameters were measured so as to study the mechanism of lignocellulose degradation.The relative total carbon content of the treatment group decreased by 22.05%,while the relative total nitrogen content increased by 76.77%than that of the control group(CK).The contents of cellulose and hemicellulose also decreased by 36.97%and 39.77%,respectively.Images of SEM and AFM showed that clear hole appeared on the surface of the wheat straw,and the roughness increased from 0.97%to 43.83%.Fourier infrared analysis showed that the absorption peak area of the functional groups C-O-H and C-O-C decreased gradually,indicating that cellulose and hemicellulose related substances had been greatly degraded.The X-ray analysis results showed that the crystallinity of the wheat straw decreased from 43.8%to 28.3%,indicting that the structure of wheat straw became loose in this process.The strain Z5 could decompose cellulose,hemicellulose and other polysaccharides from the surface of wheat straw by secreting various extracellular hydrolytic enzymes.A thermophilic bacteria B5 was also isolated from composting and later identified as Geobacillus stearothermophilus according to its morphology,physical,chemical characteristics,and genetic sequence analysis.The results of carbon source spectrum tests indicated that B5 could utilize various kinds of carbon sources,including sugars,amino acids,hexose acids,carboxylic acids,esters and fatty acids.B5 can grow in the pH ranging from 4.0 to 9.5,which is similar to the pH value of most composting materials.It can also grow at a broad temperature,ranging from 40℃ to 75℃.The optimal temperature for B5 is 60℃,which means B5 could grow well at composting thermophilic phase.The whole genome analysis showed that the chromosome of B5 was a single closed circular structure without plasmid.The genome size was 3.37 Mb,encoding 3371 genes.Compared with four reported cellulose decomposing strains,2202 homologous genes were detected,and they encoded similar numbers of sugar transporters and glycoside hydrolase genes.But the number in major facilitator superfamily(MFS)of B5 was higher,indicating greater transport capacity for small soluble materials.In addition,100 CAZymes were predicted in the genome of B5,among which glycoside hydrolase(GH)family and glycosyltransferase(GT)family members were the most abundant.These two family members play an important role in the cleavage of polymer substrates.Subsequently,the transcriptome responses of B5 at the different temperature(40℃,60℃ and 70℃)were analyzed with rice straw as the only carbon source.The expression level of the heat shock proteins was significantly improved when the temperature approached the critical value.They acted as chaperone proteins to promote degradation of denatured proteins,thereby protecting other proteins from thermophilic stress.Pathway enrichment analyses showed that the protein synthesis was vigorous in thermophilic stress.It may due to some key enzymes related to cell survival,which might enhance resistance to high temperature.At the same time,genes related to biological processes and cell metabolism are down-regulated in various degrees,such as carbon metabolism,fatty acid metabolism and secondary metabolite synthesis.The reduction of carbon catabolism can save energy and thus enhance resistance to high temperature.Geobacillus was a kind of thermophilic genus which exist extensively in composting systems.In order to understand the roles of the genus in composting,32 genomes of Geobacillus strains were downloaded and studied by pan-genome analysis.The core genome contained 940 orthologous genes,and the pan-genome contains 14913 genes.The type of its pan-genome was open,which means the gene pools are very abundant.In COG functional analysis,amino acid transport and metabolism and carbohydrate transport and metabolism accounted for 12.35%and 8.16%of the COG classification,respectively.The most abundant COGs were COG0477,which could catalyze the transport of various substrates,including carbohydrates,lipids,peptides and other molecules;COG0366,encoding α-amylase,which could hydrolyze the α-bonds of most polysaccharide;COG0395,which was associated with the absorption of carbohydrates.The GH family members were widely distributed among Geobacillus strains,including amylase(GH13),xylanase(GH10).and 1,4-β-xylanase(GH39,GH43,GH52).Geobacillus were thermophiles,and they can propagate quickly and secret abundant glycoside hydrolases during the thermophilic phase.Organic matter was degraded quickly,especially in the late period of thermophilic phase,during which the enzyme activity in composting reached the peak.KEGG analysis showed that the mobility of genes in Geobacillus was frequent,especially for G.stearothermophilus.A large number of genes related to environmental adaptability existed in the pan-genome,and they may be related to the horizontal gene transfer(HGT),which played an important role in their evolution.The plate confrontation experiment showed that there was no antagonism between B5 and Z5 strains.In order to explore the effect of Z5 and B5 on composting efficiency,different inoculants were inoculated in composting piles composed of rice straw,vinasse and pig manure.The pile was divided into four small piles:pile A(CK),pile B(inoculated with B5),pile C(inoculated with Z5)and pile D(inoculated with both B5 and Z5).The results showed that the composting efficiency and maturation degree of the treatment groups were higher than those of the control group.At the mesophilic phase,the heating speeds of pile C and D were clearly faster than that of pile A and B.The temperature of pile B and D at the thermophilic phase were 5℃ higher than that of pile C at the same time,and 8℃ higher than that of pile A.Besides,the thermophilic phase of B and D last longer.In comparison with compile A and C,the pH values of pile B and D were lower.Besides,the contents of ammonium nitrogen and nitrate nitrogen were higher than that of pile A and C at the same time.Material content determination,infrared spectrum and X-ray photoelectron spectroscopy(XPS)analyses showed that the degradation efficiency of cellulose and hemicellulose of compile C and D were relatively higher.The excitation-emission-matrix fluorescence(EEM-FL)results showed that the pile C and D matured first,and then continued to mineralize until the end of the composting process.All above,the piles inoculated with both B5 and Z5 strains had the best composting efficiency,indicating that the conversion rate of organic matter and the composting process could be accelerated by co-culture of the two thermophiles.In summary,our study indicated that A.fumigatus Z5 strain can secret abundant extracellular enzymes and decompose lignocellulose from the surface of rice straw.The function of G.stearothermophilus B5 and Geobacillus genus were analyzed by bioinformatics approaches,and transcriptome analysis revealed the regulatory mechanism of B5 in high temperature.Finally,the effects of Z5 and B5 strains on improving composting efficiency were verified by composting experiment. |
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