Microbial Community Succession And The Mechanisms Of Solid Waste Composting By Ligninolytic Microorganism

Composting, as an economic and effective technology for solid waste treatment, is one of important research subjects. But the application of traditional composting are limited due to the biodegradative recalcitrance of lignin and the stresss of heavy metal in composting materials. So the improvement of composting efficiency and heavy metal control are very important. Since microbial community are responsible for organic matter degradation during composting, the study on microbial community succession is a basis of composting technology and theory researches. To improve composting efficiency and control heavy metal pollution, exploring a new composting technology and theory based on microbial community study are needed. It would promote the development of composting technology, and be of theoretical and practical value for efficient and rapid composting treatment.This dissertation discussed microbial community succession, and investigated the effects of ligninolytic microorganisms and enzymes on microbial activities, lignin degradation and humus formation in composting. The control of heavy metal are also considered in this study. According to these researches, we developed a new and efficient composting techonology based on ligninolytic microorganisms or enzymes. This dissertation is composed of four sections.The first section describes the research on the relationship between microbial community successtion and lignocellulose degradation.(1) Microbial populations and their relationship to bioconversion during composting were studied by quinone profiling. The obvious changes in microbial population were observed. Fungi indicated by Q-9(H2) and Q-10(H2) were considered to be the most important hemicellulose and cellulose-degrading microorganisms during thermophilic stage. MK-7and Q-10, indicative of certain bacteria and fungi respectively, were responsible for lignin degradation at the thermophilic stage, whereas the mesophile ligninolytic fungi (Aspergillus niger, Penicillium, and etc.) indicated by Q-9is limited. The highest lignin degradation ratio and good cellulose degradation were found at the cooling stage and were correlated with Q-9, MK-7and long-chain menaquinones attributed to mesophilic fungi, bacteria and actinomycetes, respectively.(2) MK-9(H2) positively correlated with MK-9MK-9(H6) and MK-10(H6) respectively, which suggests coexistence and possibly even cooperativity among the corresponding microorganisms. Significant positive correlation was also suggested for the bacteria indicated by MK-5(H2) and the actinomycetes containing MK-9or MK-9(H2) as major quinone. RFLP analysis showed there were no obvious changes in genetic diversity of bacterial community during composting, and bacterial community is very active during thermophilic stage.(3) Principal component and correlation analyses showed a significant positive correlation between lignin degradation ratio and the total content of Q-9、MK-7、MK-8(H2)、MK-9、MK-9(H2)、MK-9(H6)、MK-9(H8)和MK-10(H6), which indicated that the microbial community consisting of mesophilic fungi, bacteria and actinomycetes played a key role in lignin degradation. And a positive correlation between the latter7quinones and cellulose degradation was found.(4) Lignin degradation significantly affected hemicellulose and cellulose degradation. The inferior position of ligninolytic fungi in composting might be responsible for slow degradation of lignin. It is expected that inoculating ligninolytic fungi would accelerate lignin degradation and promote composting efficiency.The second section focuses on the study of mechanisms of the composting technology by adding ligninolytic microorganism or enzymes.(1) The characteristics and degradation ability of ligninolytic fungus (white-rot fungus) were studied. Temperature and substrate moisture were confirmed to be the main effect factors. The optimal treament conditions are:5days of fungi incubation,0.8%of inoculum,0.3%of Tween80addition,37℃and85%of water content of substrate. This fungus could destroy lignin structure by the cleavage of aromatic ring and the transformation of long-bond hydrocarbons of giant molecule to the short-bond. The study also applied relational analysis to the enzyme production process of white-rot fungi, and constructed GM(1,1) model which could be used to predicted enzyme production to reduce experimental quantity.(2) Composting of lignocellulosic waste by ligninolytic fungi was studied. By this composting method, microbial activities were enhanced, and lignin degradation ratio reached43.9%, and more completed degradation of organic matter was also found.(3) The higher humification degree (54.1%) and total microbial biomass were observed during the composting process with the inocula of ligninolytic fungi than those in tradition composting. While the low diversity and evenness of microbial community were found. It might be because the lignin-degrading fungi and actinomycetes respectively indicated by Q-9and long-chain menaquinones increased, and bacteria with nitrogen fixation and lignin-degrading bacteria indicated by Q-8and MK-7increased, whereas other microorganisms decreased. Composting method with fungi inocula changed microbial community structure obviously, especially from day16to28, enhanced the effect of lignin-degrading fungi and actinomycetes.(4) The transfer and adsorption behaviours and degradation mechanisms of ligninolytic enzymes (LiP-MnP) were studied. The transfer abilities of ligninolytic enzymes on straw and chaff are better than vegetable residues and soil. The adsorption on these four substrates belonged to favourable adsorption, which ensured the effective contact between enzymes and substrates. Degradation mechanisms of ligninolytic enzymes included:benzyl alcohol unit oxidation, methoxy and methyl group removal, C=C and ether bond and Ca-Cβ cleavage, and aromatic ring cleavage and replacement. Therefore, alcohol hydroxyl,-CH2,-OCH3,-CH3, ether bond, C=C, and aromatic ring in lignin decreased. Vanillic acid and ferulic acid unit in lignin were decomposed, and lignin dimers were degraded into low-weight matters.(6) Carbon source utilization and lignin degradation by microbial community were studied during composting with LiP-MnP enzymes. Addition of LiP-MnP enzymes significantly promoted organic matter degradation and carbon source utilization. It was found by Biolog analysis that the enzymes mainly affected the utilization of17carbon source. The utilization of pyruvic acid methyl ester, a-cyclodextrin, D-mannitol, D-galacturonic acid, itaconic acid and L-asparagine were enhanced, whereas the utilization of D,L-a-glycerol phosphate, L-threonine, glycyl-L-glutamic acid and putrescine were weakened. Lignin degradation was significantly promoted by LiP-MnP enzymes. There might be two reasons:the enzymes catalyzed lignin degradation, and enhanced the utilization of lignin degradation products such as phenolic compound and carboxylic acid.The third section describes the research on composting of Pb-polluted substrates by inocula of ligninolytic fungi.(1) Mycelial growth and the degradation of lignocellulosic waste by ligninolytic fungus Phanerochaete chrysosporium under lead stress were studied. P. chrysosporium could grow in liquid media with400mg L-1Pb(II), which might be due to its two possible responses:dense and tight twist of hyphae, and secretion from mycelia to resist Pb. During solid-state fermentation, fungal colonization capability was positively correlated with the removal efficiency of soluble-exchangeable Pb when its content was higher than8.2mg kg"1dry mass. Carboxymethyl cellulase activity and cellulose degradation were inhibited at different initial Pb concentrations, whereas low initial Pb concentrations strengthened xylanase and ligninolytic-enzyme activities and the hemicellulose and lignin degradation.(2) Degradation of Pb-polluted lignocellulosic waste and the restrain of Pb hazards by P. chrysosporium were studied. P. chrysosporium degraded43.1%of lignocellulose, formed humus and reduced active Pb ions, even at the concentration of400mg kg-1dry mass of Pb. The highest lignocellulose degradation (56.8%) and organic matter loss (64.0%) were found at Pb concentration of30mg kg-1, and selective lignin biodegradation was enhanced. Scanning electron micrographs with energy spectra showed that Pb was immobilized via two possible routes:adsorption and cation exchange on hypha, and the chelation by fungal metabolite.(3) Treatment of Pb-polluted solid waste by composting with ligninolytic fungi was studied. Microbial biomass and CO2production increased, whereas soluble-exchangeable Pb content was reduced to0%. water-soluble organic C/N ratio might be not a suitable evaluating indicator for the composting of metal-polluted waste, while lignin and coarse fibre were suggested.(4) Pb-polluted soil could be remediated by composting with ligninolytic fungi, and microbial activities and carbon source utilization were enhanced. Active Pb form was transformed into the residual and Fe-Mn oxidation forms. It might be due to adsorption on fungi mycelia and complexation of humus.The four section focuses on the model construction for relationship between enzyme activities, microbial community and lignin degradation respectively.(2) It was confirmed that ANN model could be used to predicted lignin degradation by the input of LiP and MnP activities.(2) ANN model could predicted lignin degradation by the input of community information. These showed the advantage of ANN application in complex relationship resolution, which could be a reference for quantification of effect of enzymes and microorganisms on lignin degradation.This dissertation revealed microbial community succession and the mechanisms of solid waste composting by ligninolytic microorganisms, which could provide microbial information for the understanding of composting process and benefit the studies of complex microbial inoculants. It is expected to lay the foundation for the development of a new composting method with high efficiency, which could conquer some deficiency existed in traditional composting technology.