| Aerobic thermophilic composting is an economical and environmental way for agricultural solid wastes utilization. The materials evolution, microbial diversity and maturity assessment which are related to the quality of composts and mechanism of composting are always being investigated. However, all the indices reported to evaluate the compost maturity are too complicated to be widely used by small-and-medium-sized compost plants. Moreover, many studies of microbial diversity focus on small laboratory-scale composting processes without pay attention to the windrow composting that used extensively by compost plants, and their methods applied may be a little defective too. Thus, the materials evolution, microbial diversity and maturity assessment were studied during the windrow composting of dairy manure and rice stalk, and the main results are as follows:1. The results of physico-chemical and biological indice shows that the whole composting process could be divided into three distinct phages including the mesophilic phase (0-12day), thermophilic phase (22-52day) and cooling phase (62-112day), and the organic matter degradation and water evaporation were more active in the mesophilic and thermophilic phases, while the nitrification and humification mainly took place during the cooling phases. It is interesting to note that compost maturity was not described by a single property and therefore was best assessed by measuring two or more parameters according to the results of physico-chemical and biological indices analysis. Using GI as the standard of correlation analysis, it is found that the indices of C/N、OM loss and NH4+-N/NO3-N were more reliable to assess the maturity of compost produced from dairy manure and rice stalk, and the compost could be considered mature62days later based on all the physico-chemical and biological indices.2. The result of Excitation emission matrix-fluorescence regional index (EEM-FRI) reveals that the protein-like substances decreased while the concentration of fulvic-like and humic-like matters increased during composting process; additionally, the EEM-FRI which is determined fast and simply could be used to assess the maturity of compost produced from dairy manure and rice stalk according to the correlation analysis between other maturity indices. The Fourier-transform infrared (FTIR) spectra of all the nine dairy manure compost samples from different stages of composting exhibited similar peaks and the only difference was the intensity of each peak.3. An effective method suitable for the DNA extraction and purification from compost produced from dairy manure and rice stalk was developed through the comparation of five different DNA extraction methods based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and microbial diversity analysis; by this method, the cell lysis efficiency and the yields of purified DNA achieved were94.6%and33.19μg/g dw respectively, and the purified DNA was clean enough for successful PCR and real-time PCR amplifications in the presence of1μg/μl BSA. In addition, more DGGE bands and16S rRNA gene copies were obtained.4. The populations of mesophilic and thermophilic aerobic microorganisms at various stages were determined by the traditional dilution plating technique, both of the mesophilic and thermophilic populations increased after the beginning of composting, then decreased significantly with the temperature increase and increased again at the end of the thermophilic phage. The maximum populations of mesophilic bacteria and actinomycetes appeared at day22and the values of1g CFU/g were9.33and7.18respectively, while obtained at day32for the thermophilic bacteria and actinomycetes and the values of1g CFU/g dw were7.17and5.78respectively. The maximum populations of mesophilic fungi was achieved at day12, while at day22for thermophilic fungi, and the values of1g CFU/g were5.12and3.24respectively.Denaturing gradient gel electrophoresis (DGGE) was applied to investigate community structure and dynamics during the composting process. Continuous variation was observed with the temperature increase during the first12days, and the cluster analysis indicated that the whole composting process could be divided into three continuous phages including0-12,22-52and62-112which were in accordance with the phases divided according to temperature. Additionally, the microbial diversity of the thermophilic phase showed a higher similarity with that of the cooling phase than of the mesophilic phase. The phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were ubiquitous during composting, and the sequenc results shows that the bands M and X both affiliated with the genus of Bacillus, while the band S shows100%similarity with Streptomyces sp.(EF012138), moreover, most of other sequences were affiliated with uncultured. The EEM-FIR parameters of PH,n and PIV,n showed high correlation with the the evolution of ribotypes that indicated the bacterial community structure and dynamics from DGGE analysis, indicating their potential use for evaluating community structure and dynamics, and a further linear regression analysis demonstrated that PⅣ,n was the most suitable indictor of the community structure and dynamics during the composting process.Four16S rRNA clone libraries of samples from day0,12,42and112during composting were constructed and a total of779randomly selected clones were sequenced successfully, all the sequences had been deposited in the GenBank sequence database under accession numbers JQ336994-JQ337772. The Shannon-Wiener indices of the four clone libraries were2.64,3.25,3.18and4.05respectly. The phyla Firmicutes, Proteobacteria, Bacteroidetes and Chloroflexi were ubiquitous, while Actinobacteria was dominant only at the thermophilic stage (day42); moreover, sequences belonging to the candidate phylum BCR1were fist determined in sample of day112. In the library of sample day42, the genus Arthrobacter (21.74%), belonging to the phylum of Actinobacteria, and the Bacillus and Bacillus-related genera including Soilbacillus, Aneurinibacillus, Marinibacillus and Ureibacillus (8.70%), belonging to phylum of Firmicutes, showed the largest number of clones, whereas in the library of sample day112, the Bacillus and Bacillus-related genera (10.25%) became the unique dominant group. But new genera, including Flavobacterium, Parapedobacter, Nitrosomonas, Ochrobactrum and Rhizobium appeared; it is interesting to note that as composting progressed, the sequences belonging to unclassified groups continuously increased.5. Two strains T1, T2with high cellulase activities were isolated from the fresh compost sample of day82. Based on morphological, physiological and biochemical characteristics and16SrDNA sequences, they were identified as Bacillus cereus and Bacillus subtilis respectively. Tthrough the alignment of the endoglucanase genes of Bacillus subtilis downloaded from NCBI database, the primers were designed, and the endoglucanase gene of the isolated Bacillus subtilis T2strain was cloned, and showed99%similarity to the endoglucanase genes reported. Moreover, the endoglucanase gene was successfully expressed in the E. coli BL21(DE3) with PET28a as the expression plasmid, and high endoglucanase activity was determined in the crude enzyme solution. |
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