Study On Microbial Dynamic Changes In Dry Anaerobic Diges-tion Of Corn Stover By FISH

Anaerobic digestion (AD) had been generally employed to convert waste organic matter to methane and carbon dioxide under anaerobic condition. It is a complicated biochemical process involves variety of microorganism. The community, quantity, growth speed and metabolism activity of the microbes are different during different digestion stages. Interaction of the microbial population and the balance between their metabolism activities directly affects the performance of anaerobic digestion. In this paper, changes of community structure and dynamic variations of the microbes during the dry anaerobic digestion process were studied and the effects of temperature on them were investigated. The study discussed the relationship between microbial community and characteristics of the substrate, in order to optimize the technology of straw biogas dry fermentation. Finally it will provide theory for improving the efficiency of gas production.1. In order to extract the DNA from the microbes in the substrate of dry anae-robic digestion、physical、chemical、biological and the combination of these me-thods were used to break the cell wall of the microbes. The purity and yield of the DNA extracted by different methods were compared. The results showed that the effect of combination of freezing and thawing processing and enzyme treatment is the best. The purity of DNA (A260/A280) is1.392±0.027and its yield is0.143±0.016mg/g, which was significant higher compared with those of the other meth-ods. The purification of crude DNA extracted by bacterial DNA purification kit was increased by34.6%than adding adsorbent PVP (paraformaldehyde). But the A260/A280was still below1.8that cannot meet the requirement of PCR analysis for microorganism’s quantification. 2. Sample pretreatment method for qualification of the microbes in dry anaero-bic digestion by FISH was optimized. The results showed that using absolute ethanol as cleaning fluid, area and abundance of fluorescence signal of the FISH sample was significantly improved than PBS. Adding sterilized glass beads in the process of sample stirring can improve the dispersion of microbe cells and then enhance the accuracy of the microbial quantification by the FISH technical for dry anaerobic digestion. When centrifuging with13000r/min for3min, the conversion rate is increased, and it will ensure the integrity of cell morphology and lay the foundation for the experiment of subsequent FISH hybridization further.3. Fixing time of paraformaldehyde, fixing time of heat, dehydration time of ethanol, hybridization temperature and hybridization time of FISH were optimized by5factors quadric orthogonal regression. The results showed that the optimal hybridization conditions for sample of dry anaerobic digestion of corn stover were that fixing with paraformaldehyde for18h, heat fixing for15min, dehydration with ethanol for3min, hybridization at44℃and hybridization for1.5h. Under this hy-bridization conditions, the area and abundance of fluorescent signal were the lar-gest, and the bacteria distinguish clearly.4. Effects of temperature on the characteristics of dry anaerobic digestion and variations of microbial community structure were studied. The relationship bet-ween properties of the matrix and microbial community structure was analyzed. The results show that dry anaerobic digestion process at37℃and55℃started up quickly. The whole process of digestion did not have obvious accumulation of acids. Both of pH value and alkalinity changed smoothly in this reaction system. Also pH value always maintained at the appropriate range which is suitable for methanogens. At20℃, the growth of methanogens was slow and it can not convert organic acids produced into methane in time, which caused the accumulation of acids. Excessive accumulation of acids inhibited growth and metabolism of metha- nogens further, which resulted in a low level of methane in the whole fer menta-tion process. There are obvious differences in the microbial community structure between37℃and55℃. Methanogens used ethyl changed obviously in37℃, which is more than the amounts of methanogens used hydrogen. However, in55℃, methanogens used ethyl played a leading role in the process of fermentation. This results show that the main bacteria produced methane were methanogens used eth-yl and methanogens used hydrogen in37℃and55℃respectively. In the early stage of digestion, the change of all kinds of methanogens were not obvious, acids-producing bacteria play a leading role. Organic compounds were broken up into small molecule acids, then the small molecule acids were broken up into acetic acid, propionic acid and butyric acid further. Methane producing bacteria is to the largest number of microorganism in the24th days of low temperature fermentation, in the12th days of medium temperature fermentation and in the6th days of high temperature fermentation. Thus it can be seen that as temperature increases, it can shorten the fermentation periods reached the peak earlier, and improved the effi-ciency of fermentation and the rate of gas production greatly.