| Biogas is a renewable clean energy, due to the long and cold winter in northernChina, there are over-acidification during fermentation and gas production rate andother issues of biogas production. In response to this phenomenon, this paper usingtraditional microbiology, ecology and modern molecular biology techniques, from freshbiogas slurry isolated and purified Clostridium butyricum, and later expanded itstraining transfer to anaerobic fermentation system to become dominant flora, pre-fermentation by Clostridium butyricum, the hydrolysis product of the fermentation stageis to control acid-based mixture, and then to improve the system of biogas productionrate and amount of biogas purposes.(1) By anaerobic Hungate technique, the slurry in the enrichment culture ofmicroorganisms, specifically isolated, screened a Clostridium, and further verificationexperiment, the experiment includes a carbon, indole reaction, gelatin liquefaction andhydrolysis of starch and other physiological and biochemical experiments and16SrDNAsequence analysis identified it as Clostridium butyricum.(2)By single factor experiments to study the initial pH, fermentation time,temperature, inoculum size, carbon and nitrogen sources on expanding culture ofClostridium butyricum impact. Optimal conditions were optimized as follows: initial pHvalue of8.0, the culture temperature is37.0℃, inoculum was5.0%. Fermentationmedium composition: tryptone10g/L, ammonium bicarbonate10g/L, glucose10g/L,the salt solution40ml, Cysteine0.5g/L, resazurin0.5mL/L (0.1%), the configurationprocess strictly anaerobic.(3)Establish and optimize fluorescence quantitative RT-PCR method to monitor thedynamic changes of the biogas fermentation process of Clostridium butyricum. Inquantitative10-fold dilutions of the standard template for the amplification and thestandard curve established. The results showed that among the various concentrations ofstandard amplification curve presents a good gradient distribution, Ct values and theinitial template copy number of the standard curve at1.9×101~1.9×106. Between copieshas a good linear relationship, linear regression equation is a function expression Y=-3.56X+57.55(on behalf of the logarithm of the initial template copy number, Y behalf Ct value), and the copy number of Ct values starting template correlation of0.997. By dynamically monitoring and analysis, trends within the system dynamicschanges of Clostridium butyricum and butyrate production coincide.(4) Pig manure as fermentation substrate, it was pre-fermented by Clostridiumbutyricum to enhance the butyric acid content and reduce the propionic acid content inthe biogas fermentation acidification phase. During pre-fermentation, Plackett-Burman(P-B) design were adopted to screen significant factors, and Central CompositeDesign(CCD) were used to optimized the pre-fermentation condition.The optimizationexperiment results for the moisture content, inoculation time and temperature by CCDwere determined to be62.04%、27.22h and37.13℃, respectively. Under the optimumcondition, the result of verification experiment showed that21.13g/kg was themaximum butyric acid yield. The pre-fermentation experiment and maximum butyricacid yield of21.55g/kg was obtained, with a corresponding mathematical model wasestablished. This proved that statistical method was a powerful tool for the optimizationof butyric acid fermentation.(5)The pre-fermented manure was used for biogas production, biogas productionincreased by19.02%in comparison with the control (without its pre-fermentation). |
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