Breeding Of The Acetogenic Bacteria With Special Efficiency Under Waste Activated Sludge Based On Mesophilic/Thermophilic Phase Anaerobic Digestion System

The issue of reduction and reuse about waste activated sludge (WAS) has been a problem in urban development. The acetogenic bacteria with special efficiency in the anaerobic fermentation can produce stores of volatile short-chain fatty acids(VSCFAs), which can be used as easily used organic carbon source for subsequent anaerobic fermentation. In order to provide the technical support for engineering applications and reusable applications of WAS, the research would breed the acetogenic bacteria in bottom matter, explore the characteristics of acidification of the acetogenic bacteria, and parse its populative structure, in the condition of using WAS as the basal object and basing on the Mesophilic/Thermophilic Phase Anaerobic Digestion System (M.-T.PADS).With35days’ anaerobic fermentation in the M.-T.PADS, the research arrives the following conclusions:pH has gradually stabilized at7.0; organic matter of WAS would be taked as soluble total organic carbon(STOC) to gradually hydrolyze out, and ultimately be maintained at2000mg/L; at the same time, VSCFAs has been floated in the80mg/L～130mg/L, and stabilized at an average of100mg/L. On the basis of the above conclusion, M.-T.PADS has indicated a relatively stable state on anaerobic acidification.According to metabolic characteristics of the anaerobic acetogenic bacteria in M.-T.PADS, the research utilizes sodium propionate and sodium butyrate as the carbon of rich medium, to achieve enrichment of the bacteria. The research has found that the mixed bacteria can effectively achieve metabolic conversion of the complex macromolecular acids, when the pH is7.1, also the metabolic capability can reach strongest in the fluid enrichment medium containing supernatant fluid of mesophilic anaerobic reactor. Meanwhile, the study eventually isolates four anaerobic acidic bacteria strains by diluted separation and anaerobic incline, containing NJUST19, NJUST20, NJUST21and NJUST22. The study realizes NJUST19strain can produce235mg/L acetic acid after7days’ culture in50mL culture medium, which has optimalizing metabolic capability, so the strain can be used as optimal acetogenic bacteria in the subsequent anaerobic fermentation of WAS.The investigation has separately performed the analysis of microbial structure about the four anaerobic acetogenic bacteria, through the technology of PCR-Clone. The research illustrates this four single bacteria both are gram positive bacteria. And meanwhile, the SEM of bacterium has separately shown morphological characteristics of the four bacteria. Such as, NJUST19strain:Clostridium,(9.4～10.4)μm×(3.5～4.5)μm; NJUST20strain:Bacillus, (5.4～8.5)μmx(3.5-4.6)μm; NJUST21strain:Sphericity, D≈(4.0～4.5)μm; NJUST22strain: Bacillus,(8.7～10.3)μmx(5.2～6.1)μm. After BLAST contrast in the GenBank website the research has found that the sequences of DNA range from1447to1471bp, and the four strains have its certain specificity. Therefore, the four bacteria can be named as Clostridium sp. NJUST19(JX975447), Bacillus sp. NJUST20(JX975448), Microbacterium sp. NJUST21(JX975449), Bacillus sp. NJUST22(JX975450).From the metabolic characteristics for acidification of the NJUST19strain, the research reveals that NJUST19can sufficient use starch, glycerin, carbohydrate and organic acid to accomplish the metabolic transformation from macromolecule to VSCFAs, especially acetic acid. Moreover, the strain can produce262.9mg/L formic acid,422.4mg/L acetic acid by amylolysis; the strain can break down maltose into228.4mg/L formic acid and205.4mg/L acetic acid; Propionic acid can be decomposed into143.9mg/L acetic acid; also glycerin(glyceryl alcohol) can be decomposed into97.1mg/L acetic acid and67.8mg/L butyric acid. Therefore, NJUST19has shown a wide range of carbon utilization. And meanwhile, from the the effects of metabolic transformation with NJUST19in organic nitrogen of peptone, carbamide and inorganic nitrogen of NH4Cl and NaNO3, the study has indicated that the NJUST19can produce355mg/L acetic acid when takes NH4C1as inorganic nitrogen; also produce350mg/L acetic acid when takes peptone as organic nitrogen. So, the research separately take peptone/NH4Cl as the optimal organic/inorganic nitrogen source for the metabolic activity of NJUST19strain.By investigating the effects of environmental factors for the metabolic activity of NJUST19strain, the research shows that the acidic environment (pH<6.5) would restrain the growth of NJUST19strain, accordingly, weakly alkaline (8.5≤pH≤9.5) could assist the metabolism of NJUST19strain; the metabolic capability for acidification of NJUST19strain in thermophilic condition is much higher than the mesophilic stage, so under its metabolic characteristics, initially conclude the NJUST19strain belongs in thermophilic bacterium. In conclusion, the optimal environmental conditions of NJUST19strain would be defined as: pH=9.0, mesophilic condition is30℃, thermophilic condition is65℃, C/N=30. Under this conditions, would make NJUST19strain to accomplish the efficient acid production in metabolic activity.