A Microbial Ecological Study On The Mechanism Of Acetate Accumulation During Anaerobic Incubation Of Sewage Sludge

Acetate as an important methanogenic precursor produced at the acidogenesis steps has received significant attention in recent years. The main driving force for investigating the production of acetate is the higher economic values, owing to its wider range of applications in the chemical industry. The bioprocess strategy that results in the production of acetate not only achieves the objective of controlling pollution and reducing sludge volumes but also efficiently explores the resource of organic substance in organic waste. In recent years, mixed culture biotechnology (MCB) has become a very promising direction of environmental engineering. Owing to the use of undefined mixed cultures, process development in MCB can only be based on natural/ecological selection by manipulating the operation of the bioprocess or by varying the source of the natural inoculum. In order to achieve the maximum and stable production of acetate in acidogenic reactors, methanogenic inhibitors are commonly used selection pressures. By addition of inhibitors, short-chain organic acids and other intermediate metabolites cannot be consumed by methanogenic archaea without affecting the acid-producing microorganisms.Here we tested the effects of BES and chloroform on a natural bacterial community by using environmental microbial molecular techniques to study the relationship between microbial community structure and acetate accumulation, and to elucidate the contribution of different microbial groups with respect to the flow of carbon and electrons, and then lay the theoretical foundation to enhance acetate production by using anaerobic digestion. For our study we chose anaerobically incubated sewage sludge, which is a well-studied model system with respect to the bacterial community structure and biochemical processes. The main research results of this dissertation were shown as follows:1. Development of molecular assays indirectly monitors homoacetogensInitial experiment revealed that, with FTHFS primer set, 55% of clones, other than authentic acetogens, fell into the cluster of sulfate reducing bacteria and other non-acetogenic microorganisms by sequencing of quantitative PCR products. Novel primers flanking the FTHFS fragment were designed and tested. High specificity and sensitivity for estimation of the abundance of acetogens were confirmed analysis of a collection of acetogens, clone libraries and melting curves. The utility of the assay was validated and used in quantifying the FTHFS gene present in different anoxic and oxic habitats. A detection limit of approximately 500 target molecules per assay was achieved for different length fragments quantification, which corresponded roughly to 10⁴ targets per gram of dry samples. The abundance of FTHFS gene recovered by fhs1 assay was in the order of magnitude of 10⁵ up to 10⁷ copies per gram of dry weight sample, and the maximum calculated abundance of acetogens relative to Eubacteria was 0.6%-0.9%, confirming the low proportion of acetogens to total bacteria in environments.2. Inhibitory effect of methanogenic inhibitors on the specific activity of mesophilic anaerobically digested sludge The objective of this study was to evaluate the inhibitory effect of BES and chloroform towards the main anaerobic microbial populations involved in various metabolic steps of anaerobic digestion processes, such as acidogenic fermentation, syntrophic acetogenesis, and methanogenesis. The results of short-term batch bioassays indicated that the toxicity varied widely depending on the microbial population. Hydrogenotrophic methanogens appeared to adapt rapidly to toxicants. The concentrations of BES and chloroform causing 50% metabolic inhibition (IC₅₀) were 8.7 mM and 0.009%, respectively. However, the IC₅₀ of acetoclastic methanogens were 5.8 mM and 0.006%, respectively. The mechanisms responsible for the microbial toxicity of methanogenic inhibitors towards glucose, propionate and butyrate-utilizing microorganisms were fairly different. Chloroform treatment significantly reduced the glucose-fermenting and fatty acid-degrading activity, while high concentrations of BES only caused a slightly thermodynamic inhibition. Taking into account the acid-producing and methane inhibiting efficiency, the optimal concentration of BES and chloroform we chose were 50 mM and 0.05%, respectively.3. Effect of inhibitors on the quantity and diversity of acetate-consuming communityThe T-RFLP fingerprinting and the real-time PCR results suggested that inhibition treatments influenced not only the quantity but the diversity of archaeal community. As the acetoclastic Methanosaetaceae was more sensitive than hydrogenotrophic Methanobacteriales and Methanomicrobiales, the niche vacated by Methanosaetaceae was gradually filled with hydrogenotrophic methanogens. Thus, even though the acetoclastic methanogen was inhibited by relatively low concentration toxicant, acetate could still be utilized syntrophically by consortia of acetate oxidizing bacteria and hydrogenotrophic methanogen.4. Succession of bacterial populations under suppressed methanogenesisMultivariate T-RFLP data statistical analyses suggested that both the toxicants not only inhibited methanogenic activity but also considerably altered the bacterial community structure. Although by no means all, species of low % G+C Gram-positive bacteria (Clostridiales), high % G+C Actinomycetes and uncultured Chloroflexi showed relatively greater tolerance of CHCl₃, whereas BES cluster was characterized by prevalence of hydrogen-oxidizing species (Moorella sp. and Geobacter hydrogenophilus). Gram-negative bacteria such as Bacteroidetes and Synergistes tended to be indirectly suppressed by undissociated organic acids. It was also noteworthy that up to half of hydrolytic and fermentative species were insensitive, since their T-RFs showed not significant correlation to environmental variables (species fit < 0.15).5. Secondary fermenters under suppressed methanogenesisChloroform not only inhibits methanogenesis, but also inhibits homoacetogenesis. The fhs gene copy numbers exponentially dropped from the very beginning and then stayed at about 3.0×106 mL-1 until the end. With BES, however, the favorable thermodynamic condition (ΔG < 20 to -24 kJ mol) coupled with the increase of fhs gene copy numbers (1.5-folds, P = 0.02) indicated that homoacetogenesis from H2/CO)2 was stimulated under suppressed methanogenesis. For further understanding the dynamics of syntrophic acetogenic bacteria (SAB) under suppressive conditions, fluorescent in situ hybridization targeting 16S rDNA genes were performed. In both treatment groups, the numbers of SAB decreased significantly over time. The densities of syntrophic propionate-oxidizing bacteria (SPOB) and syntrophic fatty acid-β-oxidizing bacteria (SBOB) were 13.3 and 7.9-fold lower on average than those in the non-inhibited samples (P < 0.001). Unlike CHCl₃, BES only cause slightly lower cell counts in both SPOB and SBOB (1.5 and 2.0-fold; P = 0.02), probably due to the relatively low H2 partial pressures. The syntrophic acetogenic reaction in BES incubations were at values close to thermodynamic equilibrium (ΔG≈0 kJ/mol).6. Organic carbon flux and mass balance analysisBy using stable isotope and radioactive isotope techniques, we found that apparent fractionation factor (α_app) values of uninhibited sludge samples were between 1.00-1.01. These highαapp are characteristic for methane production exclusively from acetate. With a low concentration of methanogenic inhibitor, values ofα_app gradually increased to 1.03-1.0⁴, indicating methane production was migrating to CO₂ reduction pathway. Carbon balance analysis and [U-¹⁴C] glucose degradation experiments confirmed that when methanogenesis was specifically inhibited, the syntrophic interaction between hydrogen-producing fatty acid degraders and hydrogen-utilizating homoacetogens was strengthened. Moreover, [2-¹⁴C] acetic acid degradation experiments showed that the activity of acetate oxidizing bacteria was considerablely high under the condition of low H₂ partial pressure.