| Anaerobic digestion is a useful technology for energy recovery, which helps reduce the dependence on fossil fuels, mitigate the emission of greenhouse gases, and lower the risks of environmental pollution. In addition, the digestion residues, normally containing various nutrients, can be used as agricultural fertilizers. Different factors influence the performance of anaerobic digesters and several of them can cause the reactor upset or failure. Accumulation of ammonia, produced from the degradation of protein-rich materials, is one such detrimental factor in the anaerobic digesters used to treat waste such as livestock and poultry manure. However, the inhibitory effects of ammonium on individual methanogens and the microbiology mechanism of the syntrophic propionate and butyrate oxidation remain poorly understood.To investigate the inhibitory effects of ammonium on methanogens in anaerobic digester sludge, we collected a sludge sample from a full-scale swine manure digester and conducted a laboratory microcosm experiment. The responses of methanogenesis and methanogenic populations to ammonium addition were determined by analyzing the rate of CH4production, the13C isotope fractionation of CH4and CO2, and the copy number and transcripts of methyl-coenzyme M reductase encoding genes (mcrA) in the sludge incubations. The rate of CH4production substantially reduced with increased addition of ammonium. The analysis of natural13C abundances of CH4and CO2indicated that the aceticlastic methanogenesis Methanosaetaceae was more sensitive than hydrogenotrophic methanogenesis. Quantitative PCR analysis revealed that mcrA copy number decreased by one order of magnitude in the treatment with a large amount of ammonium (10g NH4+-N L-1). T-RFLP analysis of mcrA compositions showed that the structure of the methanogen community remained highly stable, with Methanosaetaceae dominating the methanogen community in all incubations. The composition of mcrA transcripts, however, showed a substantial response to the addition of ammonium.To investigate the effects of ammonium on microbes involved in syntrophic oxidation of propionate in anaerobic digester sludge, we used the same samples and culture conditions with the last experiment and conducted a laboratory microcosm experiment. During the propionate stable isotope probing (SIP) experiment, the [U-13C] propionate was added in anaerobic sludge with the treatments of different ammonium concentration (0,3and7g NH4+-N L-1). RNA-SIP, T-RFLP and clone library analysis of archaeal and bacterial16S rRNA compositions showed that the bacteria Smithella and archaea Methanosaetaceae and Methanospirillaceae participated in the syntrophic propionate oxidizing methanogenesis. There was a community succession between hydrogenotrophic methanogenes Methanospirillaceae and Methanomicrobiales under higher ammonium concentration. In ammonium stress experiment, the sludge were anaerobically incubated under different ammonium treatments (0、3、7and10g NH4+-N L-1) for3month. The responses of syntrophic propionate oxidizing methanogenesis and microbial populations to ammonium addition were determined by analyzing the rate of propionate degradation, the copy number and transcripts of bacteria16S rRNA gene and mcrA in the sludge incubations. The rate of propionate degradation and CH4production substantially reduced with increased addition of ammonium. The syntrophic propionate oxidizing bacteria (Smithella) and the methanogenes (Methanosaetaceae and Methanospirillaceae) were sensitive to ammonium. The suppression of ammonium on hydrogenotrophic methanogen Methanospirillaceae might be the direct cause of inhibition in syntrophic propionate oxidation.To investigate the effects of ammonium on microbes involved in syntrophic oxidation of butyrate in anaerobic digester sludge, we used the same samples and culture conditions with the last experiment and conducted a laboratory microcosm experiment. During the butyrate SIP experiment,[U-13C] butyrate was used in anaerobic sludge with the treatments of different ammonium concentration (0,3and7g NH4+-N L-1). RNA-SIP, T-RFLP and clone library analysis of archaeal and bacterial16S rRNA compositions showed that the bacteria Syntrophomonaceae and archaea Methanosaetaceae and Methanospirillaceae participated in the syntrophic butyrate oxidizing methanogenesis. In ammonium stress experiment, the sludge were anaerobically incubated under different ammonium treatments (0、3、7and10g NH4+-N L-1) for3month. The responses of syntrophic butyrate oxidizing methanogenesis and microbial populations to ammonium addition were determined by analyzing the rate of butyrate degradation, the copy number and transcripts of bacteria16S rRNA gene and methyl-coenzyme M reductase encoding genes (mcrA) in the sludge incubations. The rate of butyrate degradation and CH4production substantially reduced with increased addition of ammonium. The syntrophic butyrate oxidizing bacteria Syntrophomonaceae and the methanogenes Methanosaetaceae and Methanospirillaceae were inhibited with increased addition of ammonium. Methanogens were more sensitive to ammonium than syntrophic butyrate oxidizing bacteria. The sensitivity of aceticlastic methanogenes Methanosaetaceae to ammonium might play a negative role on the butyrate oxidation. The suppression of ammonium on hydrogenotrophic methanogen Methanospirillaceae might be the direct cause of inhibition in syntrophic butyrate oxidation.In conclusion, high ammonium concentration was demonstrated to have comprehensive inhibitive effects on syntrophic bacteria and methanogenic archaea, which involved in syntrophic oxidation of propionate and butyrate in anaerobic digester sludge. The suppression of ammonium on hydrogenotrophic methanogen Methanospirillaceae might be the direct cause of inhibition in syntrophic oxidation of propionate and butyrate. |
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