Methanogenesis And Nitrogen Removal In The Biological Stabilization Ponds For Large Scale Swine Wastewater Treatment

Biological stabilization ponds (BSPs) are widely used for large scale swine wastewater treatments due to their simple operation, effective cost and low maintenance requirements. However, with high input of chemical organic demand (COD) and ammonia, problems of large methane emissions and low nitrogen removal rates were presented in BSPs. So far, the research on methane production and nitrogen removal process is still lacking. Only few researches were focused on the methane fluxes and nitrogen removal rates. Studies on microbial mechanisms and their influencing factors have not been reported. Therefore, the research on microbial mechanism related to methane production and nitrogen removal in BSPs is urgently needed, in order to provide a theoretical basis for predicting methane emissions from BSPs and optimizing the application of BSPs in swine wastewater treatment. In this study, water and sediment samples were collected from BSPs in seven large scale pig farms located in different latitudes of China. Combined with analysis including physiochemical analyses, quantitive PCR and Miseq high-throughput sequencing techniques were used to detect the changes of microbial functional gene copy numbers and community structure related to methanogenesis and nitrogen removal process in water and sediments samples from BSPs in different latitudes and BSPs of different stages in the same location. As a result, the main factor that influenced methane production process of BSPs was elucidated. Besides, the mechanisms about the influence of sulfate reduction on nitrogen removal efficiency in BSPs was proposed. The main conclusions are summarized as follows:(1) Results showed that methanogen abundance and methanogenic activities were considerably high for all the sediments of first stage BSPs in different latitudes, indicating that BSPs could be a strong potential methane emission source. According to the measured methanogenic and methane oxidation activity in the sediments and the statistical number of BSPs in China,1.78-2.68 Tg estimated methane will be emitted from sediments of BSPs in China annually, which accounts for 0.56-0.84% of global anthropogenic methane emissions. Besides, according to the analysis on physiochemical characteristics and methanogenic microbial activity and abundance of sediments from four stage BSPs in Zhejiang province, the influent COD loading rates was verified to have the most significant impact on methane production process of BSPs. From the perspective of methane emission control, BSPs for swine wastewater treatment are not suitable for application in treating anaerobic digestion effluent but more likely to be utilized as the tailwater treatment process. Therefore, the influent organic loading rates should be decreased through optimizing the design of BSPs and solid-liquid separation process should be strengthened before BSPs.(2) Mean annual temperature (MAT) was considered to be the most important factor impacting on the methanogenic process of the first stage BSPs in different latitudes of China. Methanogenic microbial activity and abundance both showed strong increasing trends with the increasing MAT. For the sediments of BSPs in HM area, archaea was dominated by hydrogenotrophic methanogens (WCHD3-30), while archaea in the sediments of BSPs in LM area was dominated by acetoclastic methanogens (Methanosaetaceae). With the increasing MAT, the changes of bacteria community structure in BSPs matched the changing pattern of archaea. Bacteria in the sediments of BSPs in HM area was dominated by acetate oxide bacteria with hydrogen production function while for the sediments of BSPs in LM area, most bacteria was directly related to macromolecular organic matter degradation and acetic acid production processes. Besides, RDA analysis about microbial community structure and environmental factors showed that MAT was the most significant influencing factor on archaea community structure in the sediments of the first stage BSPs. This study elucidates the microbial mechanism of MAT impacting on the methanogenic process in BSPs and thus provides theoretical basis for the change of methane emissions from BSPs under future global warming condition.(3) The study on nitrogen concentrations of the influent and effluent in the first stage BSPs from different swine wastewater treatment showed that ammonia removal rate of the first stage BSPs was relatively low, with the highest removal rate less than 20%. Quantitative PCR was used to analyze the abundance of functional genes related to nitrogen cycle (nifH, AOA amoA, AOB amoA, nirS and nirK). Results suggested that a large number of nitrogen fixing microbes, while only few ammonia oxidizing microorganisms were found in the water and sediment samples of the first BSPs. Besides, it was worth noting that the sulfate concentration in first stage BSPs was significantly positively correlated with nifH abundance while significantly negatively correlated with AOB amoA abundance. Therefore, low ammonia removal rate of the first BSPs might possibly be attributed to a strong ammonia production but weak ammonia oxidation process.(4) According to the analysis of physiochemical characteristics, the abundance of functional genes of nitrogen and sulfur cycle in water and sediment samples in four stage BSPs of Zhejiang Province and the experiment on the addition of sulfate reduction inhibitor, sulfate reduction process has been proved as a key factor that leads to low ammonia removal rate in the first stage BSPs. With the increasing BSPs series, sulfate concentration, sulfate reduction functional genes (dsrB) abundance in sediment and water samples and sulfide concentration in water samples decreased, indicating that sulfate reduction process was weaken, while ammonia nitrogen removal rate showed an increasing tendency. RDA and Pearson correlation analysis showed that sulfide concentration in the water samples of BSPs was the most important factor influencing the overall changes of functional genes abundance related to nitrogen and sulfur cycle and sulfide concentration was also significantly negatively correlated with the abundance of major functional genes related to nitrogen removal process (AOB amoA abundance and nirS abundance in the water). Therefore, high sulfide concentrations would significantly inhibit nitrogen removal process in the BSPs. Besides, nifH and dsrB gene abundances showed a strong correlation and similarity. The presence of sulfate reduction may promote the nitrogen fixation and then increase the concentrations of ammonia nitrogen in the water and sediment samples. The incubation experiment of inhibitor addition verified that ammonia nitrogen and total nitrogen removal rates were increased by 5.1-9.5% and 3.0-6.0% when sulfate reduction in ZJ1 had been suppressed, which suggested that 20.9-32.8% of ammonia nitrogen and 12.0-25.5% of total nitrogen removal would be inhibited by sulfate reduction in ZJ1. Given that sulfate reduction process significantly inhibits nitrogen removal efficiency of BSPs, BSPs are not suitable for nitrogen removal application in the treatment of anaerobic fermentation effluent with high sulfide concentration and aquaculture wastewater, food wastewater and pharmaceutical wastewater containing high concentration of sulfate.