Proteomic Analysis Of Denitrifying Sulfide Removal Of Pseudomonas Sp. C27

Denitrifying sulfide removal(DSR) in Liming river in Daqing in Heilongjiang Province were analysed in this study. The results showed, due to nitrogen and sulfur pollutants in river, the microbial community was alternated with water flow from upstream to downstream, and then sulfate reduction, autotrophic denitrifying and heterotrophic denitrifying reactions were orderly occurred. The results showed, in detected river section that was 3 km long, sulfate reduction and denitrifying sulfide removal processes together removed approximately 2100 tons of sulfate, 2300 tons of nitrate, and 3200 tons of chemical oxygen demand(COD) from the river water annually. However, the environment capacity and self-purification power of river was limited. The large amounts of nitrogen and sulfur pollutants discharge was bound to create water ecological damages, and then create serious environmental problem, such as eutrophication and hydrogen sulfide, etc. Therefore, the denitrifying sulfide removal for domestic sewage and industrial organic wastewater should be strengthened. To develop economic and efficient technology for denitrifying sulfide removal, in-depth study of DSR mechanism must be conducted to provide theoretical direction for developing practical technology.The in-depth study of DSR mechanism was conducted by using Pseudomonas sp. C27 and proteomics technology. The strain C27 was isolated from the DSR granules sludge of the expanded granular sludge bed(EGSB) reactor. The strain C27 is Gram-negative bacterium that can effectively conduct mixotrophic and heterotrophic denitrification reactions using organic matters and sulfide as electron donors to achieve simultaneous removal of sulfide, nitrate and organic carbon from waters, but cannot perform autotrophic denitrification without external carbon sources.The mechanisms of Pseudomonas sp. C27 to response to sulfide stress and micro-aerobic stress were analysed by using comparative proteomics(2D-PAGE combined with MALDI TOF/TOF MS/MS) and bioinformatics methods. 96 protein spots for C27 were reproducibly differentially expressed under sulfide stress. A total of 61 protein spots were successfully identified. These protein spots represented 61 unique proteins involved in carbon metabolism(21%), sulfur metabolism(19%), nitrogen metabolism(5%) and other functions(55%). 101 protein spots for C27 were reproducibly differentially expressed under micro-aerobic stress. A total of 55 protein spots were successfully identified. These protein spots represented 55 unique proteins involved in metabolism(66%), folding(18%), membrane transport(5%), translation(5%), cell motility(2%) and signal transduction(5%). By the functional analyses for these proteins, the study confirmed that the C27 strain has an effective enzyme system to conduct denitrifying sulfide removal reactions. In addition, sulfide stress enhanced energy consumption rate and rates of nitrate reduction and sulfide oxidation by C27. Conversely, sulfide stress repressed the sulfate-reducing power of C27. The micro-aeration stress elevated the rates of protein synthesis and cell growth, and enhanced cell defensive system of the strain C27.The identification and functional analyses of 116(61+55) proteins showed the molecular mechanisms of denitrifying sulfide removal process for the strain C27. On this basis, the information of total protein(1916 proteins) for C27 was revealed by using quantitative proteomics(i TRAQ combined with LC-ESI-MS/MS) and bioinformatics methods. The proteins corresponded to 21 COG functions, and involved in 16 biological processes and 10 molecular functions. Identified 26 proteins indicated the pathways for sulfur, nitrogen and carbon metabolism of the strain C27: the external acetate was converted into acetyl-Co A, and then was converted into phosphoenolpyruvate after through TCA cyclc. The synthetic phosphoenolpyruvate not only could be converted into pyruvate, but also could be converted into O-Acetyl-L-serine. The synthetic O-Acetyl-L-serine could be reacted with sulfide to produce acetate and elemental sulfur. The new pathway provided alternative growth strategy for C27 in C+N+S medium, giving this strain advantage in surviving in competing with other DSR strains. In addition, the presence of the new pathway was confirmed by DSR experiments at different C/N ratios. The mechanisms of Pseudomonas sp. C27 to response to insufficient carbon source was revealed. The related proteins of C27 could be regulated actively to cope with the disadvantages.