| Ammonia is one of the major pollutions of wastewater, caused water quality to deteriorate and massive death of aquatic life. Bio-treatment method is recognized most economic, effective and friendly to the environment. But traditional Bio-treatment method needs two processions, i.e. aerobic nitrification and anoxic denitrification, which makes this method more complex and expense, therefore it has become an issue for scientific research to screening of heterotrophic nitrification bacteria with the capacity of simultaneous nitrification/denitrification, and modifying the functional genes.A heterotrophic nitrifying bacteria was isolated which could remove ammonia efficiently. By means of morphologic observation, physiological characteristics study and 16S rRNA gene sequence analysis, the strain was identified as the species of Sinorhizobium. The result of mechanism products analysis indicated that little nitrite or nitrate was produced during the removing of ammonia, and ammonia can be removed both through assimilation and alienation.Physiological and biochemical characteristics of NP1 were studied here. (1) The ability for NP1 to use organic carbon sources was arranged in order as: sodium acetate > sodium succinate > sodium citrate > sucrose > lactose. (2) NP1 has high ammonia removal efficiency at low temperatures. At the temperature range of 20-30℃, ammonia removal efficiency has no significant difference. At the temperature of 37℃, ammonia removal efficiency declined about two thirds. (3) At the initial pH value of 5.0-8.0, NP1 showed good ammonia removal efficiency. (4) NP1 can degrade ammonia efficiently at the C/N range of 1-10. (5) In suitable conditions, nearly all the ammonium could be removed in two days by NP1.Gene fragments of ammonia monooxygenase, nitrite reductase and Periplasmic nitrate reductase was cloned. The result of sequence analysis showed that it has high sequence similarity to putative ammonia monooxygenase gene of Sinorhizobium sp., has no sequence similarity to the gene of denitrifying bacteria and has low sequence similarity to the heterotrophic nitrification bacteria which had been reported. It is indicated that there is greater differences between the amo of NP1 strain and other denitrifying bacteria. Transmembrane Helices Signal Analysis showed that there are multiple transmembrane regions in the NP1 putative AMO gene sequence, this consistent with the report that AMO is a transmembrane protein. The result of Conserved Domain predict showed that this sequence also contains a AmoA protein family conserved domain.We designed primers at the conservative regions to amplify gene fragments of denitrification associated enzymes, The amplified gene fragments of nitrite reductase and periplasmic nitrate reductase proved the existence of denitrification enzymes in strain NP1. |
