Isolation and identification of a photosynthetic bacterium strain and removal of ammonia nitrogen and nitrite by the bacterium species in water

Abstract:

[Background] China is a large aquaculture country. Ammonia nitrogen and nitrite are the main nitrogen source pollutants in water. Excessive ammonia nitrogen in water can damage the nervous system, liver and kidney system, body surface and internal organs filled with blood of aquatic animals. Excessive nitrite reduces the ability of carrying oxygen by red blood cells, and immune power of fish and shrimps, results in various diseases, and even suffocation. Some photosynthetic bacteria with the function of removal of ammonia nitrogen and nitrite in water are environmental friendly. [Objective] The photosynthetic bacterium strain (No. SP3) isolated from the mixed bacteria samples from aquaculture ponds in Guangdong province will be identified for its scientific species and removal ability of ammonia nitrogen and nitrite in water so as to provide the target bacterial strain for the biological removal of the water body. [Methods] The photosynthetic bacterium strain (SP3) was isolated from mixed bacteria samples from aquaculture ponds in Guangdong province using double-layer plate method. The strain SP3 was identified by 16S rRNA gene sequence analysis, gram staining, carbon source utilization and inorganic electron donor utilization experiments. The optimal culture conditions of strain SP3 were determined by detection of OD600 in different pH and various NaCl concentrations. The utilization of strain SP3 for different nitrogen sources (amonium chloride, sodium nitrite) could be confirmed by measuring the change trends of OD600 in different concentrations of the two nitrogen sources during the period of 7 days. The ability of strain SP3 to get rid of ammonia nitrogen and nitrite in water were determined by Nessler?s reagent colorimetric method and spectrophotometry. The nitrite reductase gene (nirS) was cloned by means of Genome Walking method, and the expression dynamics of nirS in the removal process of ammonia nitrogen and nitrite nitrogen were studied by real-time RT-PCR. [Results] The isolated strain SP3 is Gram-negative, short rod-shaped, and can utilize acetate, pyruvate, pyruvate, propionate, butyrate, lactate, fumarate, succinic acid, malate, fructose and glucose as carbon sources, but can not use ethanol and propanol as carbon sources. It can employ Na2S2O3·5H2O, Na2S·9H2O and Na2SO3 as inorganic electron donors. The strain SP3 is closely similar to Ectothiorhodospira shaposhnikovii (99% similarity) based on the 16S rRNA gene sequence analysis. The suitable pH is 6.0–8.5, and the suitable salinity is 0–3% for SP3. The growth status of strain SP3 using ammonia nitrogen chloride as a single nitrogen source was significantly better than that using sodium nitrite as a single nitrogen source. Under the condition of an initial bacterial concentration of 8.6×109 cfu/mL, the ammonia nitrogen at an initial concentration of 84.15±0.58 mg/L was removed in water for 7 days was 79.45±0.29 mg/L, and the removal percentage reached 94.42%, meanwhile, the sodium nitrite at an initial concentration of 2 mg/L degraded in water for 5 days was lower than the detection limit (0.003 mg/L). The relative mRNA expression level of nirS in the strain SP3 was upregulated during the period of removal of ammonia nitrogen and nitrite. [Conclusion] The strain SP3 was identified as E. shaposhnikovii, and had the strong ability of removing ammonia nitrogen and nitrite. Therefore, it is a promising photosynthetic bacterium strain in application of aquaculture water and wastewater treatment.