一株光合细菌的分离鉴定及该菌对氨氮和亚硝态氮的去除作用

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

摘要:

【背景】中国是水产养殖大国，氨氮、亚硝态氮是水体中主要的氮源污染物。水体氨氮超标不仅会损伤水生动物的神经系统和肝肾系统，还会导致体表及内脏充血。亚硝态氮过高会阻碍血液运载氧气能力，导致鱼虾缺氧、免疫力下降，从而引发肠炎、烂鳃，甚至窒息死亡。部分光合细菌有去除水体氨氮、亚硝态氮的能力，且对环境友好无二次污染。【目的】从广东养殖水体分离、纯化、筛选出生物活性好的光合细菌(编号SP3)进行种属鉴定，优化培养条件，检测其去除水体氨氮和亚硝态氮的能力，为养殖水体去除氨氮和亚硝态氮提供目标菌株。【方法】用双层平板法从混合菌液中分离得到光合细菌，通过革兰氏染色、碳源利用试验、对无机电子供体的利用试验以及16S rRNA基因序列分析对目标菌株进行种属鉴定；测定菌株SP3在不同pH、不同浓度NaCl条件下的OD600，优化培养条件；通过测定7 d内SP3菌株在不同浓度氨氮(氯化铵配制)和亚硝态氮(亚硝酸钠配制)中OD600的变化趋势，确定该菌株对不同氮源的利用情况；用纳氏比色法、分光光度法测定SP3菌株降解水体氨氮和亚硝态氮的能力。Genome walking扩增获得亚硝酸盐还原酶基因(nirS)，通过荧光定量PCR研究nirS在氨氮、亚硝态氮去除过程中的表达动态。【结果】筛选出的菌株SP3为革兰氏阴性菌，短杆状；能以醋酸盐、丙酮酸盐、丙酸盐、丁酸盐、乳酸盐、富马酸盐、琥珀酸盐、苹果酸、果糖、葡萄糖作为碳源，不能以乙醇和丙酮作为碳源；能利用硫化钠、硫代硫酸钠、亚硫酸钠作为无机电子供体；16S rRNA基因序列分析表明其与沙氏外硫红螺菌(Ectothiorhodospira shaposhnikovii)序列相似度为99%；菌株SP3适宜pH为6.0?8.5，适宜盐度为0?3%；菌株SP3以铵盐作为氮源时生长状态明显优于亚硝酸盐；以初始菌液浓度8.6×109 cfu/mL、起始氨氮量84.15±0.58 mg/L的条件培养7 d，水体氨氮累计去除量为79.45±0.29 mg/L，氨氮累计去除率达到94.42%；在同样菌浓度和2 mg/L亚硝酸钠的条件下培养5 d，水体亚硝态氮含量低于检测限0.003 mg/L。在菌株SP3去除氨氮、亚硝态氮过程中nirS相对表达量上调。【结论】菌株SP3为沙氏外硫红螺菌(E. shaposhnikovii)，能有效去除水中氨氮和亚硝态氮，具有净化水质作用，在水产养殖和污水处理中有广阔应用前景。

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.