耐低温好氧反硝化菌Aeromonas sp.的分离鉴定及脱氮条件优化

Isolation and identification of a cold-tolerant and aerobic denitrifying bacterium Aeromonas sp. and optimization of denitrification conditions

摘要:

【目的】从冬季活性污泥和河水底泥中分离筛选得到耐低温好氧反硝化菌，并对影响脱氮的关键因子进行优化，以提高低温条件下的脱氮效果。【方法】采用富集纯化法分离筛选耐低温好氧反硝化菌株。通过形态观察和16S rRNA基因系统发育分析等方法对筛选菌株进行菌种鉴定。以NO₃^–-N去除率为响应目标，采用Box-Behnken试验设计及响应面回归分析法优化影响脱氮效果的关键因子(C/N、温度、pH和摇床转速)，确定最佳培养条件。【结果】从东北寒冷地区冬季河水底泥样品中分离得到的一株耐低温好氧反硝化菌Z6，菌落呈白色半透明圆形，菌体细胞为短杆状，大小为(0.8–1.6)μm×(0.6–0.8)μm，革兰氏染色阴性。与气单胞菌属(Aeromonas)的16S rRNA基因序列高度同源，鉴定该菌为气单胞菌。采用响应面分析方法得到菌株Aeromonas sp.Z6的最佳脱氮条件为：C/N 5.9，温度12℃，pH 6.8，摇床转速155 r/min，在此条件下对NO₃^–-N的去除率为89.72%，与预测值(90.34%)无显著差别。【结论】首次报道气单胞菌(Aeromonassp.)在低温条件下具有高效反硝化脱氮性能，为寒冷地区低温废水的生物脱氮提供思路、借鉴和菌种资源。

Abstract:

[Objective] To isolate and screen out the cold-tolerant and aerobic denitrifying bacteria from activated sludge and river sediment in winter, and optimize the key factors that influence denitrification, so as to improve the effect of denitrification at low temperature. [Methods] Cold-tolerant and aerobic denitrifying bacteria were isolated and screened out by enrichment and purification method. Morphological observation and 16S rRNA gene sequence phylogenetic analyses were used to identify the species of the newly isolated strain. Taking the removal rate of NO₃^–-N as the response target, the key factors affecting denitrification, including carbon-nitrogen ratio, temperature, pH and shaking speed, were optimized by Box-Behnken design and response surface regression analysis to determine the optimal culture conditions. [Results] A cold-tolerant and aerobic denitrifying bacterium Z6 was obtained from the river sediment in the cold area of northeast China in winter. The round colony of the strain was white and translucent, and the cell was short rod-shaped, with the size of (0.8–1.6)μm×(0.6–0.8) μm. Gram staining was negative. The strain Z6 was identified as Aeromonas sp. due to the high homology with the 16S rRNA gene sequences of the genus Aeromonas sp.. Based on the response surface analysis, the optimal denitrification conditions of Aeromonas sp. Z6 were 5.9 of carbon-nitrogen ratio at pH 6.8 and 12 ℃ with 155 r/min shaking table speed. Under the optimum conditions, the removal rate of NO₃^–-N was 89.72%, which was not significantly different from the predicted rate (90.34%). [Conclusion] Aeromonas sp. had high denitrification performance under low-temperature conditions, which provided references for biological denitrification of low-temperature wastewater in cold areas.