Solation and degradation effect of two ammonifying bacteria
： 2018 - 11 - 10
： 2019 - 04 - 09
： 2019 - 04 - 17
174 9 0

Abstract & Keywords
Keywords: organic nitrogen; ammonifying bacteria; isolation and identification; degradation effect
Abstract:Background, aim, and scope In recent years, with the rapid development of economy, a large number of nutrients such as nitrogen, phosphorus and other organic substances enter the water, causing eutrophication of the water, and water resources problems are serious. Nitrogen removal in eutrophic water has always been a research hotspot. Up to now, there have been a lot of studies on the removal of inorganic nitrogen such as ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water, among which the research on the ammoniation process of organic nitrogen is relatively less. Materials and methods In this experiment, samples were collected from the bottom sediments of Taihu Lake. After repeated screening, two strains of ammoniated bacteria were isolated, and named AB-1 and AB-2 respectively. The strains were identified by the morphological features observation, physiological and biochemical characteristics analysis, 16S rDNA molecular identification and homology analysis. In addition, the growth characteristics and degradation effects of two strains of ammoniated bacteria were compared. (1) The OD value was measured at 600 nm wavelength every 2 hours after incubation in constant temperature and dark oscillation at 28℃ for 120 r/min, and the growth characteristics were determined. (2) Inoculate 1% of the inoculated amount into organic nitrogen, incubate continuously for 70 hours under the same conditions, and measure the changes of $$\mathrm{\rho }（\mathrm{N}{\mathrm{H}}_{4}^{+}-\mathrm{N}）$$and$$\mathrm{ }\mathrm{\rho }（\mathrm{T}\mathrm{N}）$$to compare the degradation effects of the two strains. Peptone was used as the sole organic nitrogen source in the experiment, so the content of organic nitrogen could be expressed by the difference between TN and$$\mathrm{ }\mathrm{N}{\mathrm{H}}_{4}^{+}-\mathrm{N}$$ in the initial culture medium, and the degradation rate of organic nitrogen could be expressed by the increase of $$\mathrm{N}{\mathrm{H}}_{4}^{+}-\mathrm{N}$$. Results Through morphological analysis and 16S rDNA molecular identification, strains AB-1 and AB-2 were identified as Pseudomonas migulae.and Arthrobacter sp., respectively. Under the same culture conditions, the growth potential of strain AB-2 was greater than that of strain AB-1, and the maximum OD600 was 1.063and 0.851, respectively. Besides, two strains of ammoniating bacteria had strong ammoniation ability. Among them, the removal efficiency of organic nitrogen by strain AB-2 was higher than that of ammoniating bacteria AB-1, the removal rate was 83.15%, and that of strain AB-1 was only 67.33%. Discussion Compared with other studies on the degradation of organic nitrogen by ammoniating bacteria, it was found that the degradation rate of organic nitrogen by the two strains was slightly higher. This may be because the ammoniating ability of the two strains was improved by multiple screening in the process of selecting strains. Furthermore, the initial concentration of organic nitrogen in this study was lower, which indicated that the strains screened were indeed effective in degrading organic nitrogen. In addition, by monitoring the growth of two strains of ammoniating bacteria and the change of $$\mathrm{N}{\mathrm{H}}_{4}^{+}-\mathrm{N}\mathrm{ }\mathrm{ }$$content, it was found that strain AB-2 could degrade organic nitrogen for a long time, which indicated that the strain could be used as a candidate strain for high-efficiency degradation of organic nitrogen, and it was feasible to apply it to lake eutrophication treatment to improve water quality and environment. However, the conditions, mechanism, enzymatic activity and application methods of ammonia nitrogen degradation need to be further explored and studied. Conclusions In the study, two strains of ammonifying bacteria AB-1 and AB-2 were isolated from sediment of Taihu Lake. Both of them had strong ammoniating ability. Among them, strain AB-2 had higher removal efficiency of organic nitrogen than strain AB-1, with removal rate of 83.15% and strain AB-1 67.33%. Compared with other studies, the initial concentration of organic nitrogen in this study is lower, which indicates that the strains selected are indeed effective in degrading organic nitrogen. Recommendations and perspectives The removal rate of organic nitrogen of AB-2 strain isolated in this study can reach more than 83%, which provides a scientific basis for solving the problem of nitrogen pollution in Taihu Lake and improving the water quality and environment of Taihu Lake. At the same time, it has certain application value to lake eutrophication management.

1   材料与方法
1.1   样品来源

1.2   试剂与检测方法

DON培养基：蛋白胨0.1 g ∙ L−1，K2HPO4·3H2O 0.4 g ∙ L−1，MgSO4·7H2O 0.05 g ∙ L−1，NaCl 0.5 g ∙ L−1，FeSO4·7H2O 0.005 g ∙ L−1，CaCl2·H2O 0.01 g ∙ L−1，蒸馏水1 L，pH7.5。培养基于121℃下灭菌30 min。

1.3   主要仪器与设备
1.4   菌株的分离纯化

1.5   菌株鉴定
1.5.1   形态及生理生化鉴定

 加入试剂 Additional Reagents 操作 Operation 2.5%戊二醛溶液 固定2—4 h 1%PBS缓冲液（pH7.4） 清洗3次 1%锇酸溶液 4—5 h 1%PBS缓冲液（pH7.4） 清洗3次 乙醇梯度脱水（30%、50%、70%、85%、95%各一次，100%乙醇2次） 20—30 min/次 乙酸异戊酯置换2次 20 min/次
1.5.2   16S rDNA分子鉴定

 步骤 Procedure 循环数 Cycle Number 温度（℃） Temperature 时间 Time 预变性 32 94 5 min 变性 94 45 s 退火 46 45 s 延伸 72 2 min 延伸 1 72 5 min
1.6   菌株的生长特征测定

1.7   菌株降解能力研究
1.7.1   菌株对不同营养物质的吸收能力

1.7.2   菌株对有机氮的降解实验

2   结果与分析
2.1   菌株的分离纯化

2.2   菌株鉴定
2.2.1   形态及生理生化鉴定
（1）菌株形态特征

（2）生理生化特征

Fig.1 SEM photos of strain AB-1

Fig.2 SEM photoes of strain AB-2
2.2.2   16S rDNA分子鉴定

Fig.3 Phylogenetic tree of strain AB-1

Fig.4 Phylogenetic tree of strain AB-2
2.3   菌株生长曲线测定

Fig.5 Growth curves of two ammonifying bacteria
2.4   菌株降解有机氮实验结果
2.4.1   菌株对不同营养物质的吸收能力

Fig.6 The utilization of different substances by two ammonifying bacteria
2.4.2   菌株对有机氮的降解能力比较

Fig.7 Degradation of organic nitrogen by strain AB-1

Fig.8 Degradation of organic nitrogen by strain AB-8
3   讨论

4   结论

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WANG Hong

XU Jie

RUAN Aidong

XU YAOfei

WEI Qi

LI Siyan

National Natural Science Foundation of China (51378175)

Journal of Earth Environment