低温异养硝化菌的筛选、鉴定及降解特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1315

摘要/Abstract

摘要：

从冬季北京化工大学污水处理站活性污泥、江苏省某污水处理厂生物转盘污泥和实验室的好氧颗粒污泥中筛选出3株较好的低温异养硝化的菌株制成混合菌剂,解决低温条件下异养硝化菌硝化能力差的问题。应用传统分离纯化培养技术筛选菌株,纳氏试剂分光光度法评价菌株的异养硝化能力,通过形态观察、生理生化特性和16S rDNA测序对菌株进行种属鉴定,设计正交试验和单因素试验,确定菌剂复配比并进行固定化,探究低温异养硝化的最优条件。在13℃下共筛选出3株脱氮效果好的菌株,经鉴定均属于不动杆菌（Acinetobacter sp.）,复合菌剂最佳复配比为1:1:1,进行固定化后在碳源为柠檬酸钠、pH为8、NH₄⁺-N浓度为50 mg/L、NaCl浓度为10 g/L-20 g/L时,其氨氮去除效率可达95.86%,对高氨氮,高盐度废水具有较高耐受性,在低温废水处理方面具有潜在的应用价值。

关键词: 低温异养硝化, 分离鉴定, 硝化

Abstract:

Three strains capable of low temperature heterotrophic nitrification were selected from the activated sludge of the sewage treatment station of Beijing University of Chemical Technology in winter,the sludge of rotating biological contactor of a sewage treatment plant in Jiangsu province and aerobic granular sludge in a laboratory to solve the problem of poor nitrification ability of heterotrophic nitrifying bacteria at low temperature. Applying traditional separation and purification culture technology to screen strains,and Nessler’s reagent spectrophotometric method to evaluate the heterotrophic nitrification ability,the strains were identified by morphological observation,physiological and biochemical characteristics and 16S rDNA sequencing. Orthogonal test and single factor test were designed to determine the compound ratio of bacteria and immobilization to explore the optimal conditions of heterotrophic nitrification at low temperature. Three strains with good nitrogen removal efficiency were screened at 13℃. All of them were identified as Acinetobacter sp,the optimal ratio of inoculating amount of three strains was 1:1:1. After immobilization,when the carbon source was sodium citrate,the concentration of pH was 8,the concentration of NH₄⁺-N was 50 mg/L,and the concentration of NaCl was 10-20 g/L,the removal efficiency of ammonia nitrogen reached 95.86%. The bacterial agent presents high tolerance to high ammonia nitrogen and high salinity wastewater,and thus has a broad application prospect in the field of low temperature wastewater treatment.

Key words: low temperature heterotrophic nitrification, separation and identification, nitrification

李珍阳, 姜润, 刘琳, 李思琦, 王晓慧. 低温异养硝化菌的筛选、鉴定及降解特性研究[J]. 生物技术通报, 2021, 37(10): 45-56.

LI Zhen-yang, JIANG Run, LIU Lin, LI Si-qi, WANG Xiao-hui. Screening of Low-temperature Heterotrophic Nitrifying Bacteria and Their Physiological and Biochemical Characteristics[J]. Biotechnology Bulletin, 2021, 37(10): 45-56.

图/表 16

表1 培养基成分表

Table 1 Medium composition

培养基 Medium	成分Composition
活化培养基Activation medium	胰蛋白胨Petpone 5 g·L^-1,酵母浸粉Yeast extract powder 5 g·L^-1,NaCl 5 g·L^-1,蒸馏水Distilled water 1 000 mL
富集培养基Enrichment medium	NH₄Cl 0.5 g·L^-1,CH₃COONa 3.5 g·L^-1,MgSO₄·7H₂O 0.05 g·L^-1,K₂HPO₄·3H₂O 0.2 g·L^-1,NaCl 0.12 g·L^-1,MnSO₄·H₂O 0.01 g·L^-1,FeSO₄·7H₂O 0.01 g·L^-1,蒸馏水Distilled water 1000 mL
异养硝化液体培养基 Heterotrophic nitrification liquid medium	NH₄Cl 0.382 g·L^-1,CH₃COONa 2.0 g·L^-1,MgSO₄·7H₂O 0.05 g·L^-1,K₂HPO₄·3H₂O 0.2 g·L^-1,NaCl 0.12 g·L^-1,MnSO₄·H₂O 0.01 g·L^-1,FeSO₄·7H₂O 0.01 g·L^-1,蒸馏水 Distilled water 1000 mL
异养硝化液体培养基 Heterotrophic nitrification solid medium	NH₄Cl 0.382 g·L^-1,CH₃COONa 2.0 g·L^-1,MgSO₄·7H₂O 0.05 g·L^-1,K₂HPO₄·3H₂O 0.2 g·L^-1,NaCl 0.12 g·L^-1,MnSO₄·H₂O 0.01 g·L^-1,FeSO₄·7H₂O 0.01 g·L^-1,蒸馏水 Distilled water 1 000 mL,琼脂Agar powder 0.12 g·L^-1

表2 PCR反应体系

Table 2 PCR reaction system

反应物Reactant	体积Volume/μL
10×Ex Taq buffer	5.0
2.5 mmol/L dNTP Mix	4.0
10p Primer 1	2.0
10p Primer 2	2.0
5U Ex Taq	0.5
Template	2.0
ddH₂O	36.5
Total volume	50

图1 菌株对NH4+-N的去除率

Fig. 1 Removal rate of NH4+-N by various strains

图2 各菌株菌落形态

Fig.2 Colony morphology of each strain A:AJ-5;B:BJ-4;C:CJ-1

图3 革兰氏染色呈像

Fig.3 Gram chromatogram of each strain A:AJ-5;B:BJ-4;C:CJ-1

图4 菌株AJ-5的系统发育树

Fig.4 Phylogenetic tree of the bacterial strain AJ-5

图5 菌株BJ-4的系统发育树

Fig.5 Phylogenetic tree of the bacterial strain BJ-4

图6 菌株CJ-1的系统发育树

Fig.6 Phylogenetic tree of the bacterial strain CJ-1

图7 菌株生长曲线图

Fig.7 Growth curve of strain

表3 正交实验设计及结果

Table 3 Design and results of orthogonal experiment

水平 Level	因素Factors
水平 Level	A AJ-5/mL	B BJ-4/mL	C CJ-1/mL	氨氮去除率Removal rate of ammonia nitrogen/%
1	1	1	1	72.94
2	1	2	3	69.19
3	1	3	2	66.26
4	2	1	3	66.36
5	2	1	3	68.52
6	2	2	2	66.23
7	3	2	1	68.93
8	3	2	1	65.66
9	3	3	3	62.40
K1	69.46	68.32	70.13
K2	67.04	68.12	66.05
K3	65.66	65.73	65.98
R	3.80	2.59	4.15

图8 菌剂在不同碳源下OD600浓度变化（A）及NH4+-N去除率变化（B）

Fig.8 Change of NH4+-N removal rate (A) and OD600 con-centration (B) of microbial inoculum under different carbon sources

图9 菌剂在不同氮源浓度下OD600浓度变化（A）及NH4+-N去除率变化（B）

Fig.9 Change of NH4+-N removal rate (A) and OD600 con-centration (B) of microbial inoculum under different nitrogen source concentration

图10 菌剂在不同pH下OD600浓度变化（A）以及NH4+-N去除率变化（B）

Fig.10 Change of NH4+-N removal rate (A) and OD600 concentration (B) of microbial inoculum under different pH

图11 菌剂在不同盐度下OD600浓度变化（A）及NH4+-N去除率变化（B）

Fig.11 Change of NH4+-N removal rate (A) and OD600 concentration (B) of microbial inoculum under different salinities

图12 固定化菌剂氨氮的去除效率

Fig.12 Removal efficiency of ammonia nitrogen immobili-zed bacteria agent

图13 异养硝化菌的脱氮途径

Fig.13 Nitrogen removal ways of heterotrophic nitrifying bacteria

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