Screening of Low-temperature Heterotrophic Nitrifying Bacteria and Their Physiological and Biochemical Characteristics

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

Abstract

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

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.

Figures/Tables 16

References 36

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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

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

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

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

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