一株金霉素降解新菌株的分离鉴定及降解条件优化

赵慈; 张茹; 李思琦; 李文进; 宋哲华; 王晓慧; 沈鹏

doi:10.12153/j.issn.1674-991X.20210430

一株金霉素降解新菌株的分离鉴定及降解条件优化

doi: 10.12153/j.issn.1674-991X.20210430

赵慈^1,,
张茹²,
李思琦²,
李文进³,
宋哲华³,
王晓慧²,
沈鹏^1, ,

1.
中国环境科学研究院
2.
北京化工大学水处理环保材料工程技术研究中心
3.
北京京环新能环境科技有限公司

基金项目: 国家重点研发计划项目（2018YFC1801501）

详细信息

作者简介:
赵慈(1983—)，女，工程师，硕士，研究方向为环境规划、评价与管理，zhaoci07@163.com

通讯作者:
沈鹏(1977—)，男，工程师，主要从事区域循环经济、生态工业和清洁生产的理论与方法研究，shenpeng@craes.org.cn

中图分类号: X172
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- PDF下载量: 11
- 被引次数: 0
出版历程
- 收稿日期: 2021-08-18
- 网络出版日期: 2022-11-25

Isolation, identification and degradation conditions optimization of a new bacterial strain degrading chlortetracycline

1.
Chinese Research Academy of Environmental Sciences
2.
Beijing Water Treatment Environmental Protection Material Engineering Technology Research Center, Beijing University of Chemical Technology
3.
Beijing Jinghuan New Energy Environment Technology Co., Ltd.

摘要

摘要:
四环素类抗生素在畜牧业中的广泛应用对人类和动物具有潜在的危害。以金霉素制药厂污泥为原料，从中分离出一株能够高效降解金霉素的纯菌株，命名为ZL-1。经形态学观察、革兰氏染色和16S rDNA鉴定，表明该菌株属于革兰氏阴性菌、不动杆菌属（Acinetobacter sp.）。通过正交试验研究了碳源、温度、pH、初始金霉素浓度、接种量对菌株ZL-1降解金霉素效果的影响。结果表明，温度、接种量和初始金霉素浓度对该菌株降解金霉素的影响较大。以正交试验的结果为依据，采用响应面法优化该菌株对金霉素的降解条件，确定了最优条件为金霉素初始浓度134.864 mg/L，温度34.409 ℃，接种量5.223%（体积比）。在最佳降解条件下，金霉素的实际降解率为93.70%，预测降解率为93.723%，表明预测模型的预测值与实际的降解效果较贴合。
- 金霉素 /
- 生物降解 /
- 降解特性 /
- 正交试验 /
- 响应面优化
Abstract:
The extensive application of tetracycline antibiotics in stock farming has brought potential hazards to people and animals. A well-functioning chlortetracycline-degrading pure bacteria strain was isolated from the sludge of the chlortetracycline pharmaceutical factory. The strain was named ZL-1 and identified to belong to gram-negative bacteria and Acinetobacter sp. based on the morphological observation, gram staining and 16S rDNA sequencing identification. Furthermore, the effects of carbon source, temperature, pH, initial chlortetracycline concentration and inoculation amount on the chlortetracycline degradation performance of ZL-1 were analyzed by the orthogonal test. The results showed that the temperature, inoculation amount, and initial chlortetracycline concentration had a greater impact on the chlortetracycline degradation efficiency. Based on the results of the orthogonal test, the chlortetracycline degradation conditions of the bacteria were optimized by the response surface method, and the optimal conditions for degrading chlortetracycline were determined. The optimal conditions were the initial chlortetracycline concentration 134.864 mg/L, the temperature 34.409 ℃, and the inoculation concentration 5.223%. Under the optimal conditions, the actual degradation efficiency of chlortetracycline was 93.70%, whereas the predictive value was 93.723%, indicating that the proposed model had high accuracy.
- chlortetracycline /
- biodegradation /
- degradation characteristics /
- orthogonal test /
- response surface optimization

HTML全文

图 1 初筛降解菌株对金霉素的降解率

Figure 1. Degradation efficiency map of primary screened strains

下载: 全尺寸图片幻灯片

图 2 菌株ZL-1形态

Figure 2. Morphological map of strain ZL-1

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图 3 菌株ZL-1系统发育树

Figure 3. Phylogenetic tree of strain ZL-1

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图 4 各因素及其交互作用的响应面

Figure 4. Factors and their interaction in response surface chart

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表 1 试验仪器

Table 1. Experimental apparatus

试验仪器	型号	生产厂家
立式压力蒸汽灭菌锅	YM5A	上海三申医疗器械有限公司
恒温培养摇床	ZWY-2102C	上海智城分析仪器制造有限公司
生化培养箱	LRH-150F	上海一恒科学仪器有限公司
净化工作台	SW-CJ-2FD	苏州净化设备有限公司
冷冻冰箱	BC-93TMPF	青岛海尔股份有限公司
高速台式冷冻离心机	TGL-16	湘仪离心机仪器有限公司
冷冻干燥机	BK-FD10T	山东博科科学仪器有限公司
SEM场发射扫描电镜	G300	德国Zeiss有限公司
扫描电镜溅射镀膜仪	SC7620	英国Quorum有限公司
显微镜	N-400M	宁波永新光学股份有限公司
PCR	MiniAmp™ Plus	Thermo Fisher Scientific有限公司
紫外可见分光光度计	UV 756CRT	上海佑科仪表有限公司
液相色谱仪	1200	Agilent Technologies有限公司

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表 2 正交试验设计

Table 2. Orthogonal test design

水平	因素
水平	pH	温度/℃	金霉素初始浓度/(mg/L)	碳源种类	接种量/%
1	5	20	20	CH₃COONa	1
2	6	25	50	淀粉	2
3	7	30	100	柠檬酸钠	4
4	8	35	150	丁二酸钠	6
5	9	40	200	葡萄糖	8

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表 3 正交试验结果

Table 3. Orthogonal test results

试验序号	因素水平						降解率/%
试验序号	pH	温度	金霉素初始浓度	碳源种类	接种量	空白对照	降解率/%
试验1	1	1	1	1	1	1	53.51
试验2	1	2	2	2	2	2	58.83
试验3	1	3	3	3	3	3	71.53
试验4	1	4	4	4	4	4	70.75
试验5	1	5	5	5	5	5	61.11
试验6	2	1	2	3	4	5	63.79
试验7	2	2	3	4	5	1	71.54
试验8	2	3	4	5	1	2	63.41
试验9	2	4	5	1	2	3	64.61
试验10	2	5	1	2	3	4	58.23
试验11	3	1	3	5	2	4	60.88
试验12	3	2	4	1	3	5	69.93
试验13	3	3	5	2	4	1	61.33
试验14	3	4	1	3	5	2	64.26
试验15	3	5	2	4	1	3	51.77
试验16	4	1	4	2	5	3	57.34
试验17	4	2	5	3	1	4	63.66
试验18	4	3	1	4	2	5	64.48
试验19	4	4	2	5	3	1	66.38
试验20	4	5	3	1	4	3	59.96
试验21	5	1	5	4	3	2	57.76
试验22	5	2	1	5	4	3	63.08
试验23	5	3	2	1	5	4	74.02
试验24	5	4	3	2	1	5	63.73
试验25	5	5	4	3	2	1	52.14

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表 4 各因素不同水平下降解率的均值及极差

Table 4. Mean and extreme difference of degradation efficiency in different levels of factors

因素	不同水平下降解率的均值					极差
因素	水平1	水平2	水平3	水平4	水平5	极差
pH	63.146	64.316	61.634	62.364	62.146	2.682
温度	58.656	65.408	66.954	65.946	56.642	10.312
金霉素初始浓度	60.712	62.958	65.528	62.714	61.694	4.816
碳源种类	64.406	59.892	63.076	63.260	62.972	4.514
接种量	59.216	60.188	64.766	63.782	63.782	6.438
空白对照	60.980	61.065	61.382	65.508	64.608	4.528

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表 5 Box-Behnken试验因素与水平

Table 5. Box-behnken test factors and levels

因素	因素水平
因素	−1	0	1
A/℃	25	30	35
B/(mg/L)	50	100	150
C/%	2	4	6

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表 6 菌株ZL-1响应面分析结果

Table 6. Response surface analysis result of strain ZL-1

标准顺序	试验顺序	因素A	因素B	因素C	降解率/%
1	11	2	2	3	68.307
2	7	4	2	3	94.250
3	8	2	4	3	70.318
4	17	4	4	3	92.644
5	15	2	3	2	72.953
6	6	4	3	2	93.851
7	10	2	3	4	84.010
8	4	4	3	4	97.152
9	9	3	2	2	89.965
10	16	3	4	2	88.469
11	13	3	2	4	95.401
12	2	3	4	4	90.353
13	14	3	3	3	92.039
14	3	3	3	3	92.048
15	12	3	3	3	91.077
16	5	3	3	3	91.628
17	1	3	3	3	91.512

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表 7 菌株ZL-1降解金霉素的最小拟二乘法分析

Table 7. Minimum quasi-multiplied method for degrading chlortetracycline of strain ZL-1

项目	平方和	自由度	均方	F	P	显著性
模型	1215.280	9	135.030	24.750	0.0002
A	846.860	1	846.860	155.220	<0.0001	***
B	4.710	1	4.710	0.860	0.3837
C	58.740	1	58.740	10.770	0.0135	**
AB	3.270	1	3.270	0.600	0.4643
AC	15.040	1	15.040	2.760	0.1408
BC	3.160	1	3.160	0.580	0.4718
A²	216.350	1	216.350	39.650	0.0004	***
B²	40.790	1	40.790	7.480	0.0292	**
C²	26.300	1	26.300	4.820	0.0642	*
残差	38.190	7	5.460
失拟相	37.530	3	12.510	76.140	0.0006
纯误差	0.660	4	0.160
总和	1253.480	16
注：*为极显著，为较显著，*为显著，空白为不显著。

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参考文献(30)

[1]	高立红, 史亚利, 厉文辉, 等.抗生素环境行为及其环境效应研究进展[J]. 环境化学,2013,32(9):1619-1633. doi: 10.7524/j.issn.0254-6108.2013.09.004 GAO L H, SHI Y L, LI W H, et al. Environmental behavior and impacts of antibiotics[J]. Environmental Chemistry,2013,32(9):1619-1633. doi: 10.7524/j.issn.0254-6108.2013.09.004
[2]	秦松岩, 李杭, 山丹, 等.四环素类抗生素生产废水处理现状与研究进展[J]. 天津理工大学学报,2016,32(2):50-54. doi: 10.3969/j.issn.1673-095X.2016.002.012 QIN S Y, LI H, SHAN D, et al. The present situation and research progress in the treatment of tetracycline antibiotic manufacturing wastewater[J]. Journal of Tianjin University of Technology,2016,32(2):50-54. doi: 10.3969/j.issn.1673-095X.2016.002.012
[3]	吴晓霞, 周晓燕, 李久彤, 等.基于背景荧光猝灭-免疫层析法黄曲霉毒素B₁检测卡的研制[J]. 分析试验室,2017,36(2):189-193. WU X X, ZHOU X Y, LI J T, et al. Determination card of AFB₁ based on background fluorescence quenching immunochromatographic assay[J]. Chinese Journal of Analysis Laboratory,2017,36(2):189-193.
[4]	GONZÁLEZ-PLEITER M, GONZALO S, RODEA-PALOMARES I, et al. Toxicity of five antibiotics and their mixtures towards photosynthetic aquatic organisms: implications for environmental risk assessment[J]. Water Research,2013,47(6):2050-2064. doi: 10.1016/j.watres.2013.01.020
[5]	LIN J S, PAN H Y, LIU S M, et al. Effects of light and microbial activity on the degradation of two fluoroquinolone antibiotics in pond water and sediment[J]. Journal of Environmental Science and Health, Part B,2010,45(5):456-465. doi: 10.1080/03601231003800222
[6]	ZHUANG M, ACHMON Y, CAO Y P, et al. Distribution of antibiotic resistance genes in the environment[J]. Environmental Pollution,2021,285:117402. doi: 10.1016/j.envpol.2021.117402
[7]	卢运战, 祁克宗, 朱良强.四环素类药物残留检测方法研究进展[J]. 家禽科学,2006(10):36-39. doi: 10.3969/j.issn.1673-1085.2006.10.017
[8]	代安娜, 陈硕.基于生物传感技术的新型环境污染物多指标分析仪[J]. 科技创新与品牌,2012(5):77.
[9]	CHEE-SANFORD J C, MACKIE R I, KOIKE S, et al. Fate and transport of antibiotic residues and antibiotic resistance genes following land application of manure waste[J]. Journal of Environmental Quality,2009,38(3):1086-1108. doi: 10.2134/jeq2008.0128
[10]	宋冉冉, 国晓春, 卢少勇, 等.东洞庭湖表层水体中抗生素及抗性基因的赋存特征与源分析[J]. 环境科学研究,2021,34(9):2143-2153. doi: 10.13198/j.issn.1001-6929.2021.04.27 SONG R R, GUO X C, LU S Y, et al. Occurrence and source analysis of antibiotics and antibiotic resistance genes in surface water of East Dongting Lake Basin[J]. Research of Environmental Sciences,2021,34(9):2143-2153. doi: 10.13198/j.issn.1001-6929.2021.04.27
[11]	沈怡雯, 黄智婷, 谢冰.抗生素及其抗性基因在环境中的污染、降解和去除研究进展[J]. 应用与环境生物学报,2015,21(2):181-187. SHEN Y W, HUANG Z T, XIE B. Advances in research of pollution, degradation and removal of antibiotics and antibiotic resistance genes in the environment[J]. Chinese Journal of Applied and Environmental Biology,2015,21(2):181-187.
[12]	张惠东, 刘玉忠.水中四环素类污染物及吸附去除研究进展[J]. 科技创新与应用,2020(28):6-9,13. ZHANG H D, LIU Y Z. Research progress of tetracycline pollutants in water and their adsorption and removal[J]. Technology Innovation and Application,2020(28):6-9,13.
[13]	PENG L, REN Y Q, GU J D, et al. Iron improving bio-char derived from microalgae on removal of tetracycline from aqueous system[J]. Environmental Science and Pollution Research,2014,21(12):7631-7640. doi: 10.1007/s11356-014-2677-2
[14]	PEIRIS C, GUNATILAKE S R, MLSNA T E, et al. Biochar based removal of antibiotic sulfonamides and tetracyclines in aquatic environments: a critical review[J]. Bioresource Technology,2017,246:150-159. doi: 10.1016/j.biortech.2017.07.150
[15]	ZHAO C, DENG H P, LI Y, et al. Photodegradation of oxytetracycline in aqueous by 5A and 13X loaded with TiO₂ under UV irradiation[J]. Journal of Hazardous Materials,2010,176(1/2/3):884-892.
[16]	RIMOLDI L, MERONI D, CAPPELLETTI G, et al. Green and low cost tetracycline degradation processes by nanometric and immobilized TiO₂ systems[J]. Catalysis Today,2017,281:38-44. doi: 10.1016/j.cattod.2016.08.015
[17]	YUAN C, HUNG C H, LI H W, et al. Photodegradation of ibuprofen by TiO₂ co-doping with urea and functionalized CNT irradiated with visible light: effect of doping content and pH[J]. Chemosphere,2016,155:471-478. doi: 10.1016/j.chemosphere.2016.04.055
[18]	DALMÁZIO I, ALMEIDA M O, AUGUSTI R, et al. Monitoring the degradation of tetracycline by ozone in aqueous medium via atmospheric pressure ionization mass spectrometry[J]. Journal of the American Society for Mass Spectrometry,2007,18(4):679-687. doi: 10.1016/j.jasms.2006.12.001
[19]	PEI S Z, SHEN C, ZHANG C H, et al. Characterization of the interfacial joule heating effect in the electrochemical advanced oxidation process[J]. Environmental Science & Technology,2019,53(8):4406-4415.
[20]	MARTÍNEZ-HUITLE C A, PANIZZA M. Electrochemical oxidation of organic pollutants for wastewater treatment[J]. Current Opinion in Electrochemistry,2018,11:62-71. doi: 10.1016/j.coelec.2018.07.010
[21]	ZHAO X D, LI X J, ZHANG X L, et al. Bioelectrochemical removal of tetracycline from four typical soils in China: a performance assessment[J]. Bioelectrochemistry,2019,129:26-33. doi: 10.1016/j.bioelechem.2019.04.016
[22]	ZHANG J C, GIORNO L, DRIOLI E. Study of a hybrid process combining PACs and membrane operations for antibiotic wastewater treatment[J]. Desalination,2006,194(1/2/3):101-107.
[23]	张浩, 罗义, 周启星.四环素类抗生素生态毒性研究进展[J]. 农业环境科学学报,2008,27(2):407-413. doi: 10.3321/j.issn:1672-2043.2008.02.001 ZHANG H, LUO Y, ZHOU Q X. Research advancement of eco-toxicity of tetracycline antibiotics[J]. Journal of Agro-Environment Science,2008,27(2):407-413. doi: 10.3321/j.issn:1672-2043.2008.02.001
[24]	敖蒙蒙, 魏健, 陈忠林, 等.四环素类抗生素环境行为及其生态毒性研究进展[J]. 环境工程技术学报,2021,11(2):314-324. doi: 10.12153/j.issn.1674-991X.20200096 AO M M, WEI J, CHEN Z L, et al. Research progress on environmental behaviors and ecotoxicity of tetracycline antibiotics[J]. Journal of Environmental Engineering Technology,2021,11(2):314-324. doi: 10.12153/j.issn.1674-991X.20200096
[25]	王梓竹, 刘泽, 胡胜杰, 等.四环素降解菌的筛选及其降解特性研究[J]. 饲料研究,2020,43(10):64-68. doi: 10.13557/j.cnki.issn1002-2813.2020.10.017 WANG Z Z, LIU Z, HU S J, et al. Screening and degradation characteristics of tetracycline-degrading bacteria[J]. Feed Research,2020,43(10):64-68. doi: 10.13557/j.cnki.issn1002-2813.2020.10.017
[26]	陶美, 贺玉龙, 王林, 等.四环素降解菌的筛选及其降解特性[J]. 应用与环境生物学报,2018,24(2):384-389. doi: 10.19675/j.cnki.1006-687x.2017.06040 TAO M, HE Y L, WANG L, et al. Screening and degradation characteristics of a tetracycline-degrading bacterial strain[J]. Chinese Journal of Applied and Environmental Biology,2018,24(2):384-389. doi: 10.19675/j.cnki.1006-687x.2017.06040
[27]	赵永斌. 3种四环素类抗生素降解菌的筛选及降解特性的研究[D]. 太谷: 山西农业大学, 2015.
[28]	郑茂佳. 四环素降解菌的筛选及其对养殖废水的净化能力[D]. 大连: 辽宁师范大学, 2018.
[29]	LI W, ZHAO L C, SUN Y S, et al. Optimization of pressurized liquid extraction of three major acetophenones from Cynanchum bungei using a Box-Behnken design[J]. International Journal of Molecular Sciences,2012,13(11):14533-14544.
[30]	AGRAWAL M, SARAF S, PRADHAN M, et al. Design and optimization of curcumin loaded nano lipid carrier system using Box-Behnken design[J]. Biomedicine & Pharmacotherapy,2021,141:111919. □