一种苯酚降解菌Pseudoxanthomonas sp. BF-6的分离鉴定及其降解特性及途径研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0124

摘要/Abstract

摘要：

苯酚是最常见的有机污染物,为了获得降解苯酚的细菌,并探究其降解苯酚的特性和途径,采用连续富集和稀释平板分离,从农药厂活性污泥中得到一株能以苯酚作为唯一碳源生长的菌株BF-6,通过 16S rRNA 序列分析和生理生化试验将该菌株鉴定为假黄单胞菌（Pseudoxanthomonas sp.）。单因素试验结果表明,菌株BF-6在温度为20-37℃和pH5.0-10.0范围内均可很好的降解苯酚;在此条件下,5%接种量的菌株BF-6在36 h内可将100 mg/L苯酚降解97.79%,96 h可将200 mg/L苯酚降解97.58%。经苯酚诱导后,菌株BF-6细胞粗酶液表现出苯酚羟化酶和邻苯二酚1,2-双加氧酶活性,表明菌株BF-6降解苯酚的途径为邻位途径。本研究为低浓度苯酚的微生物降解提供了新的菌种资源。

关键词: 苯酚, 假黄单胞菌, 分离鉴定, 降解特性, 降解途径

Abstract:

The aim of this work is to isolate phenol-degrading bacteria and to explore the characteristics and pathways of phenol degradation. Continuous enrichment and dilution plate method were applied to isolate the phenol-degrading bacteria,and single factor test was to study the characteristics of phenol degradation. The metabolic pathway of degrading by the strain phenol was deduced by determining the activities of phenol hydroxylase and catechol dioxygenase. A phenol-degrading strain BF-6,growing with phenol as the sole carbon source,was isolated and named as BF-6. Strain BF-6 was identified as Pseudoxanthomonas sp. by using 16S rRNA sequence analysis and physiological and biochemical tests. Strain BF-6 degraded phenol well in the temperature range of 20-37℃ and pH range of 5.0-10.0;under this condition,strain BF-6 with 5% inoculum degraded 100 mg/L phenol by 97.79% in 36 h and 200 mg/L phenol by 97.58% in 96 h. Cell extracts of strain BF-6 showed enzymatic activity of phenol hydroxylase and catechol 1,2-dioxygenase after induction by phenol,indicating that the strain degraded phenol via ortho-pathway. In conclusion,phenol-degrading bacterium BF-6 is identified as Pseudoxanthomonas sp.,and it degrades phenol under a wide range of temperature and pH conditions. Phenol is degraded via ortho-pathway by strain BF-6. This study provides a new strain for microbial-degration of low-concentration phenol.

Key words: phenol, Pseudoxanthomonas sp., isolation and identification, degradation characterization, degradation pathway

卫晓博, 侯颖, 程豪杰, 秦翠丽, 牛明福, 徐建强. 一种苯酚降解菌Pseudoxanthomonas sp. BF-6的分离鉴定及其降解特性及途径研究[J]. 生物技术通报, 2021, 37(10): 72-80.

WEI Xiao-bo, HOU Ying, CHENG Hao-jie, QIN Cui-li, NIU Ming-fu, XU Jian-qiang. Isolation,Identification of Phenol-degrading Pseudoxanthomonas sp. BF-6 and Its Degradation Characteristics and Pathway[J]. Biotechnology Bulletin, 2021, 37(10): 72-80.

图/表 10

图1 菌株BF-6的菌落和菌体形态 A:菌株BF-6在LB平板上形成的菌落;B:菌株BF-6光学显微镜下形态（1 000×）

Fig.1 Colony and morphology of strain BF-6 A:Colonies of strain BF-6 on LB plate;B:morphology of strain BF-6 under optical microscope（1000×）

表1 菌株BF-6部分生理生化试验结果

Table 1 Results of physiological and biochemical test of strain BF-6

项目Item	结果Result	项目Item	结果Result
葡萄糖Glucose	+	头孢克肟Cefixime	+
蔗糖Sucrose	+	头孢克洛Cefaclor	+
乳糖Lactose	+	头孢拉定Cefradine	+
木糖Xylose	-	呋喃唑酮Furazolidone	+
甘露醇Mannitol	-	杆菌肽Bacitracin	+
甲醇Methanol	-	阿奇霉素Azithromycin	-
乙醇Ethanol	+	四环素Tetracycline	-
牛肉膏Beef paste	+	卡那霉素Kanamycin	-
蛋白胨Peptone	+	诺氟沙星Norfloxacin	-
酵母粉Yeast powder	+	革兰氏染色Gram stain	-
尿素Urea	+	甲基红试验Methyl red test	-
（NH₄）₂SO₄	+	V-P试验V-P test	-
KNO₃	+	过氧化氢酶试验Catalase test	+
NaNO₂	+	淀粉水解酶试验Starch hydrolase test	-
青霉素Penicillin	+	反硝化试验Denitrification experiment	-
阿莫西林Amoxicillin	+	明胶液化试验Gelatin liquefaction test	-
氨苄西林Ampicillin	+	最适生长温度Optimal growth temperature	37℃
链霉素Streptomycin	+	最适生长pH Optimal growth pH	8.0

图2 基于16S rRNA基因序列构建的菌株BF-6系统发育树

Fig.2 Phylogenetic tree constructed by the neighbor-joining analysis based on 16S rRNA gene sequences of strain BF-6 and related species

图3 菌株BF-6以苯酚为唯一碳源的生长曲线及苯酚降解曲线

Fig.3 Growth curve of strain BF-6 and degradation curve of phenol by using phenol as sole carbon source

图4 温度对菌株BF-6降解苯酚的影响

Fig.4 Effect of temperature on degradation of phenol by strain BF-6

图5 pH对菌株BF-6降解苯酚的影响

Fig.5 Effect of pH on the degradation of phenol by strain BF-6

图6 苯酚浓度对菌株BF-6降解苯酚的影响

Fig.6 Effect of initial concentration on the degradation of phenol by strain BF-6

图7 接种量对菌株BF-6降解苯酚的影响

Fig.7 Effect of inoculum quantity on the degradation of phenol by strain BF-6

图8 金属离子对菌株BF-6降解苯酚的影响

Fig.8 Effect of metal ions on the degradation of phenol by strain BF-6

图9 苯酚羟化酶（A）和邻苯二酚双加氧酶（B）活性测定

Fig.9 Determination of phenol hydroxylase（A）and catechol dioxygenase（B）

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