一株十二烷基硫酸钠高效降解菌的分离鉴定、降解特性及代谢途径研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0347

摘要/Abstract

摘要：

为获得更丰富的十二烷基硫酸钠（sodium dodecyl sulfate，SDS）降解菌资源，从本实验室筛选保存的复合菌系SDS1中，分离出1株以SDS为唯一碳源生长的高效降解菌D2，结合形态学观察、生理生化特征及16S rRNA序列分析，结果显示D2为帕拉伯克霍尔德菌（Paraburkholderia tropica）。实验结果证实，在温度30℃、pH 7、盐度0.1%（W/W）、附加氮源硝酸钠+氯化铵的条件下培养48 h，D2对初始浓度1 200 mg/L的SDS降解率达到98.3%。采用离子色谱法、气相色谱法、液相色谱法对D2在SDS降解过程中的主要代谢产物进行检测与鉴定，初步推测出其对SDS的降解途径可能为SDS→硫酸盐+1-十二烷醇→十二烷醛→十二烷酸→乙酸→CO₂+H₂O。本研究可为含SDS表面活性剂废水的高效降解处理提供微生物资源。

关键词: 十二烷基硫酸钠, 帕拉伯克霍尔德菌, 微生物降解, 分离鉴定, 降解特性, 代谢途径

Abstract:

In order to obtain more abundant resources of sodium dodecyl sulfate（SDS）-degrading bacteria，a highly efficient SDS-degrading strain D2 with SDS as the only carbon source was isolated from the composite strain SDS1 screened and preserved in our laboratory. According to its morphological characteristics，physiological and biochemical experiments and 16S rRNA gene sequence molecular analysis，D2 was identified as Paraburkholderia tropica. The experimental results confirmed that the degradation rate of SDS by D2 increased to 98.3% at a temperature of 30℃，pH 7，salinity 0.1%（W/W）when the initial concentration of SDS was 1 200 mg/L，and an additional nitrogen source of sodium nitrate + ammonium chloride for 48 h. The main metabolites of the strain in SDS degradation were detected via ion chromatography，gas chromatography and liquid chromatography. The feasible degradation pathway of SDS by D2 might be SDS → sulfate +1-dodecanol → dodecanaldehyde → dodecanoic acid → acetic acid → CO₂ + H₂O. This study may provide microbial resources for the efficient degradation of SDS-containing surfactant wastewater.

Key words: sodium dodecyl sulfate, Paraburkholderia tropica, microbial degradation, isolation and identification, degradation characteristics, metabolism pathway

牛鸿宇, 舒倩, 杨海君, 颜智勇, 谭菊. 一株十二烷基硫酸钠高效降解菌的分离鉴定、降解特性及代谢途径研究[J]. 生物技术通报, 2022, 38(12): 287-299.

NIU Hong-yu, SHU Qian, YANG Hai-jun, YAN Zhi-yong, TAN Ju. Isolation, Identification, Degradation Characteristics and Metabolic Pathway of an Efficient Sodium Dodecyl Sulfate-degrading Bacterium[J]. Biotechnology Bulletin, 2022, 38(12): 287-299.

图/表 14

图1 初始浓度下菌株D2对SDS的降解率

Fig. 1 Degradation rate of SDS by strain D2 under initial concentrations

图2 菌株D2各传代对SDS的降解率

Fig. 2 Degradation rate of SDS by each generation of strain D2

图3 菌株D2菌落特征 A：革兰氏染色形态图；B：菌株在LB固体培养基中的形态特征（平板划线）

Fig. 3 Colony characteristics of D2 strain A：Gram staining morphology. B：Morphological characteristics of strain in LB solid medium（streaked inoculation）

表1 菌株D2的生理生化特征

Table 1 Physiological and biochemical characteristics of D2 strain

特征 Charateristics	检测结果 Result
利用柠檬酸盐 Use of citrate	+
糖发酵 Glucose fermentation	+
甲基红 Methyl red	-
尿素水解 Urea hydrolysis	+
产氨 Ammonia	+
苯丙氨酸脱氢酶 Phenylalanine dehydrogenase	-
吲哚 Indocyanine	-
产硫化氢 Hydrogen sulfide	-

表2 D2菌株16S rRNA基因序列分析鉴定

Table 2 Identification of D2 strain by 16S rDNA gene sequence analysis

物种 Species	NCBI登录号 NCBI accession no.	相似度 Percent of similarity
Paraburkholderia tropica strain DSM 15359	NR_028965.1	99.93%
Paraburkholderia bannensis strain NBRC 103871	NR_113178.1	98.68%
Burkholderia humi Srinivasan strain JCM 18070	NR_132708.1	98.53%

图4 菌株D2及其相关菌株系统发育树 Paraburkholderia oxyphila：嗜酸伯克霍尔德菌；Paraburkholderia guartelaensis：瓜拉伯克霍尔德菌；Paraburkholderia unamae：副伯克霍尔德菌；Paraburkholderia bannensis：假诺伯克霍尔德菌；Paraburkholderia tropica：帕拉伯克霍尔德菌；Burkholderia humi：胡氏伯克霍尔德菌；Paraburkholderia eburnea：象牙白伯克霍尔德菌；Paraburkholderia metalliresistens：抗金属伯克霍尔德菌；Paraburkholderia nodosa：根瘤伯克霍尔德菌

Fig. 4 Phylogenetic tree of strain D2 and its related strains

图5 环境因素对菌株D2降解SDS的影响 A：温度；B：pH；C：培养时间；D：NaCl浓度；E：氮源（1：硝酸钠；2：氯化铵；3：硝酸钠和氯化铵；4：脲素；5：蛋白胨）。采用ANOVA Duncan法进行分析，不同小写字母表示组间差异具有统计学意义（P<0.05）

Fig. 5 Effects of environmental factors on the degradation of SDS by strain D2 A：Temperature. B：pH. C：Incubation time. D：NaCl concentration. E：Nitrogen source（1：Sodium nitrate. 2：Ammonium chloride. 3：Sodium nitrate and ammonium chloride. 4：Urea. 5：Peptone）. ANOVA Duncan method was used for analysis and different letters means significant difference at P<0.05

表3 正交实验设计

Table 3 Orthogonal experimental design

水平 Level	因素Factors
水平 Level	A 温度 Temperature/℃	B pH	C 时间 Time/h	D 盐度NaCl concentration/%
1	25	6	30	0.05
2	30	7	42	0.1
3	35	8	48	0.3

表4 正交实验结果

Table 4 Orthogonal experimental results

实验号 Experiment No.	因素 Factors				SDS降解率Degrada-tion rate of SDS/%
实验号 Experiment No.	A	B	C	D	SDS降解率Degrada-tion rate of SDS/%
1	2	1	2	2	81.98
2	2	2	3	1	80.29
3	2	3	1	3	57.44
4	3	3	2	1	83.84
5	1	1	1	1	77.13
6	3	2	1	2	91.27
7	1	3	3	2	49.79
8	1	2	2	3	57.30
9	3	1	3	3	67.20
K₁	198.59	225.70	189.07	204.80	T=646.24
K₂	253.54	233.02	215.91	224.90
K₃	194.12	187.52	241.27	216.55
X₁	66.20	75.23	63.02	68.27
X₂	84.51	77.67	71.97	74.97
X₃	64.71	62.51	80.42	72.18
R	19.81	15.17	17.40	6.70

图6 菌株D2降解SDS的离子色谱图

Fig. 6 Ion chromatograms of degrading SDS by strain D2

图7 菌株D2降解SDS的气相色谱图

Fig. 7 Gas chromatograms of degrading SDS by strain D2

图8 菌株D2降解SDS的十二烷酸液相色谱图

Fig. 8 Dodecanoic acid liquid chromatograms of degrading SDS by strain D2

图9 菌株D2降解SDS的乙酸液相色谱图

Fig. 9 Acetic acid liquid chromatograms of degrading SDS by strain D2

图10 菌株D2对SDS的降解途径

Fig. 10 Degradation pathway of SDS by strain D2

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