一株菲降解细菌产生生物表面活性剂特性的研究

doi:10.16258/j.cnki.1674-5906.2021.08.015

生态环境学报 ›› 2021, Vol. 30 ›› Issue (8): 1683-1694.DOI: 10.16258/j.cnki.1674-5906.2021.08.015

一株菲降解细菌产生生物表面活性剂特性的研究

阎洁(), 余雪巍, 李鉴博, 顾海萍^*(), 郭二辉

河南农业大学林学院,河南郑州 450002

收稿日期:2021-07-09 出版日期:2021-08-18 发布日期:2021-11-03
通讯作者: * 顾海萍（1988年生）,女,博士,主要从事土壤有机污染微生物修复方面的研究。E-mail: guhaiping.1357@163.com
作者简介:阎洁（1996年生）,女,硕士研究生,主要从事土壤有机污染微生物修复方面的研究。E-mail: yanjiechenhongqin@126.com
基金资助:
国家自然科学基金项目(41701360);河南省高等学校重点科研项目计划(20B220003);河南省重点研发与推广专项(科技攻关,212102110109)

Research on the Characterization of Surfactant Produced by A Phenanthrene-degrading Strain

YAN Jie(), YU Xuewei, LI Jianbo, GU Haiping^*(), GUO Erhui

College of Forestry, Henan Agricultural University, Zhengzhou 450000, China

Received:2021-07-09 Online:2021-08-18 Published:2021-11-03

摘要/Abstract

摘要：

利用微生物修复环境中的多环芳烃（PAHs）污染近年来已受到了广泛关注。然而,生物可利用性低是该修复过程中的主要瓶颈。生物表面活性剂由于其良好的增溶性以及环境友好性而备受青睐。因此,筛选既能降解PAHs又能产生生物表面活性剂的菌株仍然是PAHs污染修复研究领域中的热点。以菲为目标污染物,通过透明降解圈和排油圈、表面张力实验从河南信阳一处多年垃圾焚烧场地筛选分离出一株具有菲降解和产生生物表面活性剂双重功能的菌株GHP1,并从菌株形态特征、生理生化特征和16S rDNA基因序列三方面对该菌株进行鉴定;采用高效液相色谱测定了该菌株对菲的降解效率,通过薄层层析和傅里叶红外变换光谱鉴定了该菌株所产生的表面活性剂的类型;此外,通过乳化性能测试进一步探究了培养时间、温度、pH和碳源对菌株培养液乳化活性的影响。实验结果表明：该菌株是Sphingobium abikonense（S. abikonense）;培养72 h对初始浓度为100 mg∙L^-1菲的降解率可达86.08%,降解半衰期约为27.12 h;初步确定该菌株所产生的生物表面活性剂为脂肽;菌株在72 h、28 ℃、pH 6.5、添加蔗糖的培养条件下培养液的乳化指数最高（59.11%）。这是首次关于S. abikonense降解菲和产生生物表面活性剂的研究,填充了国内外关于S. abikonense降解菲和产生生物表面活性剂特性的空白。该菌株既能降解菲又能产生生物表面活性剂,适应环境能力强,在修复PAHs污染方面具有巨大的应用潜力,为微生物修复PAHs的研究提供有价值的参考。

关键词: 菲, Sphingobium abikonense, 降解, 表面活性剂, 脂肽, 优化

Abstract:

In recent years, microbial remediation of polycyclic aromatic hydrocarbons (PAHs) pollution in the environment has attracted extensive attentions. However, the low bioavailability is a major bottleneck during the bioremediation process of PAHs. Biosurfactants are favored by many researchers due to the good solubilization and environmental friendliness. Therefore, isolation of PAHs-degrading strains which are capable of producing biosurfactant is still a hot spot in bioremediation of PAHs pollution. Phenanthrene was used as the target pollutant in this study. A strain GHP1 which can degrade phenanthrene and produce surfactant was isolated from a multi-year waste incineration site in Xinyang city, Henan Province through transparent degradation circle, oil displacement circle, and surface tension experiments. The strain was identified by morphological characteristics, physiological and biochemical characteristics, and 16S rDNA sequencing. In addition, high performance liquid chromatography was used to determine phenanthrene degradation by strain GHP1; thin layer chromatography and fourier transform infrared spectroscopy were used to characterize the surfactant produced by this strain. Besides, the effects of culture time, temperature, pH, and carbon sources on the emulsification activity of the culture medium were further investigated by emulsification tests. The results showed: strain GHP1 is Sphingobium abikonense (S. abikonense). The degradation rate of phenanthrene at the initial concentration of 100 mg∙L^-1 reached 86.08% after 72 h of culture, and the half-life of degradation was about 27.12 h. The highest emulsification index (59.11%) was obtained under the conditions of 72 h, 28 ℃, pH 6.5, and adding 1% sucrose. The biosurfactant produced by this strain was identified as lipopeptide. This is the first study to research phenanthrene degradation and surfactant production by S. abikonense all over the world, which filled the research gaps in this field. Since the strain GHP1 possessed the above two characteristics and strong environmental adaptability it has great application potential during the remediation of PAHs pollution. This study provides valuable reference for the microbial remediation of PAHs.

Key words: phenanthrene, Sphingobium abikonense, biodegradation, surfactant, lipopeptide, optimization

中图分类号:

X172

阎洁, 余雪巍, 李鉴博, 顾海萍, 郭二辉. 一株菲降解细菌产生生物表面活性剂特性的研究[J]. 生态环境学报, 2021, 30(8): 1683-1694.

YAN Jie, YU Xuewei, LI Jianbo, GU Haiping, GUO Erhui. Research on the Characterization of Surfactant Produced by A Phenanthrene-degrading Strain[J]. Ecology and Environment, 2021, 30(8): 1683-1694.

图/表 11

图1 GHP1菌落在覆有菲的MSM平板上产生的透明降解圈

Fig. 1 Transparent degradation circles produced on MSM plate covered with phenanthrene by strain GHP1 colony

图2 液体LB培养基（A）、菌株GHP1培养液（B）的排油圈

Fig. 2 Oil displacement circle of liquid LB medium (A) and strain GHP1 culture solution (B)

图3 GHP1在液体（A）、固体（B）LB培养基中的菌体形态和光学显微镜下的革兰氏染色（C）以及透射电子显微镜下的细胞形态（D）

Fig. 3 Morphology of strain GHP1 in liquid (A), solid (B) LB medium, gram staining under light microscope (C) and cell morphology under transmission electron microscope (D)

表1 GHP1菌株的生理生化特征

Table 1 Physiological and biochemical characteristics of strain GHP1

测试项目 Test items	结果 Result
需氧性 Aerobism	+
运动性 Motility	+
疏水性 Hydrophobicity	中度亲水性 Moderate hydrophilic
3-羟基丁酮产生乙酰甲基甲醇 V.P. reaction	W
甲基红试验 M.R. test	-
过氧化氢酶 Catalase	+
脲酶 Urease	-
淀粉水解 Amylolysis	+
纤维素水解 Cellulase	-
精氨酸双水解酶 Arginine dihydrolase	-
柠檬酸利用 Citrate utilization	-
赖氨酸脱羧酶 Lysine decarboxilase	-
鸟氨酸脱羧酶 Ornithine decarboxilase	-
β-半乳糖苷酶 β-galactosidase	+
H₂S产生 H₂S production	-
吲哚产生 Indole production	-
色氨酸脱氨酶 Tryptophane deaminase	-
明胶水解 Gelatinase	-
D-葡萄糖 D-glucose	-
D-甘露醇 D-mannitol	-
肌醇 Inositol	-
D-山梨醇 D-sorbitol	-
L-鼠李糖 L-rhamnose	-
D-蔗糖 D-sucrose	-
D-蜜二糖 D-melibiose	-
苦杏仁苷 Amygdaline	-
L-阿拉伯糖 L-arabinose	-

图4 基于菌株GHP1与相似菌株16S rDNA序列构建的系统发育树系统发育树分枝上的数值表示可信度,标尺表示分枝距离

Fig. 4 Phylogenetic tree based on 16S rDNA sequences of strain GHP1 and related type strains Numbers on the branches of a phylogenetic tree indicate confidence, and rulers indicate branch distance

图5 GHP1菌株对菲的降解动态数据为平均值±标准差（n=3）

Fig. 5 Degradation dynamics of phenanthrene by strain GHP1 Data are mean±standard deviation (n=3)

表2 GHP1菌株对菲降解的一级动力学方程及半衰期

Table 2 First-order kinetics equations and the corresponding half-life times of phenanthrene biodegradation by strain GHP1

指数方程 Exponential equation	t_1/2/h	R²
C=122.67e^-0.616^t	27.12	0.859*

表3 菲降解菌株及其降解率统计表

Table 3 Statistical table of phenanthrene degrading strains and their degradation rates

菌株 Strain	菲初始浓度 Initial concentration of phenanthrene/(mg∙L^-1)	时间 Time/h	降解率 Degradation rate/%	参考文献 Reference
Sphingobium abikonense GHP1	100	72	86.08	This study
Sphingobium fuliginis sp. nov.	200	24	100.00	Prakash et al., 2006
Novosphingobium TVG9-VII	100	504	95.20	Dong et al., 2011
Novosphingobium sp. PCY	100	336	75.70	Wongwongsee et al., 2013
Burkholderia cepacia PM07	50	576	26.20	Lee et al., 2005
Microbacterium F10a	50	168	98.00	Sheng et al., 2009
Microbacterium sp. BPW	100	336	70.80	Wongwongsee et al.,2013
Alcaligenes EF105546	1	24	34.40	Abd-Elsalam et al., 2009
Alcaligenes sp. SSK1B	100	336	49.70	Wongwongsee et al., 2013
Achromobacter AY189752	200	504	73.20	Tiwari et al., 2010
Achromobacter sp. SSK4	100	336	58.70	Wongwongsee et al., 2013
Pseudomonas sp. USTB-RU	100	192	86.65	Masakorala et al., 2013
Ralstonia sp. BPH	100	336	65.00	Wongwongsee et al., 2013
Bacillus pumilus 1529	100	504	100.00	Khajavi-Shojaei et al., 2020

图6 GHP1菌株产生的生物表面活性剂薄层层析

Fig. 6 Thin layer chromatography of biosurfactants produced by strain GHP1

图7 GHP1菌株产生的表面活性剂红外吸收光谱

Fig. 7 Infrared absorption spectra of biosurfactants produced by strain GHP1

图8 不同培养条件对培养液乳化性指数（E24）的影响数据为平均值±标准差（n=3）;标不同字母表示差异显著（P<0.05）。图8中A、B、C、D分别为培养时间、培养温度、培养基初始pH和碳源对培养液乳化性指数（E24）的影响

Fig. 8 The effects of different culture conditions on emulsification index (E24) of culture medium Data are mean±standard deviation (n=3); different lowercase letters mean significant difference at P<0.05 levels, respectively. In figure 8, A, B, C and D represent the influences of culture time, culture temperature, initial pH of medium, and carbon sources on the emulsification index (E24) of culture medium, respectively

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一株菲降解细菌产生生物表面活性剂特性的研究

Research on the Characterization of Surfactant Produced by A Phenanthrene-degrading Strain

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