海氏肠球菌IDO5对猪粪废水中吲哚降解条件优化及降解途径分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0071

摘要/Abstract

摘要：

在前期的研究中发现了一株吲哚高效降解菌IDO5,本研究主要对其降解猪粪废水吲哚的性质进行了测定。首先通过形态学和16S rRNA序列分析,并初步分析了IDO5对吲哚的降解效果和可作用的底物类型。其次比较了转速、温度、pH、碳源等因素对IDO5降解猪粪废水中吲哚的影响。最后利用GC-MS分析IDO5降解吲哚的产物结构和降解途径。结果表明,IDO5不仅能够利用吲哚为碳源生长,而且能够降解3-甲基吲哚、苯酚、甲酚等多种污染物,具有广泛的底物谱。菌株IDO5降解猪粪吲哚最适的条件为:温度37^oC,转速170 r/min,pH 9,在此条件下24 h内可降解猪粪废水中98.2%的吲哚,去除效率较高。甚至在额外添加100 mg/L吲哚的情况下,IDO5依然能够以93.7%的高降解率去除吲哚。IDO5在降解吲哚过程中形成红色的产物,通过GC-MS分析,菌株IDO5降解吲哚很可能是通过吲哚→靛红→邻氨基苯甲酸→邻甲基苯甲醛途径。本研究表明,菌株IDO5不仅具有较强的吲哚降解能力,而且还能作用多种吲哚类化合物,对pH适应能力也较强,能够在广泛的条件下对畜禽养殖废水中的吲哚进行有效降解,具备一定应用价值。

关键词: 海氏肠球菌, 吲哚, pH稳定性, 养殖废水

Abstract:

A highly efficient indole-degrading bacterium strain IDO5 was identified in the present study. Herein,the indole-degrading ability of IDO5 in pig manure waste water was mainly investigated. Firstly,IDO5 was identified by morphology and 16S rRNA sequence analysis,and the degrading effect of IDO5 on indole and the types of substrates were preliminary analyzed. Secondly,the effects of rotational speed,temperature,pH and carbon source on the degradation of indole in pig manure waste water by IDO5 were compared. Finally,the structure and degradation pathway of indole degraded by IDO5 were analyzed by GC-MS. The results showed that IDO5 could not only use indole as the carbon source for growth,but also degrade 3-methylindole,phenol,cresol and other pollutants,thus IDO5 had a broad substrate spectrum. The optimal conditions for IDO5 degrading indole in the pig manure were as:temperature 37^oC,pH 9 and rotary speed 170 r/min. Under these conditions,98.2% of indole in the pig manure wastewater was degraded within 24 h,i.e.,the removal rate was quite high. IDO5 removed 93.7% indole even when an additional 100 mg/L indole was added. IDO5 formed a red product during the degradation of indole. The degradation of indole by strain IDO5 may be through the pathway of indole → isatin → anthranilic acid → 2-methylbenzalde after analyzed by GC-MS. Strain IDO5 not only has strong indole-degrading ability in a wide pH range,but also can act on a variety of indole derivatives and other compounds. Therefore,strain IDO5 can effectively degrade indole in livestock and poultry breeding waste water under a wide range of conditions,and has certain application value.

Key words: Enterococcus hirae, indole, pH stability, aquaculture waste water

余琴, 马现永, 邓盾, 王永飞. 海氏肠球菌IDO5对猪粪废水中吲哚降解条件优化及降解途径分析[J]. 生物技术通报, 2021, 37(12): 113-123.

YU Qin, MA Xian-yong, DENG Dun, WANG Yong-fei. Optimization of Indole-degrading Conditions in Pig Manure Waste Water by Enteroccus hirae IDO5 and Analysis of Its Corresponding Degradation Pathway[J]. Biotechnology Bulletin, 2021, 37(12): 113-123.

图/表 13

图1 IDO5形态学特征 A:IDO5菌株平板菌落;B:菌株IDO5扫描电镜镜检结果

Fig. 1 Morphological characteristics of IDO5 A:Plate colony of IDO5 strain;B:scanning electron microscopy result of strain IDO5

图2 IDO5-16S rRNA系统发育树基于IDO5的16S rRNA基因序列建立的系统发育邻接树。系统发育树显示了各个分离株和各自属内物种之间的关系。引导节点上给出的值（以1 000次复制的百分比表示）大于50%。序列变异尺度值为0.05%。GenBank登录号在括号中给出

Fig. 2 Phylogenetic tree based on 16S rRNA sequence of IDO5 Phylogenetic neighbor joining tree of IDO5 is constructed based on 16S rRNA gene sequences. The phylogenetic tree shows the relationships between individual isolates and species within the respective genus. Bootstrap values（expressed as percentages of 1 000 replications）given at nodes are > 50%. Bar 0.05% sequence variation. GenBank accession numbers are given in parentheses

图3 IDO5菌液处理吲哚及对照的HPLC结果

Fig. 3 HPLC results of treated indole by IDO5 bacterial solution and control

表1 菌株IDO5的底物广谱性

Table 1 Substrate spectrum of strain IDO5

图4 温度对IDO5降解猪粪废水中吲哚的影响误差线表示标准差,不同字母表示处理之间差异显著（P < 0.05）。下同

Fig. 4 Effects of temperature on the degradation of indole in pig anure wastewater by IDO5 Error bars indicate the standard deviation and different letters reflect significant difference（P< 0.05）between treatments. The same below

图5 转速对IDO5降解猪粪废水中吲哚的影响

Fig. 5 Effect of rotating speed on the degradation of indole in pig manure wastewater by strain IDO5

图6 pH对IDO5降解猪粪废水中吲哚的影响

Fig. 6 Effect of pH on the degradation of indole in pig manure wastewater by strain IDO5

图7 外加碳源对IDO5降解猪粪废水中吲哚的影响

Fig. 7 Effect of exogenous carbon source on the degrada-tion of indole in pig manure wastewater by strain IDO5

图8 菌株IDO5降解猪粪水中吲哚在24 h内的变化情况

Fig. 8 Variation of indole degradation in pig manure water by strain IDO5 during 24 h

图9 菌株IDO5对不同浓度吲哚在24 h内的降解情况

Fig.9 Variation of indole degradation in different concentrations by strain IDO5 during 24 h

表2 菌株IDO5降解吲哚GC-MS目标组分谱

Table 2 Target component spectrum of indole degrading GC-MS by strain IDO5

表3 吲哚降解菌资源

Table 3 Resources of indole degrading bacteria

菌株名称 Strain name	降解特性 Degradation characteristics	参考文献 Reference
Pseudomonas aeruginosaGs	36 d时降解58.5-175 mg/L吲哚 58.5-175 mg/L indole was degraded after 36 d	[25]
Comamonas sp. IDO1	至少需要160 h降解100 mg/L吲哚 100 mg/L indole was degraded after at least 160 h	[26]
Xenophilus sp. IDO4	至少需要160 h降解100 mg/L吲哚 100 mg/L indole was degraded after at least 160 h	[26]
Arthrobacter sp. B1	40 h降解117 mg/L吲哚 117 mg/L indole was degraded after 40 h	[27]
Arthrobacter sp. SPG	36 h降解58.5 mg/L吲哚 58.5 mg/L indole was degraded after 46 h	[28]
Providencia sp.	28 h降解100 mg/L吲哚 100 mg/L indole was degraded after 28 h	[29]
Cupriavidus sp. SHE	24 h降解100 mg/L吲哚 100 mg/L indole was degraded after 24 h	[23]

图10 菌株IDO5降解吲哚途径推测

Fig.10 Pathway of indole degradation by strain IDO5

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