三株异菌脲高效降解菌株的筛选、鉴定及其降解特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1372

摘要/Abstract

摘要：

为筛选适宜青藏高原地区的异菌脲高效降解菌株资源，采用富集培养法从西藏自治区蔬菜大棚土壤中分离得到Y-20、Y-29和Y-32三株异菌脲高效降解菌株，通过形态特征、生理生化特性和16S rRNA基因序列分析对上述菌株进行初步鉴定，探讨NaCl浓度（m/V）、温度、pH值、接种量（V/V）和异菌脲初始浓度等因素对其异菌脲降解速率的影响，采用气相色谱法和PCR技术分析了上述菌株的异菌脲代谢途径及降解基因（ipaH）。结果表明，菌株Y-20、Y-29和Y-32为微杆菌属（Microbacterium）的3个不同种；在1.0% NaCl（m/V）、pH 7.0、25-30℃、5%的接种量（V/V）和100 mg/L初始异菌脲浓度的最佳条件下，上述菌株能够在8-12 h内完全降解100 mg/L的异菌脲；上述菌株能够将异菌脲降解为N-（3,5-二氯苯基）-2,4-二氧代咪唑烷和异丙基氨基甲酸，且含有高度相似的ipaH基因序列（99.14%-99.69%）。本研究为高原地区异菌脲污染环境的生物修复提供了菌株资源和理论依据。

关键词: 异菌脲, 降解, 富集培养法, 微杆菌属, 代谢途径, ipaH基因, 气相色谱法

Abstract:

To isolate the iprodione-degrading microbial resources suitable for Qinghai-Tibet Plateau, three efficient iprodione-degrading strain Y-20, Y-29 and Y-32 were isolated from the greenhouse soil in Tibet by enrichment culture method. These strains were preliminarily identified based on the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis. The effects of NaCl concentration（m/V）, pH value, temperature, inoculation amount（V/V）and initial iprodione concentration on the iprodione-degradation rates of these strains were also investigated. The metabolic pathways of iprodione and ipaH gene in these strains were analyzed using gas chromatography method and polymerase chain reaction technique, respectively. The results showed that strain Y-20, Y-29 and Y-32 were identified as three species of Microbacterium genus. Under the optimal iprodione-degrading condition of 1.0% NaCl（m/V）, pH 7.0, 25-30℃, 5% inoculation amount（V/V）and 100 mg/L initial iprodione concentration, 100 mg/L iprodione was completely degraded by these strains within 8-12 h. These strains decomposed iprodione into N-（3,5-dichlorophenyl）-2,4-dioximidazolidine and isopropyl carbamate, and had a highly similar ipaH gene（99.14%-99.69%）. This study provides the strain resources and theoretical basis for the bioremediation of iprodione-contaminated environment at high altitude area.

Key words: iprodione, degradation, enrichment culture, Microbacterium, metabolic pathway, ipaH gene, gas chromatography

潘虎, 周子琼, 田云. 三株异菌脲高效降解菌株的筛选、鉴定及其降解特性分析[J]. 生物技术通报, 2023, 39(6): 298-307.

PAN Hu, ZHOU Zi-qiong, TIAN Yun. Screening Identification and Degradation Characteristics of Three Iprodione-degrading Strains[J]. Biotechnology Bulletin, 2023, 39(6): 298-307.

图/表 9

图1 菌株Y-20、Y-29和Y-32的异菌脲降解透明圈

Fig. 1 Transparent rings of iprodione degradation by strain Y-20, Y-29 and Y-32

图2 菌株Y-20、Y-29和Y-32的菌落形态特征

Fig. 2 Morphological characteristics of strain Y-20, Y-29 and Y-32 colonies

表1 菌株Y-20、Y-29和Y-32的部分生理生化鉴定结果

Table 1 Partial physiological and biochemical identification results of strain Y-20, Y-29 and Y-32

测试项目Test item	Y-20	Y-29	Y-32	测试项目Test item	Y-20	Y-29	Y-32
邻硝基苯-半乳糖苷2-Nitrophenyl-galactopyranoside	+	+	-	Kohn明胶Kohn gelatin	+	+	-
精氨酸Arginine	-	-	-	葡萄糖Glucose	+	+	-
赖氨酸Lysine	-	-	-	甘露醇Mannitol	+	+	-
鸟氨酸Ornithine	-	-	-	肌醇Inositol	-	-	-
柠檬酸钠Sodium citrate	+	+^w	+	山梨醇Sorbitol	-	-	-
硫代硫酸钠Sodium thiosulfate	-	-	-	鼠李糖Rhamnose monohydrate	+	-	-
尿素Urea	-	-	-	蔗糖Sucrose	+	+	-
色氨酸Tryptophan（TDA）	+	+^w	+	密二糖Disaccharide	+	-	-
色氨酸Tryptophan（IND）	-	-	-	苦杏仁苷Amygdalin	+	+	-
丙酮酸盐Pyruvate	+	+	-	阿拉伯糖Arabinose	+	-	+^w

图3 基于16S rRNA基因的异菌脲降解菌株的系统进化树

Fig. 3 Phylogenetic tree of iprodione-degrading strains based on 16S rRNA gene

图4 NaCl浓度、pH值、温度、接种量和异菌脲初始浓度对菌株Y-20、Y-29和Y-32异菌脲降解率的影响及其最佳降解曲线

Fig. 4 Effects of NaCl concentration, pH value, temperature, inoculation amount and initial iprodione concentration on the iprodione-degradation rates by strain Y-20, Y-29 and Y-32 and their optimal degradation curves

图5 发酵液中异菌脲与其代谢产物含量的变化及质谱分析结果 A：0 h发酵液；B：6 h发酵液；C：12 h发酵液；D：化合物1的一级质谱图谱；E：化合物2的一级质谱图谱

Fig. 5 Change of iprodione and its metabolites in the fermentation liquor and the mass spectrometry analysis results A: Fermentation broth of 0 h. B: Fermentation broth of 6 h. C: Fermentation broth of 12 h. D: Primary mass spectrometry result of compound 1. E: Primary mass spectrometry result of compound 2

图6 菌株Y-20、Y-29和Y-32的ipaH基因扩增及其系统进化树

Fig. 6 ipaH gene amplification results of strain Y-20, Y-29 and Y-32 and their phylogenetic tree

图7 菌株Y-20、Y-29和Y-32的异菌脲降解机理

Fig. 7 Degradation mechanism of iprodione in strain Y-20, Y-29 and Y-32

表2 异菌脲降解菌株的基本特性

Table 2 Basic characteristics of iprodione-degrading strains

样品来源 Source	分离菌株种类 Species of isolates	异菌脲初始浓度 Initial concentration of iprodione	降解率 Degradation rate/%	降解时间 Degradation time	降解基因 Degradation genes
土壤 Soil	Arthrobacter sp. MA6^[13]	9.90 mg/L	86.7	7 d	ND
土壤 Soil	Pseudomonas fluorescens, Pseudomonas sp., Pseudomonas paucimobilis^[14]	8.25 mg/L	100	20-24 h	ND
土壤 Soil	Zygosaccharomyces rouxii DBVPG 6399^[24]	1 mg/L	100	9 d	ND
农田土壤 Farmland soil	Arthrobacter sp.CQH^[15]	100 mg/L	100	112 h	ND
土壤 Soil	Microbacterium sp. CHQ-1^[12]	100 mg/L	100	96 h	ND
活性污泥 Activated sludge	Microbacterium sp.YJN-G^[17,25]	100 mg/L	100	24 h	ipaH
酸性土壤 Acidic soil	Arthrobacter sp. C1, Achromobacter sp. C^2[16]	60 mmol/L	100	10 d	ND
三七根际土壤 Root-zone soil of Panax notoginseng	Bacillus sp.KMS-1^[18]	25 mg/L	41.4	7 d	ND
葡萄园种植土壤 Vineyards grow soil	Paenarthrobacter sp. YJN-5^[8,25] Paenarthrobacter sp. YJN-D	1.5 mmol/L	95	80 h	ipaH, ddaH, duaH
大棚土壤 Greenhouse soil	Microbacterium sp. Y-20、Y-29、Y-32	100 mg/L	100	8-12 h	ipaH

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