耐高温木质纤维素降解菌株的分离筛选、鉴定及降解工艺的研究

doi:10.11924/j.issn.1000-6850.casb20190600244

摘要/Abstract

摘要：

本研究旨在筛选出在高温条件下具有较强木质纤维素降解酶活性的细菌菌株,探究其降解秸秆木质纤维素的特性。从太白山林区温泉采集土壤样品并在高温条件下进行富集培养,利用脱色圈试验和比色法筛选出目标菌株,通过形态观察和16S rDNA序列分析鉴定菌株种类。对高温富集初筛所得菌株的纤维素酶、漆酶、木质素过氧化物酶和锰过氧化物酶活性进行测定。菌株X-08的纤维素酶活为0.02872 U/mL,菌株M-17的MnP、LiP和Lac酶活分别为51 U/mL、672 U/mL和192 U/mL。鉴定出菌株X-08为Anoxybacillus rupiensis,M-17为Geobacillus thermocatenulatus。采用双菌降解玉米秸秆,20天后木质素和纤维素的降解率分别达到24.51%和20.47%。研究结果为农业废弃物生物降解提供了新的细菌菌种资源,并为秸秆中木质纤维素的降解处理方法提供了新的思路。

关键词: 耐热菌, 降解木质纤维素, 筛选, 菌种鉴定, 降解工艺

Abstract:

This study aims at screening out the bacterial strains with higher lignocellulolytic degradation enzyme activity under high temperature, and to explore the characteristics of straw lignocellulose degradation by bacterial strain. Soil samples were collected around forest hot spring in Mount Taibai and were used for enrichment culture at high temperature. The target strains were selected by decolorization circle method and colorimetric method. The types of strains were identified by morphological observation and 16S rDNA sequence analysis. The activities of cellulase, laccase, lignin peroxidase and manganese peroxidase were determined. The results showed that the cellulase activity of X-08 strain was 0.02872 U/mL, and the activity of MnP, LiP and Lac in M-17 strain was 51 U/mL, 672 U/mL and 192 U/mL, respectively. The X-08 strain was identified as Anoxybacillus rupiensis and M-17 strain was Geobacillus thermocatenulatus. With double bacteria degradation, the degradation rate of lignin and cellulose was 24.51% and 20.47% respectively after 20 days. The results could provide new bacterial resources for the biodegradation of agricultural waste, and a new idea for the degradation of lignocellulose in straw.

Key words: thermotolerant bacteria, decompose lignocellulose, screening, strain identification, degradation process

中图分类号:

S182

刘心吾, 张威, 马玲玲, 吕欣. 耐高温木质纤维素降解菌株的分离筛选、鉴定及降解工艺的研究[J]. 中国农学通报, 2020, 36(21): 118-125.

Liu Xinwu, Zhang Wei, Ma Lingling, Lv Xin. Lignocellulose Degradation Strains with High-temperature Resistance: Isolation, Screening, Identification and Degradation Process[J]. Chinese Agricultural Science Bulletin, 2020, 36(21): 118-125.

图/表 10

试验组	接种菌株	降解处理天数/d
A1	X-08	4
A2	X-08	8
A3	X-08	12
A4	X-08	16
A5	X-08	20
B1	M-17	4
B2	M-17	8
B3	M-17	12
B4	M-17	16
B5	M-17	20
C1	M-17降解处理10天后接种菌X-08	20
C2	X-08降解处理10天后接种菌M-17	20
D1	未灭菌未接种菌株	20
D2	灭菌后未接种菌株	20

表1. 菌株X-08和M-17降解秸秆内木质纤维素的分组方案

图1. 菌株X-08和M-17在选择培养基上的透明圈

菌株	菌落边缘至透明圈边缘距离/mm	菌株	菌落边缘至透明圈边缘距离/mm
M-4	13	M-13	15
X-010	16	X-06	13
M-3	23	M-17	30
M-011	10	X-5	22
M-09	20	M-023	19
X-08	26	X-015	21
X-8	18	M-14	24
M-027	11

表2. 透明圈直径测定结果

菌株	酶活力/(U/mL)	菌株	酶活力/(U/mL)
X-010	0.02106	X-5	0.02523
X-08	0.02872	X-06	0.01966
X-015	0.02663	X-8	0.02315

表3. 纤维素分解菌的纤维素酶活力结果

图2. 耐高温木质素分解菌的产酶活性

图3. 基于菌株X-08和M-17亲缘关系相近菌株的16S rDNA基因序列的邻接系统发育树

菌株编号	菌株种类
X-5	Anoxybacillus geothermalis
X-08	Anoxybacillus rupiensis
X-015	Anoxybacillus rupiensis
M-3	Anoxybacillus rupiensis
M-09	Geobacillus thermocatenulatus
M-14	Anoxybacillus rupiensis
M-17	Geobacillus thermocatenulatus

表4. 筛选出的7株酶活较高的菌株的种类鉴定结果

图4. 单一菌株处理时纤维素降解率随时间的变化图中数据为X-08和M-17分别单一降解玉米秸秆粉末4、8、12、16、20天后,秸秆内纤维素的降解率的平均值和标准误,每组试验重复3次。与对照组比较,*表示差异显著(P<0.05),**表示差异极显著(P<0.01)

图5. 单一菌株处理时木质素降解率随时间的变化图中数据为X-08和M-17分别单一降解玉米秸秆粉末4、8、12、16、20天后,秸秆内木质素的降解率的平均值和标准误,每组试验重复3次。与对照组比较,*表示差异显著(P<0.05),**表示差异极显著(P<0.01)

图6. 菌株接种方式对木质素和纤维素降解率的影响图中数据为不同的处理方式降解玉米秸秆粉末20天后,秸秆内木质素和纤维素降解率的平均值和标准误,每组试验重复3次。与对照组相比较,*表示差异显著(P<0.05),**表示差异极显著(P<0.01)

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