新疆寒冷地区腐木中产纤维素酶菌株的筛选与低温产酶特性

doi:10.3969/j.issn.1004-1524.2021.08.14

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (8): 1468-1476.DOI: 10.3969/j.issn.1004-1524.2021.08.14

新疆寒冷地区腐木中产纤维素酶菌株的筛选与低温产酶特性

冯欣欣¹(), 李凤兰¹, 徐永清¹, 李磊¹, 贺付蒙¹, 冯艳忠², 袁强¹^,^*(), 刘娣²^,^*()

1.东北农业大学生命科学学院,黑龙江哈尔滨 150030
2.黑龙江省农业科学院,黑龙江哈尔滨 150030

收稿日期:2020-09-23 出版日期:2021-08-25 发布日期:2021-08-27
通讯作者: 袁强,刘娣
作者简介:刘娣,E-mail: liudi1963@163.com
*袁强,E-mail: neauyqiang@sohu.com;
冯欣欣(1996—),女,黑龙江集贤人,硕士研究生,研究方向为生物工程。E-mail: fxin1996@163.com
基金资助:
国家自然基金青年科学基金(31201470);国家科技部科技基础性工作专项(2014FY111000);哈尔滨市应用科技研究与开发项目(2015RAQXJ021)

Screening of cellulase producing strains from rotten wood in Xinjiang cold area and analysis of their characteristics of enzyme production at low temperature

FENG Xinxin¹(), LI Fenglan¹, XU Yongqing¹, LI Lei¹, HE Fumeng¹, FENG Yanzhong², YUAN Qiang¹^,^*(), LIU Di²^,^*()

1. School of Life Sciences, Northeast Agricultural University, Harbin 150030, China
2. Heilongjiang Academy of Agricultural Sciences, Harbin 150030, China

Received:2020-09-23 Online:2021-08-25 Published:2021-08-27
Contact: YUAN Qiang,LIU Di

摘要/Abstract

摘要：

为筛选出能在低温条件下高效降解纤维素的菌株,提高秸秆在低温条件下纤维素的降解速度,以新疆寒冷地区腐木为试验材料,对低温纤维素降解菌进行筛选,在4 ℃条件下筛选得到4株可在低温下生长且具有纤维素降解作用的真菌,通过形态学和分子生物学的方法对低温菌进行鉴定,分别为产黄青霉(Penicillium chrysogenum)、桔绿木霉(Trichoderma citrinoviride)2株、脉纹孢菌(Neurospora sitophila);耐冷试验表明,筛选获得的菌株都为耐冷菌。通过对4株低温菌产酶特性进行研究,结果表明,菌株产纤维素酶的最佳培养时间为9 d,培养基最适初始pH值为7,最佳温度为25 ℃,最佳接种量为5%。秸秆降解试验表明,筛选获得的4株真菌对秸秆具有降解能力,对玉米秸秆降解效果最好,酵解率都在40%以上。

关键词: 纤维素, 降解菌, 低温, 菌株鉴定, 秸秆降解

Abstract:

In order to screen out the strains which can degrade cellulose efficiently at low temperature and improve the degradation rate of cellulose under low temperature, in this study, rotten wood in cold area of Xinjiang was used as experimental material, and the low-temperature cellulose degrading bacteria were screened. Four strains of fungi which could grow at low temperature and degrade cellulose were screened at 4 ℃. Morphological and molecular biological methods were used for identification of low temperature strains. They were Penicillium chrysogenum, two Trichoderma citrinoviride strains and Neurospora sitophila. The cold tolerance test showed that all the strains were cold resistant. Studies on the characteristics of enzyme production of 4 low-temperature bacteria showed that the optimal culture time for cellulase production was 9 d, the optimal initial pH of the culture medium was 7, the optimal temperature was 25 ℃, and the optimal inoculation amount was 5%. The straw degradation test showed that these four strains of fungi had ability to degrade the straw, and had the best degradation effect on the corn straw with the fermentation rate above 40%.

Key words: cellulose, degrading bacteria, low temperature, strain identification, straw degradation

中图分类号:

S662.2

冯欣欣, 李凤兰, 徐永清, 李磊, 贺付蒙, 冯艳忠, 袁强, 刘娣. 新疆寒冷地区腐木中产纤维素酶菌株的筛选与低温产酶特性[J]. 浙江农业学报, 2021, 33(8): 1468-1476.

FENG Xinxin, LI Fenglan, XU Yongqing, LI Lei, HE Fumeng, FENG Yanzhong, YUAN Qiang, LIU Di. Screening of cellulase producing strains from rotten wood in Xinjiang cold area and analysis of their characteristics of enzyme production at low temperature[J]. Acta Agriculturae Zhejiangensis, 2021, 33(8): 1468-1476.

图/表 16

图1 腐烂的云杉树树干

Fig.1 Rotten trunk of spruce trees

表1 低温产纤维素酶菌株的初筛结果

Table 1 Screening results of low temperature cellulase producing strains

菌株编号 No. of strains	培养温度Culture temperature/℃
菌株编号 No. of strains	4	10	15	25	37
F1	+	+	+	+	+
F2	+	+	+	+	+
F3	-	-	+	+	-
F4	-	-	+	+	+
F5	-	+	+	+	+
D1	+	+	+	+	+
D2	-	-	+	+	+
D3	-	-	+	+	+
D4	+	+	+	+	+
D7	-	-	+	+	+
P1	+	+	+	+	+
P2	-		+	+	+
P4	+	+	+	+	+
N3	-	-	+	+	-
N5	+	+	+	+	+
N7	-	-	+	+	-
T2	+	+	+	+	+
T5	-	-	+	+	+
T6	-	-	+	+	+
T7	+	+	+	+	+
T8	+	+	+	+	+

图2 产纤维素酶真菌CMC-刚果红培养基上产纤维素菌的透明圈 A,P1;B,T2 ;C,N5;D,T8.

Fig.2 Transparent circle of cellulose producing fungi on CMC-Congo red medium

图3 产纤维素真菌的菌落形态 A,P1;B,T2 ;C,N5;D,T8.

Fig.3 Colony morphology of the cellulose producing fungi

图4 产纤维素真菌的显微形态 A,P1;B,T2;C,N5;D,T8.

Fig.4 Microscopic morphology of the cellulose producing fungi

图5 菌株P1基于18S rDNA序列同源性构建的系统发育树

Fig.5 Phylogenetic tree of strain P1 based on 18S rDNA sequences homology

图6 菌株T2基于18S rDNA序列同源性构建的系统发育树

Fig.6 Phylogenetic tree of strain T2 based on 18S rDNA sequences homology

图7 菌株T8基于18S rDNA序列同源性构建的系统发育树

Fig.7 Phylogenetic tree of strain T8 based on 18S rDNA sequences homology

图8 菌株N5基于18S rDNA序列同源性构建的系统发育树

Fig.8 Phylogenetic tree of strain N5 based on 18S rDNA sequences homology

图9 不同氮源对4菌株产酶的影响不同小写字母表示处理间差异显著(P<0.05)。下同。

Fig.9 Effect of differernt nitrogen fountains on cellulase production of four strains The different lowercase letters indicate significant differences among treatments (P<0.05). The same as below.

图10 不同接种量对4菌株产酶的影响

Fig.10 Effect of different inoculum on cellulase production of four strains

图11 不同温度对4菌株产酶的影响

Fig.11 Effect of different temperature on cellulase production of four strains

图12 不同培养基初始pH对4菌株产酶的影响

Fig.12 Effect of initial pH on cellulase production of four strains

图13 不同培养时间对4菌株产酶的影响

Fig.13 Effect of incubating time on cellulase production of four strains

图14 四种真菌菌株酵解玉米秸秆的失重率

Fig.14 Weight loss rate of corn straw fermented by four fungal strains

图15 四种真菌菌株酵解不同秸秆的失重率

Fig.15 Weight loss rate of different straws fermented by four fungal strains

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新疆寒冷地区腐木中产纤维素酶菌株的筛选与低温产酶特性

Screening of cellulase producing strains from rotten wood in Xinjiang cold area and analysis of their characteristics of enzyme production at low temperature

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