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浙江大学学报（农业与生命科学版）
Journal of Zhejiang University (Agriculture and Life Sciences)  2022, Vol. 48 Issue (5): 614-624    DOI: 10.3785/j.issn.1008-9209.2021.10.211
Resource utilization & environmental protection     
Screening of cellulose-degrading fungus Trichoderma longibrachiatum ZJ-10 and optimization of enzyme production conditions
Zijing LI(),Fan LIU,Sheng TANG,Qingxu MA,Kefeng HAN(),Lianghuan WU()
Zhejiang Provincial Key Laboratory of Agricultural Resources and Environment, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
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Abstract  

To improve the degradation efficiency of cellulose distributed in livestock, poultry wastes and garden wastes, a high-efficiency cellulose-degrading fungus which was from bamboo shavings, dead branches, and rotted leaves, and sheep dung was screened with Congo red staining, filter paper disintegration test and the endoglucanase [carboxyl methyl cellulose (CMC)] activity was tested. The physiological and molecular identification of the strain was carried out. The results showed that a high-efficiency cellulose-degrading fungus was screened in this study, which was identified as Trichoderma longibrachiatum by morphological observation and fungus species identification, and named as T. longibrachiatum ZJ-10. Single factor test showed that the conditions for achieving the maximum enzyme production activity showed as follows: 3% inoculation, initial pH 6.5, rotation speed of 160 r/min, 40 ℃, and cultured for 5 d. According to Plackett-Burman experimental design, Box-Benhnken steepest climbing path method and response surface methodology, the optimal enzyme production medium formula was 5 g/L NaCl, 7 g/L peptone, and 12 g/L CMC-Na. Under the optimal conditions, the CMC enzyme activity of strain ZJ-10 could reach 80.32 U/mL, which was 26.45% higher than that of the former optimization. In conclusion, strain of T. longibrachiatum ZJ-10 with strong CMC enzyme activity was screened in this study, which provides a good strain resource for the utilization of livestock, poultry and garden waste resources.

Key words： Trichoderma      cellulose      fungi      optimization of conditions      response surface methodology     
Received: 21 October 2021      Published: 02 November 2022
CLC:  X 713  
Corresponding Authors: Kefeng HAN,Lianghuan WU     E-mail: lizijing@zju.edu.cn;hkf1982@163.com;finm@zju.edu.cn
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Zijing LI
Fan LIU
Sheng TANG
Qingxu MA
Kefeng HAN
Lianghuan WU
Cite this article:

Zijing LI,Fan LIU,Sheng TANG,Qingxu MA,Kefeng HAN,Lianghuan WU. Screening of cellulose-degrading fungus Trichoderma longibrachiatum ZJ-10 and optimization of enzyme production conditions. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(5): 614-624.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.10.211     OR     https://www.zjujournals.com/agr/Y2022/V48/I5/614


纤维素降解菌长枝木霉菌（Trichoderma longibrachiatum）ZJ-10的筛选及产酶条件优化

为解决畜禽废弃物及园林废弃物中纤维素降解难的问题，本试验采用刚果红染色法、滤纸崩解试验和测定内切葡聚糖酶（羧甲基纤维素酶）活力法从竹屑、枯枝烂叶、羊粪等废弃物中筛选高效的纤维素降解真菌，并对该真菌进行分子生物学鉴定。结果表明，本研究筛选到1株高效纤维素降解真菌，经形态学观察和菌种鉴定确定其为长枝木霉菌（Trichoderma longibrachiatum），并命名为T. longibrachiatum ZJ-10。单因素试验得出菌株ZJ-10最适产酶培养条件为接种量3%，初始pH 6.5，转速160 r/min，温度40 ℃，培养5 d，此时达到最大产酶活力；通过Plackett-Burman试验设计、Box-Benhnken最陡爬坡路径方法及响应面法得出菌株ZJ-10最适产酶培养基配方为NaCl 5 g/L、蛋白胨7 g/L、羧甲基纤维素钠12 g/L。在最适条件下测定的菌株ZJ-10的酶活力可达到80.32 U/mL，比优化前提高了26.45%。综上所述，本研究筛选出1株产内切葡聚糖酶活力较强的木霉真菌T. longibrachiatum ZJ-10，为畜禽及园林废弃物资源化利用提供了良好的菌种资源。


关键词： 木霉菌属,  纤维素,  真菌,  条件优化,  响应面法 

菌株名称

Strain name

水解能力（H/C）

Hydrolysis capacity (H/C)

滤纸崩解情况Disintegration of filter paper

滤纸分解率

Decomposition rate of filter paper/%

ZJ-11.71±0.01+++4.44±0.31
ZJ-21.26±0.03++1.63±0.08
ZJ-31.71±0.05+++5.49±0.30
ZJ-41.34±0.02+1.05±0.16
ZJ-51.33±0.04++1.75±0.10
ZJ-61.33±0.12++1.71±0.15
ZJ-71.85±0.02++++14.82±0.52
ZJ-81.55±0.03+1.28±0.06
ZJ-91.63±0.06+++6.42±0.13
ZJ-101.95±0.03+++++23.58±0.80
ZJ-111.37±0.03+1.22±0.12
ZJ-121.32±0.02+1.30±0.20
ZJ-131.56±0.03+++6.88±0.20
ZJ-141.36±0.03+1.44±0.18
ZJ-151.89±0.02+++++19.77±0.63
ZJ-161.36±0.05+1.24±0.35
ZJ-171.82±0.02++++11.98±0.43
Table 1 Hydrolysis capacities of different strains, disintegration of filter paper and decomposition rate
Fig. 1 Effects of different incubation times on the enzyme production of cellulose-degrading fungi
Fig. 2 Phylogenetic tree based on strain ZJ-10 and the related rDNA ITS sequence
Fig. 3 Effects of inoculation amount (A), initial pH (B), rotation speed (C), and temperature (D) on the enzyme production of strain ZJ-10

处理

Treatment

X1X2X3X4X5X6X7

CMC酶活力

CMC enzyme activity/(U/mL)

11－1－1－111－164.60
2111－1－11158.85
3－11－11－1－1151.63
41－11－1－1－1169.15
511111－1－163.60
611－11－11－158.94
7－111－11－1－169.05
8－1－11111167.96
9－1－111－11－175.43
101－1－111－1154.90
11－11－1－111156.53
12－1－1－1－1－1－1－165.18
效应 Effect－2.62－6.448.71－1.82－0.421.47－6.30
系数 Coefficient－1.31－3.224.36－0.91－0.210.73－3.15
T－1.87－4.606.23－1.30－0.301.04－4.50
P0.132 80.009 9**0.003 3**0.262 20.776 40.351 80.010 7*
R2 (R2Adj)0.956 2 (0.879 5)
Table 2 Plackett-Burman experimental design and evaluation of the response of various factors

试验号

Test No.

NaCl/(g/L)(A)

蛋白胨

Peptone/(g/L) (B)

CMC-Na/(g/L)(C)

CMC酶活力

CMC enzyme activity/(U/mL)

123861.16
234967.36
3451071.94
4561174.17
5671272.03
6781369.19
Table 3 Determination of optimum concentration ranges of NaCl, peptone and CMC-Na
试验号Test No.ABC

CMC酶活力

CMC enzyme activity/(U/mL)

方差分析 Analysis of variation

参量

Parameter

平方和

Sum of squares

F值

F value

P值

P value

1461062.31A53.8710.100.015 5*
2661066.31B137.6125.810.001 4**
3561172.20C220.9241.430.000 4**
4551057.29AB8.701.630.242 1
5571278.54AC4.280.800.399 8
6661272.85BC1.490.280.613 6
7561175.31A240.927.680.027 7*
8471164.21B282.5415.480.005 6**
9561174.12C214.372.700.144 6
10451159.41模型 Model578.5812.060.001 7**
11671175.61误差 Error37.32
12561174.29失拟项 Lack of fit29.074.700.084 6
13571064.91纯误差 Pure error8.25
14551268.48总和 Sum615.90
15461272.99R20.939 4
16651164.91R2Adj0.861 5
17561171.98
Table 4 Results and analysis of Box-Benhnken experiment
Fig. 4 Response surface diagrams of the effects of NaCl, peptone and CMC-Na on CMC enzyme activity
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