• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

纤维素降解复合菌群的筛选构建及对水稻秸秆的降解效能

Consortia of Cellulose-degrading Bacteria for Rice Straw Composing

  • 摘要:
    目的 获得降解水稻秸秆纤维素效能较高的复合细菌群,为水稻秸秆降解复合菌剂的研发提供理论基础和技术参考。
    方法 从11种生境中采集了样品,采用羧甲基纤维素钠培养基和刚果红染色法初筛菌株;基于各菌株的羧甲基纤维素酶(Carboxymethyl Cellulosease, CMCase)、滤纸酶(Filter Paper Activity, FPA)、β-葡萄糖苷酶(β - Glucosidase, β-Gase)和葡聚糖酶外切酶(Exoglucanase, C1)等纤维素降解酶的活性并结合其对滤纸的崩解结果复筛优势单菌株,利用革兰氏染色和16S rDNA 基因序列测定等方法鉴定优势单菌株类型;依据拮抗实验和纤维素酶活测定的结果,构建和确定优势复合菌群;基于单因素实验和响应面优化设计,探明优势复合菌群的最适产酶条件;探讨优势单菌株和复合菌群对水稻秸秆的降解效能。
    结果 (1)初筛了44株细菌,复筛了4株优势纤维素降解菌JBN1、CY4-2、YF9和H17,其CMCase活性分别为30.58、29.40 、26.81、12.30 U·mL−1;(2)H17为革兰氏阴性菌且为新发现的护岸植物红树杆菌(Mangrovibacter plantisponsor);而JBN1、CY4-2和YF9均为革兰氏阳性菌,其分别为枯草芽孢杆菌(Bacillus subtilis)、粪便芽孢杆菌(B. stercori)和贝莱斯芽孢杆菌(B. velezensis);(3)复合菌群JC(JBN1 + CY4-2)和JYCH(CY4-2 + JBN1 + YF9 + H17)的产酶活力较高;(4)JC在培养时间28 h、培养温度34 ℃ 和初始pH 6.0时的 CMCase平均活性为53.80 U·mL−1;JYCH在培养时间26 h、培养温度41 ℃ 和初始pH 5.5条件下的CMCase平均活性为48.18 U·mL−1;(5)JC和JYCH对水稻秸秆的降解率分别为45.47%和37.64%,均显著高于各自的单一菌株。
    结论 复合菌群JC的产酶活性和对秸秆的降解率更高,而JYCH更耐高温和酸性环境,展现了在不同环境下降解水稻秸秆的应用价值。

     

    Abstract:
    Objective Bacteria with high efficiency cellulase for degrading rice straws were collected to improve the farm waste utilization under varied conditions.
    Method Soil samples were collected from 11 habitats to preliminarily screen for candidate microbes using carboxymethyl cellulose sodium agar plates and Congo red staining. Based on the activities of cellulose-degrading enzymes, such as carboxymethyl cellulase (CMCase), filter paper enzyme (FPA), β-glucosidase (β-Gase), and exoglucanase (C1), as well as the filter paper-disintegration, superior strains were identified using Gram staining and 16S rDNA gene sequence analysis. Composite consortia of the selected microorganisms were formed by evaluations based on the results obtained from antagonism experiments and cellulase activity assays. Single variable and response surface experiments were conducted to optimize the conditions of producing the target enzymes. Cellulose-degrading efficiency on rice straws of the selected microbes were compared.
    Result (1) Forty-four strains were selected from the initially screening. Four superior performing microbes, i.e., JBN1, CY4-2, YF9, and H17, were tested to show CMCase activities of 30.58 U·mL−1, 29.40 U·mL−1, 26.81 U·mL−1, and 12.30 U·mL−1, respectively. (2) A Gram-negative bacterium, H17, was identified as a newly discovered species of Mangrovibacter plantisponsor, while JBN1, CY4-2, and YF9 were Gram-positive Bacillus subtilis, B. stercori, and B. velezensis, respectively. (3) Composite JC (JBN1 + CY4-2) and JYCH (CY4-2 + JBN1 + YF9 + H17) consortia delivered higher enzyme producing activities than the others. (4) The JC consortium yielded an average CMCase activity of 53.80 U·mL−1 under the optimal conditions at 34°C and initial pH6.0 for 28h, and JYCH of 48.18 U·mL−1 at 41°C and pH5.5 for 26h. (5) The rice straw degradation rates by JC and JYCH were 45.47% and 37.63%, respectively. Both were significantly higher than those of the consortia composed of only one individual strain.
    Conclusion Composite JC consortium displayed higher enzyme producing capability and rice straw degrading activity, whereas JYCH was more tolerant to high temperature and acidic conditions than the others. These composite consortia of cellulose-degrading bacteria appeared to be applicable for composing the waste material under varied conditions.

     

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