竹伐桩促腐微生物的筛选及培养条件的研究
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摘要
近来竹资源的大力开发造成林地留下大量伐桩、同时竹木质纤维素原料利用率不高造成资源浪费、以及环境污染问题,高效竹木质纤维素降解菌的筛选及其降解能力的研究必将是竹伐桩促腐工作、竹木质纤维素原料高效利用的关键。本论文主要分为以下几个部分：
1.竹伐桩促腐微生物的分离筛选
采用直接稀释法和富集分离法对竹腐伐桩的样品中的微生物进行分离纯化,利用纤维素底物或木质素底物类似物的平板法对菌株进行初筛,采用菌株纤维素酶活、水溶性木质素降解率和漆酶酶活测定的方法进行复筛。共分离筛选15株微生物菌株,包括真菌菌株F2、F3、F4、F9、F10、F13、F14,放线菌菌株A1-A3和细菌菌株B1-B5。
2.菌株降解性能研究及菌种鉴定
采用固态竹屑培养基,测定了复筛菌株培养15 d时对竹纤维素和竹木质素的降解率,其中真菌菌株F2和F1O对竹纤维的降解率分别为23.96%和24.31%,均高于参照菌株绿色木霉YJ-3的19.59%；对竹木质素的降解率分别为16.92%和19.15%,均高于参照菌株黄孢原毛平革菌ME-446的16.53%。生长情况较好的细菌菌株均有一定降解能力。
为挑选有效菌株,对4株产芽孢细菌进行了16S rDNA序列测定和部分生理生化实验,根据结果初步鉴定B2-B5分别为Bacillus. sp、B. simplex、B. sphaericus、B. subtilis。对真菌菌株F2和F10进行了形态学观察,初步将F2鉴定为Trichoderma.sp,将F10鉴定为Fusarium oxysporum。结合F2的rDNA ITS序列分析将F2初步鉴定为Trichoderma harzianum。除F. oxysporum为常见植物病原菌外。其余菌株均为对植物生长有益或有抗植病能力的微生物。
为获得有效的混合菌种,首先对菌株B2-B5进行了拮抗实验,发现B5对B2-B4均有拮抗作用,B2对B3有拮抗作用。据此设计了F2+B2、F2+B4、F2+B5、F2+B3+B4和F2+B2+B4五种菌种组合,测定了混合菌株培养15 d时对竹纤维素和竹木质素的降解率,组合F2+B4和F2+B2+B4对竹纤维素降解率分别为26.64%,28.85%,对竹木质素降解率分别为21.13%,20.64%,均比F2单独培养效果好。
3.木霉F2产孢条件研究
木霉制剂一般以孢子形式应用。实验发现木霉F2在固态竹屑培养基中的最高孢子生物量远远高于PDA液态培养基。利用琼脂平板法进行的碳氮源选择实验确定以竹屑、麸皮、硫酸铵、水为固态培养基的成分。在此基础上的单因子实验确定固态竹屑培养基中竹屑与麸皮比例7：3、硫酸铵添加量0.4%、含水量75%、孢子最终接种浓度l×106spore/mL为木霉F2最佳产孢条件,在此条件下培养7 d其孢子生物量最高可达6.89x 1012spore/g。
Development and utilization of Bamboo Resources and lower material utilization rate have deteriorated the problem of bamboo stake and environmental pollution. The screening of microorganisms for degradation of lignocellulosic materials and research work of useful strains' degradation capability are the key factor for fast rotten technology on bamboo stake and bamboo material treatment. This pape is organized as follow:
1. Isolation and screening of microorganisms for bamboo stakes
we used direct dilution methods and enrichment and separation methods to screen microorganisms from rotted Bamboo stake materials. Initial screen was conducted using cellulose substrate and lignin analogues and secondary screening was conducted using enzyme activity determination and water-soluble lignin degradation rate. As a result,7 fungus strains were selected, that is F2, F3, F4, F9, F10, F13, F14,3 actinomycetes strains were selected,that is A1-A3,and 5 bacteria strains were selected,that is B1-B5.
2. Studies on strain' bamboo lignocelluloses degradation ability and strain identification
Cellulose and lignin degradation ability of screened strains were tested after 15 days' incubation in bamboo chip solid state medium. Results showed that strain F2 and strain F10 were the most efficient decomposer in tested condition.Strain F2 could decompose 23.96% of bamboo cellulose and 16.92% of bamboo lignin and strain F10 could decompose 24.31% of bamboo cellulose and 23.15% of bamboo lignin, the cellulose degradation rates of the two strains were both higher than that of the Trichoderma viride YJ-3,and their lignin degradation rates were both higher than that of the Phanerochaete chrysosporium ME-446.Bacteria strains which have higher biomass in tested condition also showed better degradation ability.
In order to obtain effective strains, we conducted studies on morphological analysis, partial 16S rDNA sequences analysis and physiology and biochemistry for four spore-producing bacterium. Bacteria strain B2, B3, B4 and B5 were respectively preliminary identified as Bacillus.sp, B. simplex, B. sphaericus, B. subtilis. Basd on morphological analysis, strain F2 and F10 were respectively preliminary identified as Trichoderma.sp and Fusarium oxysporum. Combined with the results of rDNA ITS sequences analysis, strain F2 were respectively preliminary identified as Trichoderma Harzianum. Many strains of F. oxysporum caused plant Pathogen; other tested microbial strains usually do well to plant growth or have the ability of antipathogen.
In order to screen the effective composite strains, we conducted antagonistic experiments firstly. According to the antagonistic phenomenon between bacteria strains, we designed several composites, among which the composite F2+B4 and F2+B2+B4 are the most effective decomposer after 15 days incubation in bamboo chip solid state medium. Results showed that, in tested condition, composite F2+B4 could decompose 26.64% of bamboo cellulose and 21.13% of bamboo lignin and strain F10 could decompose 28.85% of bamboo cellulose and 20.64% of bamboo lignin, The efficiency of which were both better single strain's.
3. Studies on the Condition of Sporulation of strain F2
In the third part of the experiment, the results of the design and optimization of sporulati-on medium for strain F2 show that spore biomass of strain F2 was much higher when using bamboo chip as the unique matrix of solid medium than using PDA liquid medium. Bamboo chips, ammonium sulfate and water were chosen as the components of the solid medium by comparison using agar plate method. The experiments selecting the carbon and nitrogen source selection experiments. Singles showed the optimum culture conditions for sporulation are as follows:the medium contains 25%(weight ratio) of the dry carbon matrix whose content of bamboo chip and wheat bran was seven to three,75% of nutrient solution which contains 0.4% of ammonium sulfate, the spore inoculation concentration was 1×106 spores/g after seeding. Under this culture condition, the most highest biomass is 6.89×1012 per gram of medium.
1.竹伐桩促腐微生物的分离筛选
采用直接稀释法和富集分离法对竹腐伐桩的样品中的微生物进行分离纯化,利用纤维素底物或木质素底物类似物的平板法对菌株进行初筛,采用菌株纤维素酶活、水溶性木质素降解率和漆酶酶活测定的方法进行复筛。共分离筛选15株微生物菌株,包括真菌菌株F2、F3、F4、F9、F10、F13、F14,放线菌菌株A1-A3和细菌菌株B1-B5。
2.菌株降解性能研究及菌种鉴定
采用固态竹屑培养基,测定了复筛菌株培养15 d时对竹纤维素和竹木质素的降解率,其中真菌菌株F2和F1O对竹纤维的降解率分别为23.96%和24.31%,均高于参照菌株绿色木霉YJ-3的19.59%；对竹木质素的降解率分别为16.92%和19.15%,均高于参照菌株黄孢原毛平革菌ME-446的16.53%。生长情况较好的细菌菌株均有一定降解能力。
为挑选有效菌株,对4株产芽孢细菌进行了16S rDNA序列测定和部分生理生化实验,根据结果初步鉴定B2-B5分别为Bacillus. sp、B. simplex、B. sphaericus、B. subtilis。对真菌菌株F2和F10进行了形态学观察,初步将F2鉴定为Trichoderma.sp,将F10鉴定为Fusarium oxysporum。结合F2的rDNA ITS序列分析将F2初步鉴定为Trichoderma harzianum。除F. oxysporum为常见植物病原菌外。其余菌株均为对植物生长有益或有抗植病能力的微生物。
为获得有效的混合菌种,首先对菌株B2-B5进行了拮抗实验,发现B5对B2-B4均有拮抗作用,B2对B3有拮抗作用。据此设计了F2+B2、F2+B4、F2+B5、F2+B3+B4和F2+B2+B4五种菌种组合,测定了混合菌株培养15 d时对竹纤维素和竹木质素的降解率,组合F2+B4和F2+B2+B4对竹纤维素降解率分别为26.64%,28.85%,对竹木质素降解率分别为21.13%,20.64%,均比F2单独培养效果好。
3.木霉F2产孢条件研究
木霉制剂一般以孢子形式应用。实验发现木霉F2在固态竹屑培养基中的最高孢子生物量远远高于PDA液态培养基。利用琼脂平板法进行的碳氮源选择实验确定以竹屑、麸皮、硫酸铵、水为固态培养基的成分。在此基础上的单因子实验确定固态竹屑培养基中竹屑与麸皮比例7：3、硫酸铵添加量0.4%、含水量75%、孢子最终接种浓度l×106spore/mL为木霉F2最佳产孢条件,在此条件下培养7 d其孢子生物量最高可达6.89x 1012spore/g。
Development and utilization of Bamboo Resources and lower material utilization rate have deteriorated the problem of bamboo stake and environmental pollution. The screening of microorganisms for degradation of lignocellulosic materials and research work of useful strains' degradation capability are the key factor for fast rotten technology on bamboo stake and bamboo material treatment. This pape is organized as follow:
1. Isolation and screening of microorganisms for bamboo stakes
we used direct dilution methods and enrichment and separation methods to screen microorganisms from rotted Bamboo stake materials. Initial screen was conducted using cellulose substrate and lignin analogues and secondary screening was conducted using enzyme activity determination and water-soluble lignin degradation rate. As a result,7 fungus strains were selected, that is F2, F3, F4, F9, F10, F13, F14,3 actinomycetes strains were selected,that is A1-A3,and 5 bacteria strains were selected,that is B1-B5.
2. Studies on strain' bamboo lignocelluloses degradation ability and strain identification
Cellulose and lignin degradation ability of screened strains were tested after 15 days' incubation in bamboo chip solid state medium. Results showed that strain F2 and strain F10 were the most efficient decomposer in tested condition.Strain F2 could decompose 23.96% of bamboo cellulose and 16.92% of bamboo lignin and strain F10 could decompose 24.31% of bamboo cellulose and 23.15% of bamboo lignin, the cellulose degradation rates of the two strains were both higher than that of the Trichoderma viride YJ-3,and their lignin degradation rates were both higher than that of the Phanerochaete chrysosporium ME-446.Bacteria strains which have higher biomass in tested condition also showed better degradation ability.
In order to obtain effective strains, we conducted studies on morphological analysis, partial 16S rDNA sequences analysis and physiology and biochemistry for four spore-producing bacterium. Bacteria strain B2, B3, B4 and B5 were respectively preliminary identified as Bacillus.sp, B. simplex, B. sphaericus, B. subtilis. Basd on morphological analysis, strain F2 and F10 were respectively preliminary identified as Trichoderma.sp and Fusarium oxysporum. Combined with the results of rDNA ITS sequences analysis, strain F2 were respectively preliminary identified as Trichoderma Harzianum. Many strains of F. oxysporum caused plant Pathogen; other tested microbial strains usually do well to plant growth or have the ability of antipathogen.
In order to screen the effective composite strains, we conducted antagonistic experiments firstly. According to the antagonistic phenomenon between bacteria strains, we designed several composites, among which the composite F2+B4 and F2+B2+B4 are the most effective decomposer after 15 days incubation in bamboo chip solid state medium. Results showed that, in tested condition, composite F2+B4 could decompose 26.64% of bamboo cellulose and 21.13% of bamboo lignin and strain F10 could decompose 28.85% of bamboo cellulose and 20.64% of bamboo lignin, The efficiency of which were both better single strain's.
3. Studies on the Condition of Sporulation of strain F2
In the third part of the experiment, the results of the design and optimization of sporulati-on medium for strain F2 show that spore biomass of strain F2 was much higher when using bamboo chip as the unique matrix of solid medium than using PDA liquid medium. Bamboo chips, ammonium sulfate and water were chosen as the components of the solid medium by comparison using agar plate method. The experiments selecting the carbon and nitrogen source selection experiments. Singles showed the optimum culture conditions for sporulation are as follows:the medium contains 25%(weight ratio) of the dry carbon matrix whose content of bamboo chip and wheat bran was seven to three,75% of nutrient solution which contains 0.4% of ammonium sulfate, the spore inoculation concentration was 1×106 spores/g after seeding. Under this culture condition, the most highest biomass is 6.89×1012 per gram of medium.
引文
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