兼性厌氧纤维素酶产生菌的筛选及在沼气发酵中的应用
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摘要
随着社会发展和世界人口不断增长,人类面临越来越严重的环境危机与能源危机,纤维素是地球上最丰富可再生资源,我国每年农作物秸秆除部分还田外,有相当大的部分被废弃、焚烧,造成严重的环境污染。秸秆厌氧发酵生产沼气是利用秸秆中储存的丰富生物质能的简便途径,在秸秆发酵生产沼气过程中接种厌氧纤维素酶产生菌能够提高秸秆利用率和沼气产量、质量。
本论文通过采用刚果红双层平板初筛和厌氧发酵测定酶活复筛,筛选出一株兼性厌氧纤维素酶产生菌,通过紫外线和亚硝酸单独和复合诱变选育高产纤维素酶突变株,通过单因子和正交实验确定菌株最优发酵培养基和发酵产酶条件,并对菌株所产纤维素酶的组分和酶学性质进行了初步研究。对秸秆发酵生产沼气工艺参数进行了初步优化,并将所筛选的纤维单胞菌CR-14和本研究室保藏的产酸菌B2、氨化菌J5单独和复合应用在秸秆发酵沼气生产中。
1.通过双层刚果红平板从牛瘤胃内容物、污水处理厂处理物、荷花池底淤泥分别分离出4株、34株和22株能产生透明圈的菌株,通过厌氧发酵测定酶活复筛出一株酶活较高的兼性厌氧纤维素酶产生菌。根据菌株形态特征、生理生化特征和16SrDNA同源性和系统进化树分析鉴定该菌株属于Celloulonmonas sp.,命名为CR-14。
2.利用紫外线和亚硝酸进行单独和复合诱变共选育出同原始菌株的透明圈直径和菌落直径比大的突变株87株,其中亚硝酸诱变株27株,紫外诱变株42株,复合诱变株18株。通过厌氧管发酵测定酶活复筛,有6株突变株与出发菌株比差异显著,分别为Y-11、UV-17、UV-28、UV-35、UV-Y-9、UV-Y-16,其中复合诱变株UV-Y-9酶活最高为出发菌株的1.81倍,CX酶活为11.3 U,连续传代8次后,遗传性稳定。
3.菌株CR-14最优发酵培养基:秸秆粉1.0%,MgSO4 0.1%,蛋白胨0.3%,豆饼粉0.3%,KH2PO4 0.1%,NaCl 0.08%。菌株CR-14发酵条件研究表明:培养基初始pH值7.0,30℃条件下发酵3 d发酵液酶活最高;在兼性厌氧条件下,通气量对CR-14菌株的产酶影响不显著,在专性厌氧条件下同样的发酵时间发酵液酶活明显低于有氧条件,为250 mL三角瓶装量150 mL发酵液酶活的62.9%;表面活性剂吐温80对菌株CR-14产酶没有影响。
4.菌株CR-14所产纤维素酶中主要组分酶包括C1酶、CX酶和β-葡萄糖苷酶,CX酶酶活最高,而C1酶和β-葡萄糖苷酶酶活较低。在50℃,pH 6.0的条件下酶活最高的,为该酶的最适反应条件。该酶在pH 6.0-7.5条件下稳定,能保留原活力的80%以上,在60℃以上热稳定性极差,60℃条件下保温1h,酶活仅为40℃条件下保温1 h酶活力的13%。因此,该酶应保存在pH 7.0,50℃以下的条件下。Mn2+、Fe2+能显著促进该酶的活力,Zn2+、Cu2+、Ca2+、K+则抑制了该酶的活力。
5.农业废弃物牛粪、秸秆、菌糠中,秸秆的产气潜力最大,秸秆组甲烷含量与牛粪组差异不显著,但明显高于各菌糠实验组的,从产气量和甲烷含量综合考虑,秸秆是一种较好的沼气发酵原料。通过单因子实验确定了秸秆发酵生产沼气最佳有机负荷率为45 g/L,发酵原料的碳氮比为25/1。在沼气发酵过程中单独接种纤维单胞菌CR-14和接种CR-14、产酸菌B2、氨化菌J5协同发酵都能显著提高沼气产量和质量。
With the development of society and growing of population, mankind is facing more and more serious environmental crisis and energy crisis. Cellulose is the most abundant renewable resources on the earth. In our country, most of crop culm was burned and abandoned except few reused to farmland so that it caused serious environmental pollution. Anaerobic fermentation is convenient way to use bioenergy which is stored in crop culm.Inoculate anaerobic cellulase-producing bacteria could enhance the utilization rate of crops culm, production and quality of biogas.
In this thesis, the congo red double layer flat-plate method was used to screen cellulase-producing bacteria preliminary, and secondary screening by determination enzyme activity of anaerobic fermentation broth. Selected high-yielding cellulase mutant by separate or compound mutation with UV and nitrous acid (NA), determined the optimal fermentation medium and fermentation conditions of producing cellulase by single factor and orthogonal experiment, studied the composition and enzyme nature of cellulase produced by stain. Optimized the anaerobic fermentation conditions of producing biogas with crops culm. Applicated separately Cellulomonas sp. CR-14, acidogenic bacteria B2 and ammonification bacteria or mixture of them in producing biogas with crops culm fermentation.
1. Respectively isolated 4, 34 and 22 strains which could produce transparent circle from cattle rumen contents, sludge of sewage treatment plant and lotus pool. Secondary screened a higher cellulose-producing facultative anaerobic strain by determining cellulase activity of fermentation broth.According to morphological characteristics, physiological and biochemical characteristics and 16SrDNA homology and phylogenetic tree analysis , identified it belonged to Celloulonmonas sp.,named it CR-14.
2.Selected 87 mutant strains which ratio of transparent circle and colony diamate larger than original strains with UV and NA mutagenesis including 27 NA mutant, 42 UV mutant, 18 compound mutant. Secondary srceened 6 mutants which cellulase-producing are significantly different from wild strain by determining cellulase activity of anaerobic fermentation broth. They separately are Y-11, UV-17, UV-28, UV-35, UV-Y-9, UV-Y-16, the cellulase-producing activity of compound mutant UV-Y-9 is the highest, Cx activity was 11.3 U, serial passage 8 times, the genetic stability.
3.The optimum fermentation medium of strain CR-14 were composed of 1.0% straw powder, 0.1% MgSO4, 0.3% nitrogen,0.3% soybean powder, 0.1% KH2PO4, 0.08% NaCl.The optimal fermentation conditions for strain CR-14 were that the initial pH value of fermentation medium 8.0,ferment 3 days under the conditions of 30℃. In facultative anaerobic conditions, the ventilation did not significantly affect the cellulase production of the CR-14 strains. However, in anaerobic conditions the cellulase activity was significantly lower than at the same fermentation time. The cellulase activity of anaerobic broth was 62.9% of aerobic fermentation. Surfactant Tween 80 did not affect enzyme production of strains CR-14.
4.The main components of cellulase of strain CR-14 was CX, C1 andβ-glucosidase. The activity of Cx was higher, while activity of C1 andβ-glucosidase was lower. The optimal enzyme reaction conditions was 50℃, pH 6.0. Under the conditions of pH6.0-7.5 cellulase was stability, preserved the vitality of the original 80%. Thermal stability was poor at the condition of more than 60℃,heated 1h at 60℃, the activity was only 13% of activity which heated 1h at 40℃. Mn2+, Fe2+ could significantly promote the vitality of the enzyme, however ,Zn2+, Cu2+, Ca2+, K+ would inhibit the vitality of the enzyme.
5.Considered that biogas production, methane content of biogas, biogasgas production rate during biogas fermentation.Organic loading rate 45g/L was better at the initial of fermentation.Fed-batch should be carried out in the late in order to ensure the quality and production of biogas,organic loading rate should be no more than 60g/L after feding;Regulate carbon-nitrogen ratio to 25/1 was better, that was, 2% of the total solid content.Added CR-14 strain can significantly enhance the production of methane fermentation;However, add acid bacteria B5 inhibite fermentation and reduced biogas production; Add J5 strain did not significantly enhance biogas production but can buffer the pH value of fermentation broth.
本论文通过采用刚果红双层平板初筛和厌氧发酵测定酶活复筛,筛选出一株兼性厌氧纤维素酶产生菌,通过紫外线和亚硝酸单独和复合诱变选育高产纤维素酶突变株,通过单因子和正交实验确定菌株最优发酵培养基和发酵产酶条件,并对菌株所产纤维素酶的组分和酶学性质进行了初步研究。对秸秆发酵生产沼气工艺参数进行了初步优化,并将所筛选的纤维单胞菌CR-14和本研究室保藏的产酸菌B2、氨化菌J5单独和复合应用在秸秆发酵沼气生产中。
1.通过双层刚果红平板从牛瘤胃内容物、污水处理厂处理物、荷花池底淤泥分别分离出4株、34株和22株能产生透明圈的菌株,通过厌氧发酵测定酶活复筛出一株酶活较高的兼性厌氧纤维素酶产生菌。根据菌株形态特征、生理生化特征和16SrDNA同源性和系统进化树分析鉴定该菌株属于Celloulonmonas sp.,命名为CR-14。
2.利用紫外线和亚硝酸进行单独和复合诱变共选育出同原始菌株的透明圈直径和菌落直径比大的突变株87株,其中亚硝酸诱变株27株,紫外诱变株42株,复合诱变株18株。通过厌氧管发酵测定酶活复筛,有6株突变株与出发菌株比差异显著,分别为Y-11、UV-17、UV-28、UV-35、UV-Y-9、UV-Y-16,其中复合诱变株UV-Y-9酶活最高为出发菌株的1.81倍,CX酶活为11.3 U,连续传代8次后,遗传性稳定。
3.菌株CR-14最优发酵培养基:秸秆粉1.0%,MgSO4 0.1%,蛋白胨0.3%,豆饼粉0.3%,KH2PO4 0.1%,NaCl 0.08%。菌株CR-14发酵条件研究表明:培养基初始pH值7.0,30℃条件下发酵3 d发酵液酶活最高;在兼性厌氧条件下,通气量对CR-14菌株的产酶影响不显著,在专性厌氧条件下同样的发酵时间发酵液酶活明显低于有氧条件,为250 mL三角瓶装量150 mL发酵液酶活的62.9%;表面活性剂吐温80对菌株CR-14产酶没有影响。
4.菌株CR-14所产纤维素酶中主要组分酶包括C1酶、CX酶和β-葡萄糖苷酶,CX酶酶活最高,而C1酶和β-葡萄糖苷酶酶活较低。在50℃,pH 6.0的条件下酶活最高的,为该酶的最适反应条件。该酶在pH 6.0-7.5条件下稳定,能保留原活力的80%以上,在60℃以上热稳定性极差,60℃条件下保温1h,酶活仅为40℃条件下保温1 h酶活力的13%。因此,该酶应保存在pH 7.0,50℃以下的条件下。Mn2+、Fe2+能显著促进该酶的活力,Zn2+、Cu2+、Ca2+、K+则抑制了该酶的活力。
5.农业废弃物牛粪、秸秆、菌糠中,秸秆的产气潜力最大,秸秆组甲烷含量与牛粪组差异不显著,但明显高于各菌糠实验组的,从产气量和甲烷含量综合考虑,秸秆是一种较好的沼气发酵原料。通过单因子实验确定了秸秆发酵生产沼气最佳有机负荷率为45 g/L,发酵原料的碳氮比为25/1。在沼气发酵过程中单独接种纤维单胞菌CR-14和接种CR-14、产酸菌B2、氨化菌J5协同发酵都能显著提高沼气产量和质量。
With the development of society and growing of population, mankind is facing more and more serious environmental crisis and energy crisis. Cellulose is the most abundant renewable resources on the earth. In our country, most of crop culm was burned and abandoned except few reused to farmland so that it caused serious environmental pollution. Anaerobic fermentation is convenient way to use bioenergy which is stored in crop culm.Inoculate anaerobic cellulase-producing bacteria could enhance the utilization rate of crops culm, production and quality of biogas.
In this thesis, the congo red double layer flat-plate method was used to screen cellulase-producing bacteria preliminary, and secondary screening by determination enzyme activity of anaerobic fermentation broth. Selected high-yielding cellulase mutant by separate or compound mutation with UV and nitrous acid (NA), determined the optimal fermentation medium and fermentation conditions of producing cellulase by single factor and orthogonal experiment, studied the composition and enzyme nature of cellulase produced by stain. Optimized the anaerobic fermentation conditions of producing biogas with crops culm. Applicated separately Cellulomonas sp. CR-14, acidogenic bacteria B2 and ammonification bacteria or mixture of them in producing biogas with crops culm fermentation.
1. Respectively isolated 4, 34 and 22 strains which could produce transparent circle from cattle rumen contents, sludge of sewage treatment plant and lotus pool. Secondary screened a higher cellulose-producing facultative anaerobic strain by determining cellulase activity of fermentation broth.According to morphological characteristics, physiological and biochemical characteristics and 16SrDNA homology and phylogenetic tree analysis , identified it belonged to Celloulonmonas sp.,named it CR-14.
2.Selected 87 mutant strains which ratio of transparent circle and colony diamate larger than original strains with UV and NA mutagenesis including 27 NA mutant, 42 UV mutant, 18 compound mutant. Secondary srceened 6 mutants which cellulase-producing are significantly different from wild strain by determining cellulase activity of anaerobic fermentation broth. They separately are Y-11, UV-17, UV-28, UV-35, UV-Y-9, UV-Y-16, the cellulase-producing activity of compound mutant UV-Y-9 is the highest, Cx activity was 11.3 U, serial passage 8 times, the genetic stability.
3.The optimum fermentation medium of strain CR-14 were composed of 1.0% straw powder, 0.1% MgSO4, 0.3% nitrogen,0.3% soybean powder, 0.1% KH2PO4, 0.08% NaCl.The optimal fermentation conditions for strain CR-14 were that the initial pH value of fermentation medium 8.0,ferment 3 days under the conditions of 30℃. In facultative anaerobic conditions, the ventilation did not significantly affect the cellulase production of the CR-14 strains. However, in anaerobic conditions the cellulase activity was significantly lower than at the same fermentation time. The cellulase activity of anaerobic broth was 62.9% of aerobic fermentation. Surfactant Tween 80 did not affect enzyme production of strains CR-14.
4.The main components of cellulase of strain CR-14 was CX, C1 andβ-glucosidase. The activity of Cx was higher, while activity of C1 andβ-glucosidase was lower. The optimal enzyme reaction conditions was 50℃, pH 6.0. Under the conditions of pH6.0-7.5 cellulase was stability, preserved the vitality of the original 80%. Thermal stability was poor at the condition of more than 60℃,heated 1h at 60℃, the activity was only 13% of activity which heated 1h at 40℃. Mn2+, Fe2+ could significantly promote the vitality of the enzyme, however ,Zn2+, Cu2+, Ca2+, K+ would inhibit the vitality of the enzyme.
5.Considered that biogas production, methane content of biogas, biogasgas production rate during biogas fermentation.Organic loading rate 45g/L was better at the initial of fermentation.Fed-batch should be carried out in the late in order to ensure the quality and production of biogas,organic loading rate should be no more than 60g/L after feding;Regulate carbon-nitrogen ratio to 25/1 was better, that was, 2% of the total solid content.Added CR-14 strain can significantly enhance the production of methane fermentation;However, add acid bacteria B5 inhibite fermentation and reduced biogas production; Add J5 strain did not significantly enhance biogas production but can buffer the pH value of fermentation broth.
引文
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