荒漠草原地区生物结皮的微生物群落与产漆酶细菌分离纯化
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摘要
为了解生物结皮的微生物组成及影响碳素循环的微生物,文中在采用高通量技术研究西夏王陵生物结皮的微生物群落结构的基础上,采用选择培养基从生物结皮中分离产漆酶的微生物。研究结果表明:细菌的多样性显著高于真菌;变形菌门、酸杆菌门、蓝细菌门、放线菌门、拟杆菌门是绝对优势的细菌门类,子囊菌门和担子菌门是绝对优势的真菌门类;微鞘藻属、藓杆菌属、鞘氨醇单胞菌属、甲基杆菌属在细菌中占比较大的属,链格孢属、菌刺孢属、热子囊菌属、亚脐菇属等是主要的真菌,这些类群在稳定生物结皮结构、碳氮循环、有机物代谢等方面发挥了重要作用。以愈创木酚为唯一碳源从生物结皮中获得能利用愈创木酚的细菌10株,基于丁香醛联氮氧化的颜色反应,筛选出产漆酶能力最强的菌株C01和C06,基于16S rDNA序列的分子鉴定表明,两菌株均为芽孢杆菌。
High-throughput technology was employed to explicit the bacterial and fungal community composition based on the V3+V4 regions of 16 S rDNA and ITS1 region of 18 S rDNA in the biological soil crusts collected from the Western Xia Imperial Tombs. To obtain the microorganisms affecting the carbon cycle in the biological soil crust, isolation, screening, and identification of laccase producing microorganisms from the soil crust were further carried out. Results showed the diversity of bacteria was significantly higher than that of fungi. The overall phylum-level composition of bacterial community was dominated by Proteobacteria, Acidobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes, while the fungal community was dominated by Ascomycota and Basidiomycota. On the genus-level, the bacteria were divided into 145 different genera and dominated by Microcoleus, Sphingomonas, Bryobacter, Methylobacterium. On the class-level, the fungi were divided into 83 different genara and dominated by Alternaria, Mycocentrospora, Thermoascus, Omphalina, et al. The predominant groups maybe play an important role in stabilizing the structure of biological soil crusts, carbon and nitrogen cycling, and the metabolism of organic matter. The selective culture medium using guaiacol as the sole carbon source was applied to isolate the laccase producing strains. Plate-screening of the target strains was conducted based on the oxidation of syringaldehyde and the selected strain was identified via 16 S rDNA sequencing. Ten strains with the potential of laccase production were isolated and two strains were screened as the laccase producing microorganisms. The phylogenetic tree of 16 S rDNA showed both the two strains belonged to Bacillus sp.
High-throughput technology was employed to explicit the bacterial and fungal community composition based on the V3+V4 regions of 16 S rDNA and ITS1 region of 18 S rDNA in the biological soil crusts collected from the Western Xia Imperial Tombs. To obtain the microorganisms affecting the carbon cycle in the biological soil crust, isolation, screening, and identification of laccase producing microorganisms from the soil crust were further carried out. Results showed the diversity of bacteria was significantly higher than that of fungi. The overall phylum-level composition of bacterial community was dominated by Proteobacteria, Acidobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes, while the fungal community was dominated by Ascomycota and Basidiomycota. On the genus-level, the bacteria were divided into 145 different genera and dominated by Microcoleus, Sphingomonas, Bryobacter, Methylobacterium. On the class-level, the fungi were divided into 83 different genara and dominated by Alternaria, Mycocentrospora, Thermoascus, Omphalina, et al. The predominant groups maybe play an important role in stabilizing the structure of biological soil crusts, carbon and nitrogen cycling, and the metabolism of organic matter. The selective culture medium using guaiacol as the sole carbon source was applied to isolate the laccase producing strains. Plate-screening of the target strains was conducted based on the oxidation of syringaldehyde and the selected strain was identified via 16 S rDNA sequencing. Ten strains with the potential of laccase production were isolated and two strains were screened as the laccase producing microorganisms. The phylogenetic tree of 16 S rDNA showed both the two strains belonged to Bacillus sp.
引文
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[22] Steven B,Gallegos-Graves LV,Yeager C,et al.Common and distinguishing features of the bacterial and fungal communities in biological soil crusts and shrub root zone soils[J].Soil Biology & Biochemistry,2014,69 (1):302-312.
[23] Zhang J,Viikari L.Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus[J].Bioresource Technology,2012,117:286-291.
[24] 陈香碧.西南喀斯特典型生态系统土壤细菌及降解木质素的担子菌多样性研究[D].长沙:中国科学院研究生院,2012.
[25] 雷海燕.蓝藻型地衣及其共生念珠藻的分子鉴定和系统发育分析[D].苏州:苏州大学,2009.
[26] 付伟,赵遵田,郭守玉.从近年PNAS,Nature,Science发表的涉及地衣的文章看地衣学进展[J].菌物研究,2007,5(3):176-182.
[27] 李泰仑.枯草芽孢杆菌WN02芽孢漆酶的性质及染料脱色研究[D].哈尔滨:东北林业大学,2010.
[2] 闫德仁,薛英英,赵春光.沙漠生物结皮国内研究现状[J].内蒙古林业科技,2007,33(3):28-32,38.
[3] Bowker M A.Biological soil crust rehabilitation in theory and practice:an underexploited opportunity[J].Restoration Ecology,2007,15(1):13-23.
[4] Belnap J,Gillete D A.Vulnerability of desert biological soil crusts to wind erosion:The influences of crust development,soil texture,and disturbance[J].Journal of Arid Environment,1998,39:133-142.
[5] 刘利霞,张玉清,吴斌.生物结皮对荒漠地区土壤及植物的影响研究述评[J].中国水土保持科学,2007,5(6):106-112.
[6] Madhavi V,Lele S S.Laccase:properties and applications[J].Bioresources,2009,4(4):1694-1717.
[7] 张晶,张惠文,李新宇,等.土壤微生物生态过程与微生物功能基因多样性[J].应用生态学报,2006,17(6):1129-1132.
[8] Gundlapally S R,Garcia-Pichel F.The community and phylogenetic diversity of biological soil crusts in the Colorado Plateau studied by molecular fingerprinting and intensive cultivation[J].Microbial Ecology,2006,52(2):345-357.
[9] 陈青.荒漠生物结皮微生物群落组成研究[D].银川:宁夏大学,2014.
[10] 唐凯.浑善达克沙地生物土壤结皮细菌多样性和群落结构研究[D].呼和浩特:内蒙古农业大学,2016.
[11] 张星星.内蒙古荒漠生物土壤结皮及下层土壤微生物多样性和群落结构研究[D].呼和浩特:内蒙古农业大学,2015.
[12] 丛立双.内蒙古荒漠生物结皮中细菌群落分析[D].呼和浩特:内蒙古农业大学,2010.
[13] 李靖宇,张琇.腾格里沙漠不同生物土壤结皮微生物多样性分析[J].生态科学,2017,36(3):36-42.
[14] 杜维波.西北地区土遗址植物多样性调查及其保护对策研究[D].兰州:兰州大学,2013.
[15] 葛志军,黑亚青,王琳瑛.宁夏贺兰山东麓葡萄产业集群发展研究[J].北方园艺,2015(9)163-167.
[16] 潘佳颖.贺兰山东麓葡萄基地两种类型土壤重金属分布特征与评价[D].银川:宁夏大学,2017.
[17] 王锐.贺兰山东麓土壤特征及其与酿酒葡萄生长品质关系研宄[D].杨凌:西北农林科技大学,2016.
[18] Li H,Rao B,Wang G,et al.Spatial heterogeneity of cyanobacteria-inoculated sand dunes significantly influences artificial biological soil crusts in the Hopq Desert (China)[J].Environmental Earth Sciences,2014,71 (1):245-253.
[19] 刘丽燕,吾尔妮莎·沙衣丁,阿不都拉·阿巴斯.荒漠化地区生物结皮的研究进展[J].菌物研究,2005,3(4):26-29.
[20] Kulichevskaya I S,Suzina N E,Liesack W,et al.Bryobacter aggregatus gen.nov.,sp.nov.,a peat-inhabiting,aerobic chemo-organotroph from subdivision 3 of the Acidobacteria[J].International Journal of Systematic and Evolutionary Microbiology,2010,60:301-306.
[21] 盖忠辉.一株鞘氨醇杆菌代谢咔唑的分子机制研究[D].上海:上海交通大学,2011.
[22] Steven B,Gallegos-Graves LV,Yeager C,et al.Common and distinguishing features of the bacterial and fungal communities in biological soil crusts and shrub root zone soils[J].Soil Biology & Biochemistry,2014,69 (1):302-312.
[23] Zhang J,Viikari L.Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus[J].Bioresource Technology,2012,117:286-291.
[24] 陈香碧.西南喀斯特典型生态系统土壤细菌及降解木质素的担子菌多样性研究[D].长沙:中国科学院研究生院,2012.
[25] 雷海燕.蓝藻型地衣及其共生念珠藻的分子鉴定和系统发育分析[D].苏州:苏州大学,2009.
[26] 付伟,赵遵田,郭守玉.从近年PNAS,Nature,Science发表的涉及地衣的文章看地衣学进展[J].菌物研究,2007,5(3):176-182.
[27] 李泰仑.枯草芽孢杆菌WN02芽孢漆酶的性质及染料脱色研究[D].哈尔滨:东北林业大学,2010.
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