多功能固氮菌筛选及其在土壤生态修复中的应用  

Screening of Multi-functional Nitrogen Fixing Bacteria and Their Application in Soil Ecological Restoration

作　　者：毛晓洁 王新民[2] 赵英[2] 周义清[1] 孙建光[1] 

机构地区：[1]中国农业科学院农业资源与农业区划研究所,农业部农业微生物资源收集与保藏重点实验室,北京100081 [2]国家环境保护创面生态修复工程技术中心路域生态工程有限公司,北京100082

出　　处：《生物技术通报》2017年第10期148-155,共8页Biotechnology Bulletin

基　　金：内蒙古自治区国土资源厅项目

摘　　要：以固氮酶和ACC(1-aminocyclopropane-1-carboxylate,1-氨基环丙烷-1-羧酸)脱氨酶活性为指标筛选功能菌株,研制菌剂,用于修复生荒地土壤生态。采用乙炔还原法测定菌株固氮酶活性;比色法定量测定ACC脱氨酶活性;PCR扩增得到菌株16S r RNA,通过序列分析、比对研究菌株的系统发育地位;通过形态、生理生化特征和16S r DNA序列比对鉴定菌种。结果显示,筛选到固氮酶活性大于9 nmol/(h·mg)的菌株共计47株,其中43株的固氮酶活性大于阳性对照固氮菌剂生产菌株圆褐固氮菌ACCC11103。筛选到产生ACC脱氨酶的菌株20株,ACC脱氨酶活性为0.326-21.980μmol/(h·mg)。将筛选到的功能菌株接种小白菜测试促生效应,其中47株使普通白菜(Brassica campestris)鲜重增加。16S r RNA测序鉴定显示筛选到的51株功能菌株系统发育地位上属于农杆菌、节杆菌、芽孢杆菌、伯克氏菌、鞘氨醇杆菌、屈挠杆菌、剑菌、肠杆菌、溶杆菌、微球菌、类芽孢杆菌、叶杆菌、假单胞菌、根瘤菌、中华根瘤菌、芽孢乳杆菌等16属31种。选取高效菌株7012、7134、7144和7164作为核心菌株制备了固氮多功能菌剂,应用于生荒地,试验地土壤微生物数量显著提高,土壤蔗糖酶、过氧化氢酶、脲酶、磷酸酶、纤维素酶、木聚糖酶活性极显著高于未施用菌剂的对照处理。多功能固氮菌制剂对于提高生荒地土壤生物活性、改善土壤生态环境具有重要作用和应用价值。The objective of this study is to screen multi-functional nitrogen-fixing bacteria based on nitrogenase and ACC（1-aminocyclopropane-1-carboxylate）deaminase activity,to develop inoculant and test its effect in wasteland soil ecological restoration. First,nitrogenase activity was determined by acetylene reduction assay,and ACC deaminase activity was quantitatively measured by colorimetry.Then,the 16 S r RNA of the strains was amplified by PCR,and the phylogenetic status of the strains was investigated by sequence analysisand alignment. The identification of strains was carried out based on the morphology,physiology,biochemical test results,and 16 S r RNAanalysis. As results,total 47 strains with nitrogenase activity higher than 9 nmol C2 H4/（h · mg）protein were screened,and the nitrogenaseactivities of the 43 strains were higher than that of positive control Azotobacter ACCC11103. And 20 strains producing ACC deaminase of0.326 μmol/（h·mg）to 21.980 μmol/（h·mg）were obtained. Plant growth promotion test showed that the 47 strains increased the freshweights of Chinese cabbage（Brassica campestris）. Based on 16 S r RNA sequence,the 51 selected strains were identified as Agrobacterium albertimagni,Arthrobacter pascens,Bacillus circulans,Burkholderia vietnamiensis,Chitinophaga ginsengisegetis,Dyadobacter fermentans,Ensifer adhaerens,Enterobacter aerogenes,Lysobacter yangpyeongensis,Micrococcus luteus,Paenibacillus alginolyticus,Phyllobacterium ifriqiyense,Pseudomonas fluorescens,Rhizobium alamii,Sinorhizobium adhaerens,Sporolactobacillus laevolacticus of 31 species of 16 genera. After test,4 excellent strains of 7012,7134,7144,and 7164 were manufactured as inoculant termed as NFMF（Nitrogen Fixingand Multi Function）biofertilizer,with which the field experiment showed that soil microorganism quantity increased significantly after 3 months of NFMF biofertilizer inoculation,and soil activities of sucrase,catalase,urease,phosphatase,cellulase and xylanase significantlyincreased compared with the no

关 键 词：生物固氮 ACC脱氨酶 土壤酶 生态修复 

分 类 号：S156[农业科学—土壤学]