山西矿区复垦土壤中解磷细菌的筛选及鉴定

Screening and identification of phosphate-solubilizing bacteria from reclaimed soil in Shanxi mining area

【目的】矿区复垦土壤贫瘠、有效磷含量低。解磷细菌能够将有机磷和难溶性无机磷转化为可溶性磷,促进植物对磷素的利用。因此筛选和鉴定具有解磷能力的菌株,可为解决矿区生态恢复使用的微生物肥料提供菌种资源。【方法】采用平板分离法初筛菌株,得到D/d≥1.5的菌株,然后以磷酸钙为磷源,通过液体发酵试验复筛菌株,挑选出解磷率高于巨大芽孢杆菌(Bacillus megaterium)As1.223的菌株。以磷矿粉和卵磷脂为磷源,液体发酵试验测定菌株的解磷能力及磷酸酶活性。进行菌株的生长试验以测定菌株温度适宜性、耐盐性及耐酸碱性。通过形态学、基因序列分析及脂肪酸组成分析综合进行菌株鉴定。菌落形态观察用营养琼脂平板培养基培养;菌体形态即细胞形态及其大小采用扫描电镜观察;基因序列分析采用16S r DNA序列测定,基因在线比对采用Ez Taxon数据库;使用美国MIDI公司的Sherolock全自动细菌鉴定系统对菌株进行脂肪酸组成分析。【结果】利用无机磷和有机磷平板培养基,从山西省矿区复垦区土壤样品中筛选出19株解磷微生物,其中D/d≥1.5的有7株。在以磷酸钙为磷源的液体培养试验中,4株菌的解磷率高于巨大芽孢杆菌As1.223,解磷率为7.89%12.61%,最高的为菌株Y14。4株菌对磷矿粉的解磷率为0.81%1.21%,最高的为菌株Y14。在以卵磷脂为磷源的液体培养试验中,4株菌的解磷率与酸性磷酸酶活性分别为1.79%3.07%和24.3 28.4U/L,均高于巨大芽孢杆菌As1.223;碱性磷酸酶活性为11.9 50.2U/L;菌株Y14的解磷率与磷酸酶活性均最高。4株菌均有较强的环境适应能力,以Y14的适应性最强。H22、Y11和Y34与假单胞菌属(Pseudomonas sp.)同源性在99%以上,Y14与泛菌属(Pantoea sp.)有99.79%的同源性;H22、Y11和Y34的细胞脂肪酸组成特征峰与假单胞菌属(Pseudomonas sp.)相一致,Y14与泛菌属(Pantoea sp.)相一致;H22、Y11和Y34被鉴定为假单胞菌(Pseudomonas sp.),Y14为泛菌属(Pantoea sp.)。【结论】分离、筛选到4株高效解磷菌,对于磷酸钙和卵磷脂的解磷率均高于巨大芽孢杆菌As1.223。4株菌分别隶属于假单胞菌属(Pseudomonas sp.)和泛菌属(Pantoea sp.)。菌株Y14无机磷与有机磷平板的D/d值分别为3.28与1.59,降解磷酸钙、磷矿粉、卵磷脂的解磷率分别为12.61%、1.21%、3.07%,酸性与碱性磷酸酶活性分别为28.4 U/L和50.2 U/L,均为4株菌里最高的,且环境适应能力最强,生长温度为2060℃,能耐受p H 4 11的酸碱梯度和2%7%的盐分梯度,Y14被鉴定为泛菌属(Pantoea sp.)。4株菌均具有良好的解磷能力及较强的环境适应能力,可望进一步研发成为微生物肥料生产菌种。综合D/d值、解磷率、磷酸酶活性和生长试验,本试验最终确定适合山西矿区复垦农田推广的高效解磷菌菌株为Y14。

Objectives]The rec1aimed soi1s are inferti1e and 1ack of avai1ab1e PhosPhate. PhosPhate so1ubi1izing bacteria are caPab1e of transfering inso1ub1e organic PhosPhorus and inorganic PhosPhorus into the avai1ab1e form for the uti1ization of P1ants. With the PurPose of eco1ogica1y recover the rec1aimed soi1s,PhosPhorus so1ubi1izing bacteria strains were screened and investigated for microbia1 ferti1izer Production.[Methods]Strains which had D/d va1ue ≥1. 5 were Pre-screened using P1ate screening methods,then the strains disP1aying PhosPhorus so1ubi1izing efficiency higher than Bacillus megaterium As1. 223 were Picked out with hydroPonics. The PhosPhate so1ubi1izing abi1ity and PhosPhatase activity of the iso1ated strains were assessed in 1iquid cu1ture with so1ubi1izing rock PhosPhate and 1ecithin as P sources . The growth abi1ity of strains assessed through their resistance to temPerature, sa1t and PH variations. Strains were identified by the methods of morPho1ogy,gene sequencing and ce11u1ar fatty acid comPositions ana1ysis,co1ony morPho1ogy was observed on nutrient agar,microbia1 morPho1ogy was observed by scanning e1ectron microscoPe,gene sequencing was Performed by 16S rDNA sequence ana1ysis and a1igned in EzTaxon,and ce11u1ar fatty acid comPositions ana1ysis was carried out by the Sher1ock Microbia1 Identification System( MIDI).[Results]About nineteen PhosPhorus so1ubi1izing bacteria strains are screened from the rec1aimed soi1s in Shanxi mining area with inorganic and organic PhosPhorus P1ate medium. The D/d va1ues of seven strains are ≥1. 5. In medium using Ca3( PO4 )2 as the PhosPhorus source,the PhosPhorus so1ubi1izing efficiencies of four strains are higher than that of Bacillus megaterium As1. 223 ,and the efficiencies are about 7. 89% -12. 61%. Among the four strains,Y14 has the highest efficiency. In the medium containing 1ecithin,the PhosPhorus so1ubi1izing efficiency and acid PhosPhatase activity of four strains are about 1. 79% -3. 07% and 24. 3-28. 4 U/L resPective1y,higher than those of As1. 223. The a1ka1ine PhosPhatase activities are about 11. 9-50. 2 U/L. Strain Y14 disP1ays the highest PhosPhate-disso1ving abi1ity and PhosPhatase activity. The four strains a1so have strong environmenta1 adaPtabi1ity,and the Performance of Y14 is the best. The Strains H22,Y11,and Y34 disP1ay 99%nuc1eotide homo1ogy with Pseudomonas sP. ,and the strain Y14 disP1ays 99. 79% with Pantoea sP. The ce11u1ar fatty acid comPositions of the strains H22,Y11,and Y34 are simi1ar with Pseudomonas sP. ,and the strain Y14 is simi1ar with Pantoea sP. The strain H22,Y11,and Y34 are identified as Pseudomonas sP. ,whi1e the strain Y14 is identified as Pantoea sP.[Conclusions]Four strains are screened and identified having high PhosPhorus so1ubi1izing caPacity. The four strains be1ong to Pseudomonas sP. and Pantoea sP. resPective1y. The D/d va1ues of the strain Y14 in inorganic and organic PhosPhorus P1ate medium are 3. 28 and 1. 59 resPective1y,the PhosPhorus so1ubi1izing efficiencies in medium containing Ca3( PO4 )2 ,rock PhosPhate,and 1ecithin are 12. 61%,1. 21%, and 3. 07% resPective1y,the acid and a1ka1ine PhosPhatase activities are 28. 4 U/L and 50. 2 U/L resPective1y. The growth temPerature for Y14 is from 20℃ to 60℃,PH 4 to 11 and NaC1 2% to 7%. Y14 is identified as Pantoea sP. According to the resu1ts of D/d va1ue,PhosPhorus so1ubi1izing efficiency,PhosPhatase activity and growth exPeriment,the strain Y14 is suPPosed to be the most suitab1e for PoPu1arizing in rec1amation-area of Shanxi.