一株红壤溶磷菌的分离、鉴定及溶磷特性

【目的】为了提高红壤磷素利用率,探讨溶磷菌溶磷机理。【方法】利用难溶性无机盐培养基从花生根际土壤样品中分离到一株溶磷菌C5-A,结合菌落形态特征、生理生化和16S rRNA序列确定该菌株的系统发育地位;通过菌株C5-A在NBRIP液体培养基培养过程中培养液pH变化确定其溶磷能力;利用液体发酵实验测定不同的碳源、氮源对菌株C5-A溶磷的影响;通过高效液相色谱检测C5-A在不同氮源培养液中有机酸的种类和浓度。【结果】菌株C5-A鉴定为洋葱伯克霍尔德氏菌(Burkholderia cepacia),遗传稳定性较好。在FePO4和AlPO4培养液中,菌株C5-A的溶磷量和pH变化呈显著负相关;菌株C5-A对磷酸三钙、磷酸铝、磷酸铁、磷矿粉均有较强的溶解能力,最高溶磷量分别为125.79、227.34、60.02和321.15 mg/L;菌株C5-A对不同浓度的两种磷矿粉有较强的溶解能力;分别以麦芽糖和草酸铵为碳源和氮源时溶磷量最高。高效液相色谱检测出10种有机酸,分别为草酸(葡萄糖酸)、乙酸、苹果酸、琥珀酸和5种未知有机酸,然而,乙酸而非草酸似乎是影响C5-A溶磷的重要有机酸。【结论】从红壤花生根际土壤中筛选到一株对难溶性无机盐具有较强溶解能力溶的菌株C5-A,有望为开发高效红壤微生物磷肥提供种质资源。     

溶磷菌对4种难溶性磷酸盐溶解能力的初步研究

以4种难溶性磷酸盐为培养基,发现供试菌株溶解这些磷酸盐的特性差异很大,真菌溶磷能力普遍比细菌要高得多。以NO3-为氮源时的溶磷量通常高于以NH4+为氮源时的溶磷量,只有2TCiF2对氟磷灰石及4TCiF6对磷酸铝的溶解能力以NH4+为氮源时较高。大多数菌株较易溶解CaP(氟磷灰石和磷矿粉),其次为AlP(AlPO4),而溶解FeP(FePO4·4H2O的能力都比较弱,只有曲霉2TCiF2具有较强的溶解FeP能力,尤其是当供给NO3-时,溶解FeP的活性比供给NH4+时大幅度提高。欧文氏菌4TCRi22和肠杆菌1TCRi15能大量地溶解氟磷灰石,而两株节杆菌对磷矿粉的溶解能力最强。供试菌株的溶磷作用可能是由于分泌的有机酸与金属离子络合或螯合作用所致,欧文氏菌和肠杆菌溶解难溶性磷过程中,非有机酸物质可能在起主要作用。     

石灰性土壤拉恩式溶磷细菌的筛选鉴定及溶磷特性

从山西石灰性土壤作物根际分离筛选出多株溶磷细菌,经过多次分离纯化得到一株溶磷能力较强的菌株W25,通过菌落形态、生理生化特性和16S rRNA序列分析,确定溶磷菌W25为拉恩式菌属.对W25溶解磷特性进一步研究表明:其对磷酸三钙、磷酸铝和磷酸铁最高溶磷能力分别为385.5、110.4、216.6 mg·L-1;在磷酸铝和磷酸铁培养液中,W25溶磷量与培养液pH的相关系数分别为0.56和0.81,呈极显著负相关;在不同碳氮源条件下,W25以葡萄糖为碳源和NH4NO3为氮源时对磷酸三钙的溶磷量最高,对碳源的利用顺序依次为葡萄糖＞乳糖＞蔗糖＞甘露醇＞淀粉,对氮源的利用顺序依次为NH4NO3 ＞NH4Cl＞(NH4)2SO4＞NaNO3＞KNO3.不同氮源对W25产生有机酸的种类影响较大,以铵态氮为氮源产生甲酸和乙酸,以硝态氮为氮源产生草酸和琥珀酸,以硝酸铵为氮源还产生柠檬酸.     

类芦根际溶磷真菌的筛选、鉴定及其溶磷能力分析

为揭示类芦(Neyraudia reynaudiana)等水土保持植物的耐低磷机制,开发溶磷菌种质资源,提高赤红壤磷素利用率,从类芦根际土壤中筛选到一株溶磷能力较强的真菌FP1,经形态学和ITS序列分析,鉴定为黑曲霉(Aspergillus niger)。3种难溶性磷酸盐液体培养基接种菌株FP1后,其pH值和溶磷量的动态变化显示,培养液的pH值均呈显著下降趋势。溶磷量与培养时间有关,除磷酸三钙外,菌株FP1对其他难溶性磷酸盐的溶磷趋势均为先上升再下降并趋于稳定。菌株FP1对不同磷源的最大溶磷率顺序为:磷酸铝(92.02%)磷酸三钙(41.62%)3种磷酸盐的混合物(35.86%)磷酸铁(19.20%)。FP1对磷酸铝和磷酸铁都具有较强的溶磷能力,表明抗逆性强的水土保持植物类芦根际土壤蕴藏着高效的溶磷微生物资源。     

溶磷菌和固氮菌溶解磷矿粉时的互作效应

采用4株溶磷菌（Lx81、Dm84、Jm92、Lx191）、和3株固氮菌（ChW5、ChW6、ChO6）单独和混合接种后测定培养液有效磷含量、pH值及总有机酸含量的方法,研究溶磷菌和固氮菌溶解磷矿粉时的互作效应。结果表明,相对于单独接种溶磷菌：Lx81与3株固氮菌分别混合培养能提高磷矿粉的溶解能力,4株溶磷菌与ChW6,Lx81、Dm84、Lx191与Ch06分别混合培养及Jm92＋ChW5组合溶磷量极显著增加（P〈0．01）;Dm84＋ChW5、Lxl91＋ChW5、Jm92＋Ch06组合的溶磷量下降（P〈0．01）。除Lx81＋ChW6、Lx81＋Ch06培养液pH值降低外,混合培养的其它组合培养液pH值均较单独接种溶磷菌时升高。有机酸测定结果表明,Lx81、Jm92与ChW5、Ch06分别混合培养、ChW6＋Lx81组合有机酸含量升高（P〈0．01）,其它7种组合的有机酸含量均较单独接种溶磷菌的值下降（P〈0．01）。溶磷菌和固氮菌单菌培养时溶磷量与pH值、溶磷量与总有机酸含量及pH值与总有机酸含量之间呈现线性相关;Dm84、Lx191与3株固氮菌分别混合培养溶磷量与pH值之间、Lx81与3株固氮菌分别混合培养溶磷量与总有机酸含量之间呈现线性相关,其它组合的溶磷量与pH值、总有机酸含量间没有相关性。溶磷菌和固氮菌混合培养对溶解磷矿粉既有协同作用也有拮抗作用。     

两株溶磷真菌的筛选、鉴定及溶磷效果的评价

【目的】从作物根围土壤中筛选高效溶磷菌株。【方法】结合溶磷圈筛选法和钼锑抗比色法评价菌株的溶磷能力;利用菌株的形态学特性、培养性状和微管蛋白β-tubulin基因序列分析方法进行菌株的鉴定;采用气相色谱-质谱法(GC-MS)对溶磷菌的产酸物质进行分析;并用平板亲和性实验测定菌株间的兼容性。【结果】筛选得到2株高效溶磷菌株P1-1、P2-2;经鉴定,菌株P1-1为黑曲霉(Aspergillus niger),P2-2为塔宾曲霉(A.tubingensis)。2株溶磷菌株的产酸物质相同,均为草酸、葡萄糖酸、乳酸和甘油酸。这2株溶磷菌与杀线虫功能菌株淡紫拟青霉(Purpureocillium lilacinum)、橄榄色链霉菌(Streptomyces olivaceus)和苍白杆菌(Ochrobactrum pseudogrignonense)兼容性好。2菌株分别在Ca_3(PO_4)_2、Zn_3(PO_4)_2、羟基磷灰石为磷源的无机磷固体培养基中25°C培养5 d,测定溶磷圈的直径(D)与菌落直径(d),通过计算其比值D/d的大小对比,以及在Ca_3(PO_4)_2、Zn_3(PO_4)_2、羟基磷灰石为磷源的液体培养基中培养5 d,测定发酵液中有效磷含量进行比较后判定,这2株溶磷菌溶解磷的能力强且效果相当。【结论】获得了2株高效的溶磷真菌。它们能活化多种难溶性磷源,同时伴随挥发性酸性物质的产生;2个菌株与1组杀根结线虫微生物菌群兼容性均良好。     

宁夏产枸杞根际溶磷菌分离及溶磷能力分析

采用常用的PKO(无机培养基)和蒙金娜有机培养基,从宁夏枸杞(Lycium barbarumL.)根际分离具有溶磷能力的菌株,并利用溶磷圈法测定溶磷菌株的溶磷能力。从宁夏枸杞根际共分离出88株具有溶磷能力的菌株,其中溶解无机磷能力较强的菌株4株,其溶磷圈直经(D)与菌落直径(d)的比值(D/d)从高至低依次是NQ2、NQ4、NQ3、NQ1,D/d值为3.02~1.85;菌株NQ2、NQ1、NQ3和NQ4对无机磷的溶磷强度分别为91.2、82.5、75.5和74.8μg.mL-1。所分离的4株具有较强无机磷溶解能力的菌株在微生物肥料的开发方面有潜在应用价值。     

贵州两处茶园溶磷青霉菌的筛选、鉴定及溶磷能力分析

为维持土壤自然完整性、活化利用土壤中难溶性磷,从贵州名茶产地都匀、贵定茶园土壤中筛选高效溶磷真菌,为制备真菌肥料提供菌种资源。利用溶磷指数(SPI)、形态特征和ITS rDNA序列筛选、鉴定菌株,并采用液体摇床培养实验测定鉴定菌株在以磷酸钙、磷酸铁或磷酸铝为唯一磷源的无机磷液体培养基中的溶磷能力。共筛选到7个高效溶磷菌落,经形态观察分属2种菌株,鉴定为微紫青霉(Penicillium janthinellum)和赭绿青霉(Penicillium ochrochloron)。液体培养基接种、摇床培养15 d,微紫青霉菌在以Ca3(PO4)2、Fe3(PO4)2或AlPO4为唯一磷源的上清液中有效磷含量分别为73.47 mg·L^-1、30.93 mg·L^-1和14.00 mg·L^-1,4℃继续保存至30d后对Fe-P和Al-P的溶解量分别达到72.20 mg·L^-1、32.84 mg·L^-1;赭绿青霉菌培养15d的溶磷量分别为30.72 mg·L^-1、4.14 mg·L^-1和1.51 mg·L^-1,30d对Fe-P和Al-P的溶解量分别达到35.19 mg·L^-1和10.98 mg·L^-1。微紫青霉菌溶解无机磷能力明显优于赭绿青霉菌,有望应用于地区缺磷茶园土壤真菌肥料的制备。     

红树林溶磷菌的初步鉴定、溶磷能力测定及其优化培养

对分离来自华南红树林的溶磷菌进行16S rDNA或ITS等基因水平上的初步鉴定, 测定其溶解无机磷的能力, 并对溶磷菌的生长培养基条件进行优化。结果表明, 溶磷真菌的溶磷效果明显强于溶磷细菌, 且溶磷真菌的溶解无机磷能力与培养液的pH呈极显著负相关, 而溶磷细菌的溶磷能力与pH没显著相关关系。单因素实验表明, 对供试菌株生长的合适碳源为麦芽糖, 氮源为尿素。通过正交实验得到的优化培养基为麦芽糖5 g/L、尿素0.05 g/L、NaCl 5 g/L、pH 5, 在30°C下培养48 h菌落总数可达6.06×     

解磷菌发酵及溶磷条件的研究

从新疆盐碱土中分离纯化得到一株高效解磷菌PS-3,为改善盐碱地磷素供应提供菌种资源。采用单因素和正交试验,对菌株PS-3的发酵条件和溶磷能力进行分析。结果表明,菌株PS-3较适发酵条件为温度35℃、pH 7和盐浓度1%,其对碳、氮源的利用顺序依次为葡萄糖>麦芽糖>果糖>蔗糖>乳糖,硝酸铵>硝酸钠>氯化铵>硫酸铵>尿素,菌株PS-3最适溶磷条件为2%葡萄糖、0.01%硝酸铵、温度35℃、初始pH 7.5、盐浓度2 g/L、接种量4%,在此条件下,其对磷酸三钙的溶解量可达100 2.95mg/L。该菌株具有一定耐盐性和良好的溶磷能力,在生物溶磷方面具有较好的应用潜力。     

一株金黄蓝状菌解磷特性及其对毛竹的促生效应

为深入了解毛竹根际微生物金黄蓝状菌(JXBR04)的解磷特性并评价其促生效应,采用液体发酵培养法研究碳源、氮源、pH、装液量及盐离子等因素对金黄蓝状菌解磷能力的影响,以及该菌株对不同难溶性磷酸盐的溶解能力,并应用温室盆栽法研究该菌株对毛竹幼苗生长的促进效应.结果表明: 金黄蓝状菌(JXBR04)分别在碳源为蔗糖、氮源为酵母粉、初始pH值3.5、装液量1/5或2/5、盐离子浓度为0 或1.0 g·L^-1时解磷能力最强;对Ca₃(PO₄)₂、CaHPO₄、FePO₄均具有较好的解磷作用,其中对CaHPO₄表现最佳,达1304.04 mg·L^-1.施用金黄蓝状菌(JXBR04)菌剂180 d后显著提高了毛竹实生苗根际土壤养分和植株体内磷含量,且地径、苗高及生物量分别比对照增长了28.1%、28.3%和51.5%.可见,金黄蓝状菌具有成为我国南方酸性土壤毛竹林环境友好型生物肥料的潜力.     

Characterization of the mineral phosphate solubilizing activity of <Emphasis Type="Italic">Serratia marcescens</Emphasis> CTM 50650 isolated from the phosphate mine of Gafsa

The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO₄), tri-calcium phosphate (Ca₃(PO₄)₂) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO₄, Ca₃(PO₄)₂, hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP.     

Trial of different nutrient media for the production of an intracellular thermolabile enterotoxin by Escherichia coli strains H74-114 and 86

The conditions of cultivation, ensuring the maximum accumulation of intracellular thermolabile enterotoxin in the cultures of two E. coli strains of different origin, have been studied. Culture media manufactured in the USSR have been selected and the conditions of cultivation, necessary for obtaining intracellular thermolabile enterotoxin in preparative amounts, have been established. Under these conditions the yield of thermolabile enterotoxin in 1.4 mg per 1 liter of culture medium for strain H74-114 and 1.0 mg per 1 liter of culture medium for strain 86.     

Hydrolysis of 4-hydroxybenzoic acid esters (parabens) and their aerobic transformation into phenol by the resistant Enterobacter cloacae strain EM

Enterobacter cloacae strain EM was isolated from a commercial dietary mineral supplement stabilized by a mixture of methylparaben and propylparaben. It harbored a high-molecular-weight plasmid and was resistant to high concentrations of parabens. Strain EM was able to grow in liquid media containing similar amounts of parabens as found in the mineral supplement (1,700 and 180 mg of methyl and propylparaben, respectively, per liter or 11.2 and 1.0 mM) and in very high concentrations of methylparaben (3,000 mg liter(-1), or 19.7 mM). This strain was able to hydrolyze approximately 500 mg of methyl-, ethyl-, or propylparaben liter(-1) (3 mM) in less than 2 h in liquid culture, and the supernatant of a sonicated culture, after a 30-fold dilution, was able to hydrolyze 1,000 mg of methylparaben liter(-1) (6.6 mM) in 15 min. The first step of paraben degradation was the hydrolysis of the ester bond to produce 4-hydroxybenzoic acid, followed by a decarboxylation step to produce phenol under aerobic conditions. The transformation of 4-hydroxybenzoic acid into phenol was stoichiometric. The conversion of approximately 500 mg of parabens liter(-1) (3 mM) to phenol in liquid culture was completed within 5 h without significant hindrance to the growth of strain EM, while higher concentrations of parabens partially inhibited its growth.     

一株新的溶磷棘孢青霉菌Z32的分离、鉴定及其土壤定殖与溶磷特性

【目的】从植物根部土壤中分离到一株高效溶磷真菌Z32,进行了分类学鉴定和土壤定殖与溶磷特性的初步研究。为溶磷微生物的应用提供新的菌株。【方法】通过形态特征、培养特征和ITSrDNA序列分析方法进行菌株鉴定。通过菌株Z32在土豆液体培养基培养过程中培养液pH的变化确定溶磷菌株的溶磷能力。利用菌株土培试验,进行菌株的土壤定殖和土壤中不同形态无机磷转化试验。【结果】菌株Z32鉴定为棘孢青霉菌(Penicillium aculeatum)。菌株Z32能在4d内完全溶解固体培养基中的磷酸三钙,18h内将土豆液体培养基的pH值从7.0降低到1.5左右。菌株Z32在20℃时的定殖效果最好,21d时,菌数达到起始的9.83倍,而且能够保持到49d不消亡。在20℃时,添加菌株Z32的土培实验在21d时,土壤中Ca8H2(PO4)6·5H2O、AlPO4和FePO4等难溶无机磷向可溶性的CaHPO4转化,CaHPO4含量增加了58.83%。49d时,土壤中的Ca8H2(PO4)6·5H2O、AlPO4和和FePO4被转化后,没有随着微生物的减少而完全被固定。【结论】筛选到一株新的溶磷菌株Z32在土壤中很好定殖,能够转化土壤中多种形态的难溶无机磷为可溶性的CaHPO4,显著增加土壤中CaHPO4的含量。同时阻滞了Ca8H2(PO4)6·5H2O、AlPO4和和FePO4等难溶无机磷的固定。     

一株耐盐日本曲霉的筛选及其溶磷促生作用

【目的】从内蒙古种植葵花的盐碱地中筛选高效溶磷真菌,为盐碱地增产节肥开发生物肥料提供溶磷菌种资源。【方法】利用ITS r DNA序列鉴定菌株、固体培养基测定耐盐性,液体摇床培养与盆栽试验结合分析菌株溶磷能力,盆栽和田间试验明确菌株M1促进作物生长和增产作用;LC-MS技术测定菌株M1在液体培养基中分泌有机酸和植物激素含量,明确菌株M1的溶磷和促生机理。【结果】溶磷菌株M1鉴定为日本曲霉(Aspergillus japonicus)。液体培养基接种菌株M1培养6 d,以Ca_3(PO_4)_2为磷源时上清液有效磷达1020.89 mg/L,溶解率为63.30%;以AlPO_4为磷源时有效磷达995.69 mg/L,溶解率为48.59%;以贵州开阳磷矿粉、江苏锦屏磷矿粉、云南晋宁磷矿粉、河北钒山磷矿粉和云南昆阳磷矿粉为磷源接种菌株M1,从晋宁磷矿粉释放的有效磷浓度最高,达到363.64 mg/L。菌株M1可耐受10%NaCl。将M1制备的菌剂分别接种于施用Ca_3(PO_4)_2、AlPO_4和开阳磷矿粉3种磷源的4种盆栽试验土壤包括北京石灰性潮土、安徽黏性潮土、安徽水稻土和山东沿海盐潮土。结果显示,菌株M1对玉米植株促生效果显著,玉米植株鲜重比对照提高2.14%–90.91%、干重增加22.15%–268.28%;土壤有效磷提高21.81–24.27 mg/kg。菌株M1与4种土壤的适配性均高于对照菌株DSM 821。田间小区花生产量结果显示,接种溶磷菌剂M1增产效果最好,花生果实产量达4.46 t/hm~2,比不接种菌剂的对照处理增加0.81 t/hm~2,增产22.19%。菌株M1在含有磷酸三钙、磷酸铝和开阳磷矿粉3种难溶磷培养液中经过6 d培养,均产生7种有机酸,其中草酸和柠檬酸含量最高,分别为616.16 mg/L和413.69 mg/L;培养液均能检测到吲哚乙酸(IAA)和玉米素,IAA含量为15.45–77.58 mg/L,玉米素浓度为0.06–0.11 mg/L。【结论】获得了一株高效溶解多种难溶磷的日本曲霉菌M1,它能显著增加土壤有效磷、促进玉米生长和花生增产,与4种典型土壤适配性好,具有良好的农业应用前景。     

不同碳源对三种溶磷真菌溶解磷矿粉能力的影响

通过液体培养法 ,对 3种溶磷真菌利用葡萄糖、果糖、蔗糖、麦芽糖、淀粉和纤维素等碳源溶解宜昌产磷矿粉的试验 ,结果表明 ,菌株P2 3在供给葡萄糖时的溶磷能力最高 ,并在一定程度上能够利用长链碳源淀粉和纤维素为营养而溶磷 ;而高效溶磷菌株P6 6和P39溶磷的最佳碳源是果糖和麦芽糖 ,该两菌株利用淀粉和纤维素的溶磷效果很小 ,甚至不溶磷。 3种溶磷真菌培养滤液 pH值、可滴定酸含量与其溶磷量之间的相关性因菌株而异 ,差别很大。菌株P2 3培养滤液pH值、可滴定酸含量与其溶磷量之间相关性很低 ,但菌株P6 6和P39培养滤液pH值、可滴定酸含量与其溶磷量之间相关性却达到极显著水平 (P <0 0 1)。结果表明 ,不同碳源对溶磷菌溶解磷矿粉能力影响很大 ,分析推断 3种菌株产生的有机酸活化磷矿粉能力为P6 6>P39>P2 3。     

Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages?

The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected.