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。     

微生物溶解磷矿粉能力与pH及分泌有机酸的关系

从玉米根际和非根际土壤中分离得到的 74株溶解磷矿粉的微生物 ,发现它们的溶磷能力差异很大 ,主要决定于菌株本身的特性 ,与其来源无关 ,真菌普遍比细菌要强。真菌的溶磷能力与其培养介质的 pH之间呈显著的负相关 ,但细菌的这种关系非常弱。二者都产生多种有机酸 ,真菌主要分泌草酸、丙二酸和乳酸 ,而细菌主要分泌草酸、酒石酸、丙二酸、乳酸和乙酸 ,不同菌株分泌有机酸的数量和种类差异很大 ,但溶磷量与有机酸总量或单个有机酸浓度之间 ,没有发现显著的相关性 ,唯独柠檬酸与真菌的溶磷量之间存在显著的相关性。说明不同菌株有完全不同的溶磷机理 ,可能多种机理并存。     

出芽短梗霉F4的溶磷能力及机理

从安徽省铜陵市铜官山尾矿库木贼根际分离筛选出的出芽短梗霉F4,以磷酸钙、磷酸铝、磷酸铁和磷矿粉4种不同磷源进行液体培养,测定培养液的pH、水溶性磷、菌体磷及有机酸含量.结果表明： 菌株F4对不同磷源的溶磷能力为：磷酸铝>磷酸铁、磷酸钙>磷矿粉,溶磷量均高于200 mg·L^-1;培养液pH在48 h内迅速下降,以磷酸铝、磷酸铁为磷源的培养液pH下降幅度明显大于磷酸钙与磷矿粉.出芽短梗霉F4产生的有机酸主要为草酸、柠檬酸和酒石酸,其中,以草酸为主.菌株的溶磷能力与有机酸无显著相关性,而与pH呈显著相关.接种出芽短梗霉F4时加入葡萄糖,尾矿中速效磷含量显著增加,说明出芽短梗霉F4在尾矿生态修复中具有潜在的应用价值.
     

棉花根际解磷菌的解磷能力和分泌有机酸的初步测定

利用特殊培养基对盐碱地棉花根际解磷菌进行了分离以及pH值和分泌有机酸能力的初步测定。利用溶磷圈法筛选出10个解磷能力较高的菌株进行深入研究,其中液体培养条件下测定了菌株的溶磷能力,有效磷在4.04~185.63 mg/L,其中wpL2溶磷量达到185.63 mg/L;测定了培养液pH值,下降到5.12~6.67,但是pH与溶磷量之间没有线性关系,测定了培养液的有机酸含量,菌株溶磷量与有机酸总量没有线性相关性,其中所分离到的解无机磷菌株均可以分泌酒石酸,除此之外,wpc1还分泌乙酸,wpc2和wpL2还分泌柠檬酸;分离到的解有机磷菌株均可分泌乙酸,除此之外,ypL1和ypc2分泌酒石酸,ypL3分泌柠檬酸,ypL2和ypc3分泌柠檬酸和丁二酸,均不能产生苹果酸。     

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。     

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。     

解磷菌JL-1对磷矿粉降解性能的研究

以不动杆菌(Acinetobacter indicus)JL-1为菌种,磷矿粉为磷源,测试解磷菌JL-1对磷矿粉的降解能力。通过单因素实验及响应面试验得到最优降解磷矿粉条件为:以葡萄糖为碳源,浓度为17 g/L,以质量比为1∶1的硫酸铵与酵母粉为氮源,浓度为1.2 g/L,磷矿粉添加量为2.0 g/L,温度为28℃,初始pH为7.0,接种量为1%,在此最优条件下检测动态解磷曲线,在60h时降解磷矿粉的效果最好,此时解磷量为4.65μg/mL,溶磷率为2.34%,菌落数达到最高,为10.65 log(CFU/mL),pH达到最低。砂培实验表明磷矿粉中溶解的有效磷的72.08%会被菌体直接释放出来,7.63%会储存在菌体细胞中,20.29%会被菌体同化。     

一株高效解磷菌的筛选及其解磷效果验证

为开发新型、安全及高效的解磷菌肥,从农田中分离筛选出多株菌,通过解磷能力验证,最终选出解磷能力最强的X-P18菌株,经16S rDNA分析鉴定为贝莱斯芽孢杆菌。同时,通过优化X-P18菌株液体发酵培养条件并测定发酵液中小分子有机酸种类,初步探究菌株的解磷机理。将该解磷菌施用于黑叶葵扇白菜盆栽,并对小白菜的基本指标进行分析。结果表明,X-P18菌株在无机磷液体培养基中,溶磷量为495.4 mg/L,具有分泌乙酸及其他少量小分子有机酸的特性。培养基初始接种量为1%、pH8.0、碳源为甘露糖、氮源为硫酸铵、培养温度为30℃时,X-P18菌株的解磷能力最佳,溶磷量为582.4 mg/L,比优化前高出17.6%。在X-P18菌剂添加量为2×10~9CFU/盆时,对黑叶葵扇白菜的促生效果最明显,鲜重增加65.5%,叶片全磷含量增加46.9%。     

一株高效溶磷产红青霉培养条件优化及其溶磷特性

为了减少农业生产中化学肥料的使用,本研究利用无机磷培养基对富磷的茶树（Camellia sinensis L.）根际微生物进行筛选,获得一株对磷酸三钙具有高效降解能力的真菌菌株JL-1,经鉴定为产红青霉（Penicillium rubens）。通过检测菌株JL-1在溶磷过程中发酵液的pH值、磷含量和有机酸含量变化发现,该菌株在无机磷培养基中,发酵液pH值与葡萄糖酸含量、pH值与磷含量以及葡萄糖酸与磷含量分别呈显著负相关、显著负相关和显著正相关,且在电子显微镜下观察到磷酸三钙颗粒表面存在被侵蚀痕迹,深入分析发现菌株JL-1通过分泌葡糖糖酸实现侵蚀磷酸三钙与溶磷的目的。通过单因素试验、Plackett-Burman设计试验、最陡爬坡试验和中心组合试验等一系列试验对培养条件进行优化发现,菌株JL-1溶解磷酸三钙的最佳碳源和氮源分别是葡萄糖和硫酸铵。葡萄糖含量、磷酸三钙含量和温度是影响菌株JL-1溶磷能力的主要因素,在葡萄糖29.8 g/L,磷酸三钙7.1 g/L,温度31.9℃的条件下,菌株JL-1在无机磷培养基中的溶磷能力达到最佳,溶磷量达到1 194.15 mg/L,是初始值的近3倍。在...     

杨树根际解无机磷细菌Mp1-Ha4的鉴定及其解磷机理

解无机磷细菌能够溶解土壤中的难溶性磷酸盐,增加土壤有效磷含量,促进植物生长。以一株杨树根际土壤中筛选得到的解无机磷细菌Mp1-Ha4为研究对象,利用分子生物学的方法对该菌株进行鉴定,测定了其对磷酸钙、磷酸铝和磷酸铁的解磷能力,并对该菌株9 d内的磷酸钙溶解动力学进行了研究。结果表明,解无机磷细菌Mp1-Ha4为一株西地西菌Cedecea sp.,其对磷酸钙的溶解能力远强于对磷酸铝和磷酸铁的溶解能力。在NBRIP液体培养基中,该菌株对磷酸钙的溶解能力达到了497.4 mg/L,在其对磷酸钙解磷过程中,培养基pH及可滴定酸度与解磷量分别呈显著负、正相关。高效液相色谱分析显示,该菌株在解磷过程中分泌了大量有机酸,主要包括α-酮戊二酸,酒石酸和苹果酸。分泌有机酸,降低环境pH可能是解无机磷细菌西地西菌(Cedecea sp.)Mp1-Ha4溶解难溶性磷酸盐的主要机制,同时该菌株对磷酸钙的高效溶解作用使其具有较大的研究和应用前景。     

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose‐ and (NH₄)₂SO₄‐based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P‐solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH₄)₂SO₄ as C and N sources allowed a higher solubilization efficiency at high pH. Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P‐poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.     

Enhanced solubilization of rock phosphate by Penicillium bilaiae in pH-buffered solution culture

Little is known about how pH-buffering capacity affects phosphorus (P) solubilization by Penicillium bilaiae. This study compared solubilization of rock phosphate (RP) by P. bilaiae in nonbuffered (pH 5.0) and buffered (pH 7.0) media. Fungal growth reached the stationary phase around day 12 and was slightly enhanced in the buffered medium. The fungus reduced solution pH from 5.0 to 4.1 in the nonbuffered medium and from 7.0 to 4.9 in the buffered medium by day 12. Phosphorus concentrations increased after day 9 more in the buffered than in the nonbuffered media (53 and 5 mg P x L(-1), respectively, on day 12). On day 12, higher concentrations of citric and oxalic acids were detected in the buffered (2.0 and 1.2 g x L(-1), respectively) than nonbuffered media (0.5 and 0.04 g x L(-1), respectively). Solubilization of RP was simulated without P. bilaiae in solutions equivalent to the nonbuffered and buffered cultures of P. bilaiae grown with RP. After a 24 h incubation, the P concentrations were of similar magnitudes to those observed in the P. bilaiae culture (18 and 47 mg P x L(-1), respectively, in the nonbuffered and buffered media). Under increased pH-buffering conditions, the enhanced production of citric and oxalic acids led to significant RP solubilization.     

Quantitative evaluation of the role of organic acid exudation in the mobilization of rock phosphate by rape

Phosphorus-deficient rape plants appear to acidify part of their rhizosphere by exuding malic and citric acid. A simulation model was used to evaluate the effect of measured exudation rates on phosphate uptake from Mali rock phosphate. The model used was one on nutrient uptake, extended to include both the effect of ion uptake and exudation on rhizosphere pH and the effect of rhizosphere pH on the solubilization of rock phosphate. Only the youngest zones of the root system were assumed to exude organic acids. The transport of protons released by organic acids was described by mass flow and diffusion. An experimentally determined relation was used describing pH and phosphate concentration in the soil solution as a function of total soil acid concentration. Model parameters were determined in experiments on organic acid exudation and on the uptake of phosphate by rape from a mixture of quartz sand and rock phosphate. Results based on simulation calculations indicated that the exudation rates measured in rape plants deficient in phosphorus can provide the roots with more phosphate than is actually taken up. Presence of root hairs enhanced the effect of organic acid exudation on calculated phosphate uptake. However, increase of root hair length without exudation as an alternative strategy to increase phosphate uptake from rock phosphate appeared to be less effective than exudation of organic acids. It was concluded that organic acid exudation is a highly effective strategy to increase phosphate uptake from rock phosphate, and that it unlikely that other rhizosphere processes play an important role in rock phosphate mobilization by rape.     

Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium

Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. niger-B. cepacia) culture. During 9 days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A. niger-B. cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 ± 0.60 to 1103.64 ± 1.21 μg PO(4) 3- mL(-1) and was 9-22% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A. niger culture, 3.1 in the co-culture, and 4.2 in the B. cepacia culture. On the third day of postinoculation, acid production by the co-culture (mean 5.40 ± 0.31 mg NaOH mL(-1)) was 19-90% greater than single cultures. Glucose concentration decreased almost completely (97-99% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO(4) under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.     

Identification and characterization of the rhizosphere phosphate-solubilizing bacterium <Emphasis Type="Italic">Pseudomonas frederiksbergensis</Emphasis> JW-SD2 and its plant growth-promoting effects on poplar seedlings

The rhizospheric bacterium JW-SD2 was identified as Pseudomonas frederiksbergensis based on phenotypic features, the Biolog Identification System and 16S rRNA sequence analysis. The phosphate-solubilizing activity, acidification in culture media, growth rate and organic acid secretion of JW-SD2 were investigated during 192 h of cultivation. The phosphate solubilized by JW-SD2 reached 7.75 mM. The decrease of pH and increase of titratable acidity were closely correlated (Pearson’s r?=??0.953 and 0.969, respectively) with the phosphate-solubilizing activity. High concentrations of gluconic, 2-ketogluconic, pyruvic, maleic and malic acids were detected before 96 h of culture, when the strain displayed a high level of phosphate-solubilizing activity, indicating that these organic acids were efficient components in phosphate solubilization. However, acetic acid did not affect phosphate solubilization as shown by a remarkable increase at 144 h of culture when the phosphate-solubilizing activity decreased. The phosphate-solubilizing ability of JW-SD2 was significantly (P?<?0.01) affected by environmental factors. Over a broad ranges of temperature (20?35 °C), pH (4?9), salinity (0?3.0 %), and volume of medium (1/5?3/5 of flask volume), the phosphate solubilized by JW-SD2 remained above 4.00 mM, demonstrating good potential in adapting to a changing environment. The inoculation experiments indicated that JW-SD2 could significantly (P?<?0.05) promote growth of poplar (Populus euramericana cv. NL-895) in both sterilized and non-sterilized soils. The effects of plant growth promotion were greater in non-sterilized than in sterilized soil. During the 150 days of the trial, the effects of plant growth promotion by JW-SD2 first increased then decreased over time, suggesting that, in field applications, the periodic supplementation of the strain into the rhizosphere should be considered.     

4株溶磷细菌和真菌溶解磷矿粉的特性

溶磷微生物广泛地分布在土壤,根际等生态环境中,了解这些微生物溶解难溶性磷酸盐如磷矿粉的特性,对于开发利用这些微生物,提高磷素利用效率具有重要作用,研究发现;真菌比细菌溶解磷矿粉的能力要强得多,培养基中的铁,镁,锰,钠等成分可以提高真菌的溶磷量,但降低了细菌的溶磷量,培养基中磷矿粉用量越高,溶磷量越低;碳源物质浓度高于3%将显地降低溶磷量,微生物能够破坏磷矿粉的结构。使其中的磷在以后的培养过程中更加容易释放出来,可见利用微生物活化磷矿粉中的磷,具有良好前景。