黑土磷素有效性的微生物调控技术及其机理研究
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摘要
针对我国土壤潜在磷源丰富而有效磷缺乏的现状,以松辽平原玉米带的黑土为供试土壤,通过室内分析与田间试验、功能菌株的筛选与产品研制、生物技术与常规技术相结合的技术路线,进行了黑土土壤微生物区系变化规律；土壤养分与酶活性的时空变化规律及其相关性；高效溶磷微生物菌株的筛选、鉴定及发酵条件；菌株的代谢产物分析及其溶磷机理；溶磷生物肥料的研制及其应用效果等研究。明确了溶磷生物肥料活化土壤难溶性磷、提高化学磷肥利用率的作用,为磷肥的合理施用提供理论依据。研究结果如下：1、黑土土壤微生物区系与酶活性变化规律
不同处理黑土微生物区系空间变化结果表明：在玉米整个生育期,细菌、真菌和放线菌的活动,主要集中在0-30cm土层内,30cm以下数量很少。不同处理黑土微生物区系时间变化结果表明：玉米播种前、拔节-抽雄期和收获后,宽窄行处理土壤中的细菌数量显著高于常规耕作；真菌变化在拔节-抽雄期常规耕作明显高于宽窄行处理；放线菌数量在苗期-拔节期和收获后宽窄行休闲种植的处理高于常规耕作,而在拔节-抽雄期则是常规耕作高于宽窄行休闲种植的处理。黑土酶活性在玉米生育期的时间变化结果表明：脲酶活性在玉米灌浆期最高；中性磷酸酶灌浆期降至最低；酸性磷酸酶和碱性磷性酶活性,在播种前宽窄行休闲种植高于常规耕作。
2、黑土土壤养分的时空变化规律
玉米整个生育期,不同处理黑土各个土层内均有较高的碱解氮含量,并且在播种前常规耕作明显高于宽窄行处理,生育中期则是宽窄行处理高于常规耕作,收获后处理间水平一致；有效磷不同处理均表现出养分表聚现象,且宽窄行休闲种植,在玉米整个生育期的不同土层内均高于常规耕作；速效钾含量随着土层的加深降低不十分明显；全氮、全磷含量随着土层的加深逐渐降低；全钾含量在播种前和收获后常规耕作高于宽窄行休闲种植,随着土层的加深降低不明显；有机质含量在玉米生育期内基本在2%-3%之间；pH值随着土层的加深有逐渐升高的趋势。
3、黑土土壤养分与酶活性的相关分析
土壤脲酶与土壤全氮、碱解氮、十壤有机质和pH值都表现了一定的相关性,与土壤磷、钾养分和土壤微生物区系没有表现出相关性。土壤磷酸酶(酸性、碱性和中性)在玉米关键生育期,与土壤全氮、全磷、有机质、pH值均显著或极显著相关,与土壤全钾含量没有表现出明显的相关性；磷酸酶在播种前与土壤微生物数量均达到极显著相关,在玉米生长后期没有表现有规律的相关性。土壤微生物数量与土壤有机质、土壤全氮和全磷呈显著或极显著正相关,与速效养分相关性不明显。
4、高效溶磷微生物菌株的筛选、鉴定、溶磷能力测定及发酵条件研究
从松辽平原丘米带的贫磷黑土中分离筛选了高效溶磷菌80余株。对筛选获得的菌株进行溶磷、产生明胶酶、蛋白酶以及纤维素酶的能力进行综合评价,获得了综合能力较强的菌株24株。通过DNA测序和16SrRNA系统发育树构建,将筛选获得的菌株分别鉴定为枯草芽孢杆菌(Bacillus subtilis Q1)、苏云金芽胞杆菌(Bacillus thuringiensis serovar kurstaki AR-10)、荧光假单胞菌(Pseudomonas fluorescens Pf0-1)、蜡状芽孢杆菌(Bacillus cereus生产禁用菌)、伯克金氏菌(Burkholderia sp. CEB01056附DNA序列及16S rRNA系统发育树)、Oxalobacteraceae bacterium NR186、肠杆菌(Enterobacter ludwigii K9)和解磷巨大芽孢杆菌(Bacillus megaterium)。
对筛选获得解磷巨大芽孢杆菌(Bacillus megaterium),进行了溶磷圈和发酵液有效磷浓度测定,结果该菌株在以磷酸钙为唯一磷源的培养基上,能够形成明显的溶磷圈；发酵培养到第4天,发酵液的有效磷含量达到420mg/kg。溶磷菌发酵反应条件优化试验表明：溶磷菌发酵液的最适培养温度为28℃,培养时间为72h,接种浓度为0.1%,最佳碳源为10g/L的葡萄糖,最佳氮源为0.5g/L的硫酸钱。
5、高效溶磷微生物菌株的溶磷机理研究
通过粒子大小分析仪,测定了溶磷菌发酵液中的磷酸钙颗粒大小,对照处理的磷酸钙粒径,主要集中在132.7nm～181.7nm之间,溶磷菌液处理的磷酸钙粒径,主要集中77.5nm～124.2nm之间,磷酸钙颗粒明显变小；通过扫描式电子显微镜观察,溶磷菌发酵液对磷酸钙表面的破坏作用,结果未经发酵液处理的正常磷酸钙表现光滑坚硬,通过菌液处理的表面凸凹不平,表明发酵菌液对磷酸钙有明显的溶蚀作用；通过高效液相色谱分析仪(HPLC)和傅里叶变换红外光谱分析仪(FTIR),对溶磷菌发酵液代谢产物进行分析,确定了溶磷菌最终的发酵产物为葡萄糖酸。
6、溶磷生物肥料的研制及其对黑土磷有效性的调控效果
选择适宜的载体和稳效助剂,将筛选获得的高效溶磷株研制成固态、液态两种剂型5种溶磷生物肥料,并进行田间应用效果试验：结果溶磷生物肥的施入,可使玉米的生物产量增加5.67%-10.02%,籽实产量增加521%—13.18%；溶磷生物肥在大豆上施用,生物产量和籽粒产量分别增加5.45%和9.24%。溶磷生物肥的施入,可明显改善黑土有效磷含量。与基质对照相比,在玉米拔节期土壤有效磷含量增加0.89mg/kg-3.53mg/kg,在玉米收获后土壤有效磷含量增加9.03mg/kg～11.91mg/kg;在大豆开花期溶磷生物肥与化肥配施,有效磷含量比单施化肥增加13.48mg/kg。溶磷菌液的施入,可使玉米整个生育期磷肥利用率达到21.75%～37.98%,可使玉米对土壤难溶性磷的吸收效率达到10.94%。
溶磷生物肥对作物(玉米、大豆)植株吸磷量,表现出了明显的促进作用,与植株的生物产量和土壤有效磷含量变化趋势相同。尤其是5号溶磷生物肥,在玉米整个生育期单株吸磷量比基质对照高241.77mg；大豆整个生育期单株吸磷量比对照高36.37mg。从对土壤磷的活化效果分析,在玉米整个生育期总吸磷量,高出不施溶磷生物肥处理22026mg,大豆整个生育期单株吸磷总量,高出不施溶磷生物肥44.75mg。充分说明了溶磷生物肥对土壤难溶性磷有明显的活化作用。
在我省不同生态区域示范应用,溶磷生物肥具有明显的增产效果：在吉林省中部(公主岭)的半湿润半丁旱地区,增产幅度在8.2%～11.7%；在西部(乾安县)的干旱地区增产幅度在4.1%～8.3%；在东部(桦甸市)的湿润冷凉区增产幅度在5.6%～13.0%。溶磷生物对不同生态区域的不同土壤类型有一定的生态适应性。
According to the present status that soil potentially phosphorus was rich, whereas available phosphorus was deficiency, black soil from Corn Belt of Songliao Plain was chosen as study object. Some field trials combined with indoor analysis, screening strains and development of the product, biological control and the cultivation technology were carried out to investigate the mechanism of soil microbiota in black soil, the spatial-temporal change law of soil nutrient and enzyme activities and the correlation, screening and identification of the PSB and their mechanisms on phosphorus solubilization, elevate the ability in phosphorus uptake, and development of phosphorus solubilization microbial fertilizer, and the effects of field experiments, etc. This project wishes to find the mechanisms of soil phosphorus bioactivation and provide theory evidence to scientific utilization of soil phosphorus and rational application phosphorus fertilizer. The research results are as follows:
1. Variation of soil microflora and enzyme activity in black soil
The activity of bacterium. Fungi, and actinomycete mainly concentrate in the0-30cm scope with different conservation tillage modes, the quantity of soil microorganisms under30cm are very few, During the different growth period of Maize. The bacterium populations in narrow-row and wide spacing practice methods were more than that of cultivation tillage methods at corn pre-planting, jointing and post-harvest stages, the changes of Fungi populations in cultivation tillage methods were more than that of narrow-row and wide spacing practice methods at jointing stage to tasseling stage, however at seedling stage to jointing stage the actinomycete populations in narrow-row and wide spacing practice methods were more than that cultivation tillage methods, At jointing stage to tasseling actinomycete populations in stage cultivation tillage methods were more than that of narrow-row and wide spacing practice methods, soil urease activity was the most highest in Maize at filling stage,and the activity of soil neutral phosphatase were the lowest, the activities of acid phosphatase and alkaline phosphatase at pre-planting stage in narrow-row and wide spacing practice methods were higher than that of cultivation tillage methods.
2. The spatial-temporal variation of soil nutrients
The higher NH4+-N was measured at each soil layer at different growth stages of maize, and that of cultivation tillage methods were higher than that of narrow-row and wide spacing practice methods at pre-planting stage.while higher available phosphorus was measured at table of the poly. For potassium content, a minor change was found at each layer. As well as, total nitrogen, phosphorous was decreased with the soil depth and minor changes were investigated in total potassium. During the growth period, organic matter content was maintained at2～3%and the pH value was positively correlated with the soil depth which demonstrates that both applications may contribute to soil acidification.
3. Correlation Analysis of black soil nutrients and enzyme activity
The correlations among soil enzyme activities, soil nutrient and variations of microflora was investigated at black soil are of central Jilin during different developmental stages of maize. Soil urease activity was significantly correlated with soil total N and total P contents (p≦0.01) at post-harvest stage and pH (p≦0.01) after filling stage. As well as. soil urease was significantly correlated with alkali-hydrolyzable N (p≦0.05) at pre-planting, jointing and post-harvest stages. However, soil urease showed no significant correlations with available P, K contents and no correlations with the amount of soil microflora, respectively. Soil phosphatase (acid, alkaline and neutral) activities were significantly correlated with soil total N (p≦0.05) and total P (p≦0.01) during the main development stages of maize. As well as, soil phosphatase (acid, alkaline and neutral) activities showed relatively significant correlations with available P, K contents and pH (negative) except filling stage. It means that, activities of soil phosphatase were mostly infected by the distribution of soil nutrient and pH values. Soil phosphatase activities were significantly correlated with soil microflora (p≦0.01) at pre-planting stages and no continuous regularity correlations were investigated at the end of maize growth. Soil enzyme activities showed relative significant correlation with soil organic matters. While no significant correlation was investigated with soil total K content.
4. Screening and identification of phosphorus solubilizing microorganisms and Determination of phosphorus solubilization capacity and fermentation conditions of phosphorus solubilizing microorganisms
The80strains of PSB were screened from phosphorus deficiency black soil samples in maize cultivation belt of northeast china.Then.24strains of microbial were isolated by their abilities in phosphorus solubilization, gelatinase, and protease and cellulose production ability. These strains were identified as Bacillus subtilis strain Q1, Bacillus thuringiensis serovar kurstaki strain AR-10. Pseudomonas fluorescens Pf0-1, Bacillus cereus, Burkholderia sp. CEB01056, Oxalobacteraceae bacterium NR186、Enterobacter ludwigii strain K9and Bacillus megateriun.
In the following study, Bacillus megaterium was selected by their strong ability in phosphorus solubilizing for this work. The soluble-P concentration continuously increased during the incubation periods and the total amount of soluble P released in culture filtrate was detected at420mg.L-1after4days of inoculation. The optimal culture condition for the phosphatase production by Bacillus megaterium was72hours of cultivation at28℃in a medium composed of0.1%inoculation concentration.1%glucose.0.5g.L-ammonium sulfate at initial pH7.0.
5. Phosphorus solubilization mechanism of phosphorus solubilizing microorganisms
The particle size of calcium phosphorus surface was found to destroyed and the size were changed to between77fnm～124.2nm. while no changes in the surface and size were found in the control with the size132.7nm～181.7nm. The bacterium was found to release gluconic acid by the HPLC and FT-IR analysis and the solubilization of hydroxyapatite in the liquid medium by a significant drop in pH to4.1from an initial pH
6. The development of phosphorus solubilizing fertilizer and the effect of phosphorus solubilizing biofertilizer on improving effectivenessof black soil phosphorus and utilization efficiency
In this study, insoluble phosphorus could be indented and screened and solubilizing bacillus megaterium and burkholderia were functional strains. The4biological fertilities from different dosage forms were experienced in the field. The vegetative growth, photosynthesis, biomass production and grain yield were improved by utility of biological fertilities. The results showed that biomass productions of maize were increased from5.67%to10.02%and grain yield were escalated from5.21%to13.18%. Under different condition of utility of phosphorus fertility, productions of biomass and grain yield increased2.47%and1.79%respectively by using biological fertility. The biomass and grain yield of soybean increased5.45%and9.24%individually by utility of biological fertility.
The condition of phosphorus in the soil were improved by the utility of dissolving phosphorus obviously at main growth stage of different crops such as maize and soybean. The effective phosphorus in the soil was increased from0.89mg/kg to3.53mg/kg at jointing of maize by utility of biological fertility. However, after harvesting of maize, effective phosphorus was increased from9.03mg/kg to11.91mg/kg to11.91mg/kg. The available phosphorus in the soil was increased13.48mg/kg dissolving phosphorus fertility than single fertility. The results showed that dissolving biological fertility could enhance utility ratio of fertility and improve the condition of phosphorus in soil. In total, the dissolving biological fertility could improve the phosphorus efficiency in field experience. The results showed that utility ratio of phosphorus was increased from21.75%to37.98%.
Application amount of chemical phosphorus fertilizer was reduced by1/3～1/2by microbial phosphorus fertilizer amendment and activation rate of insoluble phosphorus in different ecological zone of Jilin province and yield of maize were increased obviously. The scope of yield was increased from8.2%to11.7%in semi moist zone in the middle of Jilin province. The scope of yield was increased from4.1%to8.3%in semiarid zone in the west zone of Jilin province. The scope of yield was increased from5.6to13.0%in most and cold zone in the east zone of Jilin province. These results showed that dissolving biological fertility could reduce the utility of phosphorus and have better ecological function to save energy source, protect environment and increase profits.
不同处理黑土微生物区系空间变化结果表明：在玉米整个生育期,细菌、真菌和放线菌的活动,主要集中在0-30cm土层内,30cm以下数量很少。不同处理黑土微生物区系时间变化结果表明：玉米播种前、拔节-抽雄期和收获后,宽窄行处理土壤中的细菌数量显著高于常规耕作；真菌变化在拔节-抽雄期常规耕作明显高于宽窄行处理；放线菌数量在苗期-拔节期和收获后宽窄行休闲种植的处理高于常规耕作,而在拔节-抽雄期则是常规耕作高于宽窄行休闲种植的处理。黑土酶活性在玉米生育期的时间变化结果表明：脲酶活性在玉米灌浆期最高；中性磷酸酶灌浆期降至最低；酸性磷酸酶和碱性磷性酶活性,在播种前宽窄行休闲种植高于常规耕作。
2、黑土土壤养分的时空变化规律
玉米整个生育期,不同处理黑土各个土层内均有较高的碱解氮含量,并且在播种前常规耕作明显高于宽窄行处理,生育中期则是宽窄行处理高于常规耕作,收获后处理间水平一致；有效磷不同处理均表现出养分表聚现象,且宽窄行休闲种植,在玉米整个生育期的不同土层内均高于常规耕作；速效钾含量随着土层的加深降低不十分明显；全氮、全磷含量随着土层的加深逐渐降低；全钾含量在播种前和收获后常规耕作高于宽窄行休闲种植,随着土层的加深降低不明显；有机质含量在玉米生育期内基本在2%-3%之间；pH值随着土层的加深有逐渐升高的趋势。
3、黑土土壤养分与酶活性的相关分析
土壤脲酶与土壤全氮、碱解氮、十壤有机质和pH值都表现了一定的相关性,与土壤磷、钾养分和土壤微生物区系没有表现出相关性。土壤磷酸酶(酸性、碱性和中性)在玉米关键生育期,与土壤全氮、全磷、有机质、pH值均显著或极显著相关,与土壤全钾含量没有表现出明显的相关性；磷酸酶在播种前与土壤微生物数量均达到极显著相关,在玉米生长后期没有表现有规律的相关性。土壤微生物数量与土壤有机质、土壤全氮和全磷呈显著或极显著正相关,与速效养分相关性不明显。
4、高效溶磷微生物菌株的筛选、鉴定、溶磷能力测定及发酵条件研究
从松辽平原丘米带的贫磷黑土中分离筛选了高效溶磷菌80余株。对筛选获得的菌株进行溶磷、产生明胶酶、蛋白酶以及纤维素酶的能力进行综合评价,获得了综合能力较强的菌株24株。通过DNA测序和16SrRNA系统发育树构建,将筛选获得的菌株分别鉴定为枯草芽孢杆菌(Bacillus subtilis Q1)、苏云金芽胞杆菌(Bacillus thuringiensis serovar kurstaki AR-10)、荧光假单胞菌(Pseudomonas fluorescens Pf0-1)、蜡状芽孢杆菌(Bacillus cereus生产禁用菌)、伯克金氏菌(Burkholderia sp. CEB01056附DNA序列及16S rRNA系统发育树)、Oxalobacteraceae bacterium NR186、肠杆菌(Enterobacter ludwigii K9)和解磷巨大芽孢杆菌(Bacillus megaterium)。
对筛选获得解磷巨大芽孢杆菌(Bacillus megaterium),进行了溶磷圈和发酵液有效磷浓度测定,结果该菌株在以磷酸钙为唯一磷源的培养基上,能够形成明显的溶磷圈；发酵培养到第4天,发酵液的有效磷含量达到420mg/kg。溶磷菌发酵反应条件优化试验表明：溶磷菌发酵液的最适培养温度为28℃,培养时间为72h,接种浓度为0.1%,最佳碳源为10g/L的葡萄糖,最佳氮源为0.5g/L的硫酸钱。
5、高效溶磷微生物菌株的溶磷机理研究
通过粒子大小分析仪,测定了溶磷菌发酵液中的磷酸钙颗粒大小,对照处理的磷酸钙粒径,主要集中在132.7nm～181.7nm之间,溶磷菌液处理的磷酸钙粒径,主要集中77.5nm～124.2nm之间,磷酸钙颗粒明显变小；通过扫描式电子显微镜观察,溶磷菌发酵液对磷酸钙表面的破坏作用,结果未经发酵液处理的正常磷酸钙表现光滑坚硬,通过菌液处理的表面凸凹不平,表明发酵菌液对磷酸钙有明显的溶蚀作用；通过高效液相色谱分析仪(HPLC)和傅里叶变换红外光谱分析仪(FTIR),对溶磷菌发酵液代谢产物进行分析,确定了溶磷菌最终的发酵产物为葡萄糖酸。
6、溶磷生物肥料的研制及其对黑土磷有效性的调控效果
选择适宜的载体和稳效助剂,将筛选获得的高效溶磷株研制成固态、液态两种剂型5种溶磷生物肥料,并进行田间应用效果试验：结果溶磷生物肥的施入,可使玉米的生物产量增加5.67%-10.02%,籽实产量增加521%—13.18%；溶磷生物肥在大豆上施用,生物产量和籽粒产量分别增加5.45%和9.24%。溶磷生物肥的施入,可明显改善黑土有效磷含量。与基质对照相比,在玉米拔节期土壤有效磷含量增加0.89mg/kg-3.53mg/kg,在玉米收获后土壤有效磷含量增加9.03mg/kg～11.91mg/kg;在大豆开花期溶磷生物肥与化肥配施,有效磷含量比单施化肥增加13.48mg/kg。溶磷菌液的施入,可使玉米整个生育期磷肥利用率达到21.75%～37.98%,可使玉米对土壤难溶性磷的吸收效率达到10.94%。
溶磷生物肥对作物(玉米、大豆)植株吸磷量,表现出了明显的促进作用,与植株的生物产量和土壤有效磷含量变化趋势相同。尤其是5号溶磷生物肥,在玉米整个生育期单株吸磷量比基质对照高241.77mg；大豆整个生育期单株吸磷量比对照高36.37mg。从对土壤磷的活化效果分析,在玉米整个生育期总吸磷量,高出不施溶磷生物肥处理22026mg,大豆整个生育期单株吸磷总量,高出不施溶磷生物肥44.75mg。充分说明了溶磷生物肥对土壤难溶性磷有明显的活化作用。
在我省不同生态区域示范应用,溶磷生物肥具有明显的增产效果：在吉林省中部(公主岭)的半湿润半丁旱地区,增产幅度在8.2%～11.7%；在西部(乾安县)的干旱地区增产幅度在4.1%～8.3%；在东部(桦甸市)的湿润冷凉区增产幅度在5.6%～13.0%。溶磷生物对不同生态区域的不同土壤类型有一定的生态适应性。
According to the present status that soil potentially phosphorus was rich, whereas available phosphorus was deficiency, black soil from Corn Belt of Songliao Plain was chosen as study object. Some field trials combined with indoor analysis, screening strains and development of the product, biological control and the cultivation technology were carried out to investigate the mechanism of soil microbiota in black soil, the spatial-temporal change law of soil nutrient and enzyme activities and the correlation, screening and identification of the PSB and their mechanisms on phosphorus solubilization, elevate the ability in phosphorus uptake, and development of phosphorus solubilization microbial fertilizer, and the effects of field experiments, etc. This project wishes to find the mechanisms of soil phosphorus bioactivation and provide theory evidence to scientific utilization of soil phosphorus and rational application phosphorus fertilizer. The research results are as follows:
1. Variation of soil microflora and enzyme activity in black soil
The activity of bacterium. Fungi, and actinomycete mainly concentrate in the0-30cm scope with different conservation tillage modes, the quantity of soil microorganisms under30cm are very few, During the different growth period of Maize. The bacterium populations in narrow-row and wide spacing practice methods were more than that of cultivation tillage methods at corn pre-planting, jointing and post-harvest stages, the changes of Fungi populations in cultivation tillage methods were more than that of narrow-row and wide spacing practice methods at jointing stage to tasseling stage, however at seedling stage to jointing stage the actinomycete populations in narrow-row and wide spacing practice methods were more than that cultivation tillage methods, At jointing stage to tasseling actinomycete populations in stage cultivation tillage methods were more than that of narrow-row and wide spacing practice methods, soil urease activity was the most highest in Maize at filling stage,and the activity of soil neutral phosphatase were the lowest, the activities of acid phosphatase and alkaline phosphatase at pre-planting stage in narrow-row and wide spacing practice methods were higher than that of cultivation tillage methods.
2. The spatial-temporal variation of soil nutrients
The higher NH4+-N was measured at each soil layer at different growth stages of maize, and that of cultivation tillage methods were higher than that of narrow-row and wide spacing practice methods at pre-planting stage.while higher available phosphorus was measured at table of the poly. For potassium content, a minor change was found at each layer. As well as, total nitrogen, phosphorous was decreased with the soil depth and minor changes were investigated in total potassium. During the growth period, organic matter content was maintained at2～3%and the pH value was positively correlated with the soil depth which demonstrates that both applications may contribute to soil acidification.
3. Correlation Analysis of black soil nutrients and enzyme activity
The correlations among soil enzyme activities, soil nutrient and variations of microflora was investigated at black soil are of central Jilin during different developmental stages of maize. Soil urease activity was significantly correlated with soil total N and total P contents (p≦0.01) at post-harvest stage and pH (p≦0.01) after filling stage. As well as. soil urease was significantly correlated with alkali-hydrolyzable N (p≦0.05) at pre-planting, jointing and post-harvest stages. However, soil urease showed no significant correlations with available P, K contents and no correlations with the amount of soil microflora, respectively. Soil phosphatase (acid, alkaline and neutral) activities were significantly correlated with soil total N (p≦0.05) and total P (p≦0.01) during the main development stages of maize. As well as, soil phosphatase (acid, alkaline and neutral) activities showed relatively significant correlations with available P, K contents and pH (negative) except filling stage. It means that, activities of soil phosphatase were mostly infected by the distribution of soil nutrient and pH values. Soil phosphatase activities were significantly correlated with soil microflora (p≦0.01) at pre-planting stages and no continuous regularity correlations were investigated at the end of maize growth. Soil enzyme activities showed relative significant correlation with soil organic matters. While no significant correlation was investigated with soil total K content.
4. Screening and identification of phosphorus solubilizing microorganisms and Determination of phosphorus solubilization capacity and fermentation conditions of phosphorus solubilizing microorganisms
The80strains of PSB were screened from phosphorus deficiency black soil samples in maize cultivation belt of northeast china.Then.24strains of microbial were isolated by their abilities in phosphorus solubilization, gelatinase, and protease and cellulose production ability. These strains were identified as Bacillus subtilis strain Q1, Bacillus thuringiensis serovar kurstaki strain AR-10. Pseudomonas fluorescens Pf0-1, Bacillus cereus, Burkholderia sp. CEB01056, Oxalobacteraceae bacterium NR186、Enterobacter ludwigii strain K9and Bacillus megateriun.
In the following study, Bacillus megaterium was selected by their strong ability in phosphorus solubilizing for this work. The soluble-P concentration continuously increased during the incubation periods and the total amount of soluble P released in culture filtrate was detected at420mg.L-1after4days of inoculation. The optimal culture condition for the phosphatase production by Bacillus megaterium was72hours of cultivation at28℃in a medium composed of0.1%inoculation concentration.1%glucose.0.5g.L-ammonium sulfate at initial pH7.0.
5. Phosphorus solubilization mechanism of phosphorus solubilizing microorganisms
The particle size of calcium phosphorus surface was found to destroyed and the size were changed to between77fnm～124.2nm. while no changes in the surface and size were found in the control with the size132.7nm～181.7nm. The bacterium was found to release gluconic acid by the HPLC and FT-IR analysis and the solubilization of hydroxyapatite in the liquid medium by a significant drop in pH to4.1from an initial pH
6. The development of phosphorus solubilizing fertilizer and the effect of phosphorus solubilizing biofertilizer on improving effectivenessof black soil phosphorus and utilization efficiency
In this study, insoluble phosphorus could be indented and screened and solubilizing bacillus megaterium and burkholderia were functional strains. The4biological fertilities from different dosage forms were experienced in the field. The vegetative growth, photosynthesis, biomass production and grain yield were improved by utility of biological fertilities. The results showed that biomass productions of maize were increased from5.67%to10.02%and grain yield were escalated from5.21%to13.18%. Under different condition of utility of phosphorus fertility, productions of biomass and grain yield increased2.47%and1.79%respectively by using biological fertility. The biomass and grain yield of soybean increased5.45%and9.24%individually by utility of biological fertility.
The condition of phosphorus in the soil were improved by the utility of dissolving phosphorus obviously at main growth stage of different crops such as maize and soybean. The effective phosphorus in the soil was increased from0.89mg/kg to3.53mg/kg at jointing of maize by utility of biological fertility. However, after harvesting of maize, effective phosphorus was increased from9.03mg/kg to11.91mg/kg to11.91mg/kg. The available phosphorus in the soil was increased13.48mg/kg dissolving phosphorus fertility than single fertility. The results showed that dissolving biological fertility could enhance utility ratio of fertility and improve the condition of phosphorus in soil. In total, the dissolving biological fertility could improve the phosphorus efficiency in field experience. The results showed that utility ratio of phosphorus was increased from21.75%to37.98%.
Application amount of chemical phosphorus fertilizer was reduced by1/3～1/2by microbial phosphorus fertilizer amendment and activation rate of insoluble phosphorus in different ecological zone of Jilin province and yield of maize were increased obviously. The scope of yield was increased from8.2%to11.7%in semi moist zone in the middle of Jilin province. The scope of yield was increased from4.1%to8.3%in semiarid zone in the west zone of Jilin province. The scope of yield was increased from5.6to13.0%in most and cold zone in the east zone of Jilin province. These results showed that dissolving biological fertility could reduce the utility of phosphorus and have better ecological function to save energy source, protect environment and increase profits.
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