【作者】 钟传青；

【导师】 黄为一；

【作者基本信息】 南京农业大学 ， 微生物学， 2004， 博士

【摘要】 本文从解磷微生物的分离、筛选入手，从来自全国的多个土壤样品及种子表皮分离出了两百多株解磷微生物，经过初筛、复筛，最后得到了效果较好的解磷微生物菌株，其中包括细菌、酵母和霉菌，中国各地解磷微生物资源作了初步调查。通过菌落、菌体形态观察，繁殖方式判断以及生理生化试验检测，初步将解磷微生物P17菌株、P10菌株、Y3菌株与F4菌株分别鉴定为巨大芽孢杆菌（Bacillus megaterium）、短杆菌属（Brevibacterium）、红酵母属（Rhodotorula）和专霉属（Penicillium）。 不同解磷微生物溶磷效果不同。从试验结果来看，Ca₃（PO₄）₂、FePO₄、AlPO₄等难溶性磷酸盐容易被酵母、霉菌溶解，而磷矿粉容易被芽孢杆菌溶解，各种解磷微生物对不同化学结构含磷物质的亲和溶解能力不同。本研究所选的四株解磷微生物具有一定代表性，分别属于产芽孢杆菌、短杆菌、酵母和霉菌。研究表明，霉菌并不是对所有形态难溶磷的溶解能力都高于细菌，细菌、酵母、等在溶磷方面也发挥着重要作用。Ghani A（1994）和Kucey（1983）报道真菌溶磷能力高于细菌，这种说法存在一定片面性。从本研究来看，各种微生物对不同难溶磷酸盐有不同溶解效果。真菌生长、繁殖需要的碳源、能源较高，生长周期长，但在溶解难溶磷酸盐时发挥着生物量大、代谢产物多的优势；细菌P17菌株溶解磷矿粉的能力强，且代时短。因此生物磷肥应该考虑由细菌和真菌混合菌群组成。若把在溶解难溶性磷酸盐和磷矿粉方面能相得益彰的不同种类解磷微生物混合菌群作为生物磷肥，将会为农业做出更大贡献。 解磷微生物溶磷效果研究表明P17菌株接种不同来源磷矿粉为唯一磷源的发酵液中均使发酵液有效磷含量增加。可见P17菌株等解磷微生物能够有效地溶解、转化磷矿粉中的难溶磷，用微生物学途径来提高磷矿粉直接施用效果是可行的。云南海口磷矿、贵州开阳磷矿、湖北宜昌磷矿等为沉积磷块岩，而黄金卡黄磷矿、湖北黄麦岭磷矿则为变质型磷块岩，由于磷矿地质形成条件不同，磷矿粉的有效磷含量有很大差异。经P17菌株接种培养后，黄麦岭和黄金卡黄磷矿粉溶磷增幅最大，说明变质岩型磷块岩中的磷易于被解磷微生物菌株转化、溶解。通过摇瓶试验、扫描电镜观察为解磷微生物对不同来源磷矿粉的生物风化提供了证据，选出了巨大芽孢杆菌P17菌株：最适合作用的磷矿粉类型。从摇瓶试验结果可以看出，P17菌株对来源于黄麦岭、黄金卡黄的变质岩型磷矿粉有较好的溶解能力。经过P17菌株长达70d的溶解，磷矿粉的难溶磷逐渐被P17菌株溶解下来。黄麦岭、黄金卡黄磷矿粉经过P17菌株长达70d的溶解，累计有效磷含量分别由第一次接种时发酵液中的291.46mg L^-1、316.6mz L^-1增加到最终的869.71mg L_-1、837.04 mg L^-1，磷矿粉的难溶磷逐渐被P17菌株溶解下来。第十次培养后，黄麦岭磷矿粉全磷的81.02％被溶解下来，接活菌滤液中累积有效磷是对照的5.93倍，接灭活菌是对照的2.54倍。而对于黄金卡黄磷矿粉，P17菌株溶解了全磷总量的78.97％，接活菌滤液中累积有效磷是对照的5.4倍，接灭活菌是对照的2.32倍。接灭活菌由于带进了代谢产物如难挥发性南京农业大学博士学位论文:解磷微生物溶解磷矿粉和土壤难溶磷的特性及其溶磷方式研究酸等作用于磷矿粉，使滤液有效磷含量增加。磷矿粉中难溶磷以无机磷为主。属于变质磷块岩的黄麦岭磷矿粉和黄金卡黄磷矿粉适于巨大芽抱杆菌P17菌株生长及有效代谢产物的产生，从而易受侵蚀。经过P17菌株的长期作用，大部分转化为有效磷。为进一步证实上述结果，将磷矿粉进行了扫描电镜观察。继续接种培养时，接菌、接灭活菌、加空白培养基各处理间没有显著差异，说明磷矿粉中仍有少部分磷不能被巨大芽袍杆菌P17菌株溶解下来，表明解磷微生物只能在一定限度内溶解磷矿粉，不可能把磷矿粉中磷全部溶解下来。 过磷酸钙是由磷矿粉与硫酸反应制成的水溶性磷肥。其主要成分为磷酸一钙[Ca（HZPo;）:.H20]和难溶性硫酸钙。施入过磷酸钙后，土壤pH有细微下降。随着时间延长，pH下降趋势增大。过磷酸钙会与土壤中游离金属离子如c扩‘、Fes+、A13十等结合形成难溶性磷酸盐沉积下来，试图通过在土壤中接种微生物来改善被过磷酸钙固定化土壤的磷素营养。研究结果表明，接菌处理与未接菌处理的pH值有较小差异，与有效磷含量的增加无正相关。说明使难溶态磷转化成有效磷的过程中，pH降低不是唯一因素。加水对照的土样pH无明显变化。除加水对照外，土样的各种处理如接菌、接灭活菌、加空白培养基等有效磷含量是逐渐升高的。加过磷酸钙的各接菌处理土样中，10叼前，未灭菌土样有效磷高于灭菌土壤，而10叼后前者低于后者，说明施加过磷酸钙处理前期土著微生物有效菌群占主导地位，10叼后P17菌株起主要作用，所有结果均表明解磷微生物对黄棕壤中难溶态磷的转化有重要作用。接菌、接灭活菌、加培养基处理的磷酸酶活性均高于加水对照;接菌处理的磷酸酶活性高于接灭活菌与加培养基处理;接灭活菌与加培养基间无明显差异。取样前期灭菌土壤中磷酸酶活性不高，随着时间延长，解磷微生物数目增多，磷酸酶活性增加。磷酸酶为诱导酶，不仅?更多还原

【Abstract】 Severing and filtration of P-solubilizing microorganisms were studied in this article. More than two hundred P-solubilizing microbes were filtered again and compared with P-solubilizing microbes safe-deposited in our microbiological laboratory. P-solubilizing microbes with preferable P-solubilizing effects were found, consisting of bacteria, yeasts, and fungi. P-solubilizing microbes sources survey was conducted at the same time. Through colony and thalli configuration observation and physiological, biochemical experiment, P-solubilizing microbes strain P17, P10, Y3, F4 were identified as Bacillus megaterium, Brevibacterium, Rhodotorula and Penicillium, respectively.P-solubilizing effects and methods of different microbes were different Research results showed that such difficultly soluble phosphates as Ca3(PO4)2, FePO4, A1PO4 were dissolved by yeasts and fungi easily. Phosphate rock powder was solubilized better by bacillus. Different kinds of microbes could dissolve different chemical configuration of phosphates or other substances containing phosphorus. Microbes chosen in this study were representative of all P-solubilizing microbes, belonged to bacteria including bacillus, yeasts, and mildew respectively. Results showed that mildew could dissolve best not all the difficultly soluble phosphates. Bacillus and yeasts also played much important role in P-solubilizing. Some conferences reported that P-solubilizing ability of fungi was always higher than bacteria (Ghani A, 1994). This thesis is unilateral, to some extent. Results showed that different kinds of microbes have different P-solubilizing ability on phosphates. Carbon, nitrogen sources needed by fungi were more than bacteria, and generation time is longer; but biological gross and metabolistic products of fungi are more than bacteria, which do good to solubilization of phosphorus. P-solubilizing ability of phosphate rock powder is stronger and their generation time shorter. All results above showed biological phosphorus fertilizer need consist of mixture of bacteria and fungi. So bacteria and fungi can bring out the best in each other in P-solubilizing. More contribution would be done to agriculture.P-solubilizing effects study results showed that strain P17 could facilitate solution of phosphate rock powder from different sources. So strain P17 could solubilize and transform difficultly soluble phosphorus. Results suggested that microbiological methods to improve P-solubilizing effects of phosphate rock powder are probable. Phosphate rock powder from haikou in Yunnan province, Kaiyang inGuizhou province, yichang in Hubei province are sediment phosphate rock, while phosphate rock from huangmailing, huangjinkahuang are metamorphose phosphate rock. Available P content is different because of geology formation conditions. After inoculation by strain P17, available P content of hangmailing and huangjinkahuang phosphate rock powder were increased largely. Through shaking bottle experiment and SEM observation, biological efflorescence evidences of solubilization from huangmailing and huangjinkahang phosphate rock were brought. Accumulative available P were increased from 291.46 mg L-1, 316.6 mg L-1 to 869.71mg L-1 and 837.04 mg L-1 after 70d inoculation. Poorly soluble phosphate were solubilized by P17 gradually. 81.02% of whole P content of huangmailing phosphate rock powder was released as available P. Accumulating available P of inoculation treatment was 5.93 times of control. While strain P17 could solubilize 78.97% whole P of huangjinkahuang phosphate rock powder. Accumulating available P content after inoculation in filtrate was 5.4 times of ck. Sterilized bacteria inoculation could bring such metabolism product as nonvolatile organic acids into fermentation liquid, which could have effects on phosphate rock powder. Inorganic P is basis in difficultly soluble phosphate rock powder. Huangmailing and huangjinkahuang phosphate rock powder are metamorphose phosphate rock, which are adapted to growth and metabolism production of strain P17. Difficultly soluble P o更多还原