磷酸鈣與磷酸鐵皆為難溶性磷酸鹽，而溶磷菌能溶解這些難溶性磷酸鹽。本研究將溶磷菌培養在磷酸三鈣及磷酸鐵液體培養基中，並分析其生理特性、水溶性磷含量及胞外分泌物，來探討影響溶磷菌溶解磷酸三鈣與磷酸鐵的因子。
本研究自91株從土壤中分離純化的溶磷菌中篩選出四株溶磷菌，分別是Pseudomonas capecia、Acinetobacter calcoaceticus 、Bucillus subtilis及 Rhizobium sp.。分別接種於磷酸三鈣及磷酸鐵液體培養基中，孵育120小時，每12小時取樣一次，測定其菌數變化、pH變化、水溶性磷含量變化，並以HPLC分析胞外分泌物中的有機酸。四株溶磷菌在磷酸三鈣及磷酸鐵液體培養基中均會使pH降低，Acinetobacter calcoaceticus在鈣磷培養基中的溶磷量在第108小時可達511 PO43- mg L-1，為四株溶磷菌中最高者。而Rhizobium sp.在鐵磷培養基中的溶磷量在第60小時達73 PO43- mg L-1，為四株溶磷菌中最高者。同一株溶磷菌在鈣磷與鐵磷液體培養基中，胞外分泌物中有機酸的種類很接近，但是分泌量卻差異很大。
本研究並計算培養基在孵育期之pH、菌數、有機酸及unknown物質變化量等各因子與水溶性磷含量變化之相關係數r值。結果發現磷酸三鈣培養基在孵育期中之水溶性磷含量與pH有高度負相關，顯示菌種培養期間pH愈低愈有利於磷酸三鈣的溶解，而2-酮基-葡萄糖酸與unknown 2與水溶性磷含量有高度正相關，推測可能是造成溶磷的重要因子。而孵育期中磷酸鐵之溶解與pH的關係不大，2-酮基-葡萄糖酸、葡萄糖酸與unknown 2在鐵磷的溶磷初期與水溶性磷含量有高度正相關，因此推測2-酮基-葡萄糖酸、葡萄糖酸與unknown 2在鐵磷的溶磷初期可能扮演重要的角色。由試驗結果推測磷酸三鈣與磷酸鐵的溶磷機制可能並不相同。Calcium phosphate and ferric phosphate are the insoluble phosphate. Phosphate-solubilizing bacteria would solubilize these insoluble phosphates. The phosphate-solubilizing bacteria of this study were incubated in the tricalcium and ferric phosphate liquid medium individually, and analyzed the physiological properties, the content of water-soluble phosphorus, and the exudates of bacteria, and will evaluate the factors of phosphate solubilization of phosphate-solubilizing bacteria.
The four phosphate-solubilizing bacteria were screened from 91 isolates from Taiwan are Pseudomonas capecia, Acinetobacter calcoaceticus, Bucillus subtili, and Rhizobium sp.. Four bacteria were incubated in the tricalcium and ferric phosphate liquid medium individually for 120 hours, and sampled per 12 hours. The population changes, pH changes, the changes of the content of water-soluble phosphorus, and the exudates of bacteria were determined. The pH in the tricalcium and ferric phosphate liquid medium would be decreased by four phosphate-solubilizing bacteria. The content of water-soluble phosphorus of A. calcoaceticus in the tricalcium phosphate medium was 511 PO43-, mg L-1 at 108 hour, was the highest of the four bacteria, and the content of water-soluble phosphorus of Rhizobium sp. in the ferric phosphate medium was 73 PO43- , mg L-1, was the highest of the four bacteria. The exudates of the same bacterium in the tricalcium and ferric phosphate liquid medium were very similar, but the contents of the exudates were very different.
To calculate the correlation coefficient between the changes of pH, population, the content of exdutaes of the bacteria and the content of water-soluble phosphorus of the tricalcium and ferric phosphate medium during the incubation period. The results indicate there is a negative linear correlation between the changes of pH and the content of water-soluble phosphorus in the tricalcium phosphate medium. There is a linear correlation between the changes of the content of 2-keto-D-gluconic acid, unknown 2 and the content of water-soluble phosphorus in the tricalcium phosphate medium. The result implicated 2-keto-D-gluconic acid and unknown 2 are the factors of solubilizing tricalcium phosphate possibly. There is no linear correlation between the changes of pH and the content of water-soluble phosphorus in the ferric phosphate medium. There is a linear correlation between the changes of the content of 2-keto-D-gluconic acid, D-gluconic acid, unknown 2 and the content of water-soluble phosphorus in the ferric phosphate medium at early incubation period. The result implicated 2-keto-D-gluconic acid, D-gluconic acid, and unknown 2 are the important factors of solubilizing ferric phosphate possibly at early incubation period. The result implicated the mechanism of solubilizing tricalcium phosphate and ferric phosphate are different possibly.中文摘要……………………………………………………….Ⅰ
英文摘要……………………………………………………….Ⅲ
目錄…………………………………………………………….Ⅵ
表次…………………………………………………………….Ⅷ
圖次…………………………………………………………….Ⅹ
壹、前言………………………………………………………..1
貳、前人研究…………………………………………………..3
一、 土壤中無機磷的型態及其重要性……………………..3
二、 溶磷微生物之研究發展………………………………..3
三、 溶磷菌溶磷機制之探討………………………………..4
四、 溶磷菌接種至土壤中之效應………………………….11
五、 接種溶磷菌對植物之效應…………………………….11
參、材料與方法……………………………………………….14
一、 菌株之篩選與保存…………………………………….14
二、 菌株之生長及生理特性分析………………………….18
三、 液體培養基中水溶性磷含量的變化………………….18
四、 溶磷菌胞外分泌物之分析…………………………….19
肆、結果與討論……………………………………………….21
一、 受試菌株的篩選……………………………………….21
二、 受試菌株之生長及生理特性分析…………………….23
1. 受試菌株在鈣磷及鐵磷培養基中pH的變化……….23
2. 受試菌株在鈣磷及鐵磷培養基中菌數的變化………28
三、 受試菌株在液體培養基中水溶性磷含量變化………28
四、 受試菌株胞外分泌物之分析…………………………34
五、 探討各因子與受試菌株溶磷作用的關係…………….54
伍、結論………………………………………………………..65
陸、參考文獻…………………………………………………..6
