- 學位論文
耐高溫溶磷微生物分離及多功能性生物肥料製作
Thermo-Tolerant Phosphate-Solubilizing Microbe Isolation and Multi-Functional Biofertilizer Preparation
摘要
為了偵測堆肥和生物肥料堆積過程中溫和耐高溫溶磷微生物菌相變化及其溶磷活性，使用National Botanical Research Institute’s phosphate growth medium (NBRIP)、Pikovsakaya’s medium (PVK)及sucrose calcium phosphate medium (SCP)三種培養基進行研究。三種磷酸鈣培養基均可應用於偵測生物肥料和豬糞堆肥堆積過程中溫和耐高溫溶磷微生物菌相變化。但耐高溫溶磷微生物分離株則以PVK平板檢測法於25和50°C培養時均可得較快生長和較高溶三磷化鈣活性。因此PVK為偵測堆肥和生物肥料耐高溫溶磷微生物菌相及其溶三磷化鈣活性之較佳培養基。雞糞堆肥測得較高堆肥溫度及耐高溫溶磷微生物族群，而豬糞堆肥則為較低堆肥溫度及較高中溫溶磷微生物族群。由禽畜糞堆肥採集22次和生物肥料4次分別以NBRIP、PVK及SCP平板檢測法進行篩選得977株溶磷微生物分離株，挑選生長快速、產生明顯溶磷圈及高溶三磷化鈣活性參數(TCPSAI)之47株耐高溫溶磷細菌、6株耐高溫溶磷放線菌及4株耐高溫溶磷黴菌再進行NBRIP、PVK及SCP液態檢測法。PVK為耐高溫溶磷微生物表現溶三磷化鈣活性最佳的培養基。PVK於25°C培養時最適合耐高溫溶磷黴菌表現溶三磷化鈣活性和酸化培養基能力，而PVK於50°C培養時最適合耐高溫溶磷細菌和放線菌表現溶三磷化鈣活性和酸化培養基能力。耐高溫溶磷微生物產生有機酸為其主要的溶無機磷機制。耐高溫溶磷細菌表現溶三磷化鈣磷活性與其酸化培養基能力有顯著(P>0.001)負相關，但與NBRIP和PVK平板檢測法測得TCPSAI有顯著(P>0.05)正相關而與菌落和溶磷圈直徑並無明顯相關。於25和50°C培養時，8株耐高溫溶磷細菌分離株(C45、D12、F10、F18、G39、H28、O1、V1及V8)、2株耐高溫溶磷放線菌(J57和W6)及2株耐高溫溶磷黴菌(O4和P50)表現較高溶三磷化鈣活性。8株細菌分離株和4株黴菌分離株具溶三磷化鈣、磷酸鐵、磷酸鋁、hydroxyapatite以及以色列磷礦石活性，其中細菌分離株C45、F18、O1及V1與黴菌分離株O4、P50及T13於25和50°C培養時均具有溶解此5種磷酸鹽活性。細菌分離株F18於50°C培養時得較高溶三磷化鈣活性(532.5±77.2 µg ml-1)、溶磷酸鋁活性(119.9±3.1 µg ml-1)、溶磷酸鐵活性(10.2±0.4 µg ml-1)及溶以色列磷礦石活性(433.1±64.0 µg ml-1)。細菌分離株F10於25°C時得較高溶三磷化鈣活性(594.5±88.0 µg ml-1)。黴菌分離株O4於25°C時得較高溶磷酸鋁活性(106.2±7.9 µg ml-1)和溶磷酸鐵活性(92.4±5.8 µg ml-1)。放線菌分離株J57得較高溶以色列磷礦石活性(288.1±38.6 µg ml-1)。10株細菌分離株和4株黴菌分離株具中溫和耐高溫澱粉、纖維素、幾丁質及果膠質分解酵素與固氮酵素活性。細菌分離株A3、C45、O1及V1與黴菌分離株O4和T13於25和50°C培養時均具有此6種酵素能力。細菌分離株A3具較高中溫和耐高溫纖維、蛋白質及脂質分解酵素與固氮酵素活性。許多分離株於25和50°C培養時表現抗菌活性。接種耐高溫溶磷微生物於農業和禽畜糞廢棄物堆肥可加速腐熟、增加可溶性磷含量及提高品質，且增進中溫和耐高溫溶磷、纖維素分解、蛋白質分解及脂質分解微生物生長以製備多功能性生物肥料。相較於耐高溫溶磷放線菌和黴菌，接種耐高溫溶磷細菌可縮短腐熟時間、增進生物肥料品質及釋放更多可溶性磷，且以接種A3、C45、F18及V1為較佳。添加適量(1-5% w/w)松樹素可加速農業和禽畜糞廢棄物堆肥於好氣堆積下之腐熟速度，提升耐高溫溶磷和纖維素分解微生物族群及可溶性磷含量。混合接種數種耐高溫溶磷微生物或配合松樹素一起添加於農業廢棄物、廚餘、蔬果市場及禽畜糞廢棄物，可製備成多功能生物肥料。具生物堆肥製作、資源回收、農業生產及永續農業經營之應用價值。
並列摘要
In order to survey the mesophilic and thermo-tolerant phosphate-solubilizing microbial populations during compost and biofertilizer preparation, National Botanical Research Institute’s phosphate growth medium (NBRIP)、Pikovsakaya’s medium (PVK), and sucrose calcium phosphate medium (SCP) were used. These three kinds of tricalcium phosphate (TCP) media were suitable to determine the populations of mesophilic and thermo-tolerant phosphate-solubilizing microbes. Thermo-tolerant phosphate-solubilizing microbial isolates displayed the faster growth rates and the higher TCP-solubilizing activities grown at 25 and 50°C by PVK plate assay, and the growths and TCP-solubilizing activities shown by plate assay were not related with the TCP isolation medium. Therefore, PVK is definitely presumed the best medium not only for determining the populations of mesophilic and thermo-tolerant phosphate-solubilizing microbes in compost and biofertilizer preparation, but also for evaluating the TCP-solubilizing activities of thermo-tolerant phosphate-solubilizing microbes. The poultry and livestock waste composts were sampled 22 times and the biofertilizers were done 4 times in this study. Nine hundred and seventy seven phosphate-solubilizing microbes were isolated by serial dilution and pour plate method, and determined the TCP-solubilizing activities by plate assay with NBRIP, PVK, and SCP. Forty seven thermo-tolerant phosphate-solubilizing bacteria, 6 thermo-tolerant phosphate-solubilizing actinomycetes, and 4 thermo-tolerant phosphate-solubilizing fungi were further selected for the fast growths, manifest halo surrounding the colony, and high TCP-solubilizing activities indices (TCPSAI) at 25 or 50°C. PVK was the best medium with which the thermo-tolerant phosphate-solubilizing fungi could perform their TCP-solubilizing activities and medium-acidifying abilities grown at 25°C, while thermo-tolerant phosphate-solubilizing actinomycetes and bacteria could do at 50°C. Production of organic acids by the thermo-tolerant phosphate-solubilizing microbial isolates for solubilizing the inorganic phosphorus is the major mechanism. The inorganic phosphate-solubilizing activity and acidification ability of the thermo-tolerant phosphate-solubilizing bacteria showed the significantly (P>0.001) negative correlation. The TCPSAI obtained by NBRIP and PVK plate assay represented the significantly (P>0.05) positive correlation with the true TCP-solubilizing activity by broth assay, however, the diameters of colony sizes and clear zones did not. Eight thermo-tolerant phosphate-solubilizing bacterial isolates (C45, D12, F10, F18, G39, H28, O1, V1, and V8), 2 thermo-tolerant phosphate-solubilizing actinomycete isolates (J57 and W6), and 2 thermo-tolerant phosphate-solubilizing fungi manifested the high TCP-solubilizing activity by broth assay at 25 and/or 50°C. Eight bacterial and 4 fungal isolates possessed TCP-, aluminum phosphate-, iron phosphate-, hydroxyapatide-, and Israel rock phosphate-solubilizing activities, and among them, bacterial isolates C45, F18, O1, and V1, and fungal isolates O4, P50, T13 expressed this 5 kinds of inorganic phosphate-solubilizing activities at both 25 and 50°C. The bacterial isolate F18 manifested the higher Ca3(PO4)2-solubilizing activity (532.5±77.2 µg ml-1), AlPO4-solubilizing activity (119.9±3.1 µg ml-1), FePO4-solubilizing activity (10.2±0.4 µg ml-1), and Israrl rock phosphate-solubilizing activity (433.1±64.0 µg ml-1) grown at 50°C. The bacterial isolates F10 represented the higher Ca3(PO4)2-solubilizing activity (594.5±88.0 µg ml-1) growing at 25°C; the fungal isolate O4 revealed the higher AlPO4-solubilizing activity (106.2±7.9 µg ml-1), and FePO4-solubilizing activity (92.4±5.8 µg ml-1) grown at 25°C; the actinomycete isolate J57 displayed the higher Israrl rock phosphate-solubilizing activity (288.1±38.6 µg ml-1). Ten thermo-tolerant phosphate-solubilizing bacteria and four thermo-tolerant phosphate-solubilizing fungi possessed mesophilic and/or thermo-tolerant amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase activities. The bacterial isolates A3, C45, O1, and V1, and the fungal isolates O4 and T13 carried out those 6 kinds of enzyme activities grown at both both 25 and 50°C. The isolate A3 manifested the higher amylase, CMCase, protease, lipase, and nitrogenase activities. Some of thermo-tolerant phosphate-solubilizing microbial isolates also possessed anti-microbial activities. The inoculation with these thermo-tolerant phosphate-solubilizing microbes into agricultural, poultry and livestock waste composts accelerated the maturity rates, elevated the soluble phosphorus contents, improved the quality, and increased the populations of mesophilic and thermo-tolerant phosphate-solubilizing bacteria, cellulolytic microbes, proteolytic microbes, and lipolytic microbes throughout preparation of multi-functional biofertilizers. Inoculation with thermo-tolerant phosphate solubilizing bacteria could more short the maturity time, improve the quality, and more release more soluble phosphorus than actinomycetes or fungi did. The bacterial isolates A3, C45, and F18 are the ideal candidates for application. Supplement of appropriate quantity (1-5%, w/w) polyelectrolyte oxygen detoxifier (POD) into the agricultural, and poultry and livestock waste composts could speed the maturity, enhance soluble phosphorus contents, improve the quality, and propagate the populations of mesophilic and thermo-tolerant phosphate-solubilizing bacteria, and cellulolytic microbes during composting. The inoculation of mixed cultures containig thermo-tolerant phosphate-solubilizing bacteria and other functional microbes with supplement of POD can convert agricultural, animal, food wastes, and vegetable and fruit market wastes into multi-functional biofertilizers for bioresource recycling, agricultural applications, and prospective for sustainable argriculture.