臺灣博碩士論文加值系統

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。

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.

口試委員會審定書 I
謝誌 II
中文摘要 III
Abstract V
目錄 IX
表目錄 XX
圖目錄 XXXII
第一章 緒論1
1.1 磷 1
1.2 溶磷微生物 3
1.3 溶磷機制 4
1.4 溶磷微生物之應用 7
1.5 溶磷基因 11
1.6 肥料 13
1.6.1 肥料分類 13
1.6.2化學肥料 13
1.6.3有機肥料 14
1.6.4 生物肥料 16
1.7 堆肥 18
1.8 耐高溫溶磷微生物 20
1.9 禽畜糞廢棄物 21
1.10 永續農業 24
1.11 多功能耐高溫溶磷微生物與生物肥料製作 25
1.12 研究目標 29

第二章 堆肥和生物肥料溶磷菌相研究及分離方法建立 30
2.1 摘要 30
2.2 前言 31
2.3 材料與方法 32
2.3.1 堆肥樣品和生物肥料樣品來源 32
2.3.2 堆肥採集 32
2.3.3 培養基 32
2.3.3.1 營養洋菜培養基(Nutrient agar, NA) (Merck, Germany) 32
2.3.3.2 Potato dextrose agar (PDA) (Merck, Germany) 33
2.3.3.3 Pikovskaya medium (PVK) 33
2.3.3.4 Sucrose calcium phosphate medium (SCP) 33
2.3.3.5 National Botanical Research Institute’s phosphate growth medium 33
2.3.4 培養條件 33
2.3.5 溶磷微生物計數和分離 34
2.3.6 溶磷微生物分類 34
2.3.7 耐高溫PSMs的活化 35
2.3.8 平板檢測法 35
2.3.8 統計分析 35
2.4 結果與討論 25
2.4.1 豬糞堆肥堆積過程溶磷菌相 36
2.4.2 生物肥料溶磷菌相 38
2.4.2 豬糞堆肥溶磷微生物分離 39
2.4.2.1 豬糞堆肥耐高溫溶磷細菌分離株生長和溶磷活性 39
2.4.2.1.1 以NBRIP篩選之耐高溫溶磷細菌 39
2.4.2.1.2 以PVK篩選之耐高溫溶磷細菌 42
2.4.2.1.3 以SCP篩選之耐高溫溶磷細菌 44
2.4.2.2 生物肥料耐高溫溶磷微生物分離 33
2.4.2.2.1 以NBRIP篩選之耐高溫溶磷細菌 48
2.4.2.2.2 以PVK篩選之耐高溫溶磷細菌 50
2.4.2.2.3 以SCP篩選之耐高溫溶磷細菌 52
2.4.3 豬糞堆肥和生物肥料溶磷微生物分離株生長和溶磷活性 55
2.5 結論 57

第三章 禽畜糞堆肥和生物肥料溶磷菌相及溶磷微生物分離 98
3.1 摘要 98
3.2 前言 99
3.3 材料與方法 101
3.3.1 堆肥樣品和生物肥料樣品 101
3.3.2 堆肥採集方法 102
3.3.3 培養基 102
3.3.4 培養條件 102
3.3.5 溶磷微生物計數和分離 102
3.3.6 溶磷微生物分類 102
3.4 結果與討論 102
3.4.1 禽畜糞堆肥和生物肥料之性質和溶磷微生物相 102
3.4.1.1 豬糞堆肥 102
3.4.1.2 雞糞堆肥 103
3.4.1.2.1 Ty1堆肥場 105
3.4.1.2.2 Ty3堆肥場 105
3.4.1.2.3 Ml1堆肥場 106
3.4.1.2.4 Hc1堆肥場 106
3.4.1.2.5 Ch2堆肥場 106
3.4.1.3 牛糞堆肥 107
3.4.1.3.1 Ty2堆肥場 108
3.4.1.3.2 Ch3堆肥場 109
3.4.1.4 生物肥料 109
3.4.1.4.1 生物肥料Y 110
3.4.1.4.2 生物肥料Z 111
3.4.2 溶磷微生物分離 112
3.4.2.1 豬糞堆肥溶磷微生物分離 112
3.4.2.2 雞糞堆肥溶磷微生物分離 113
3.4.2.3 牛糞堆肥溶磷微生物分離 114
3.4.2.4 禽畜糞溶磷微生物分離 115
3.4.2.5 生物肥料溶磷微生物分離 116
3.5 結論 118

第四章 培養基和溫度對耐高溫溶磷微生物溶磷活性之影響 131
4.1 摘要 131
4.2 前言 132
4.3 材料與方法 133
4.3.1 培養基 133
4.3.2 培養條件 133
4.3.3 耐高溫溶磷微生物的活化 133
4.3.4 平板檢測法 133
4.3.5 液態檢測法 134
4.3.6 統計分析 135
4.4 結果與討論 135
4.4.1 培養基與培養溫度對溶磷微生物生長和菌落型態影響 135
4.4.1.1 耐高溫溶磷細菌生長和菌落型態 135
4.4.1.1.1 菌落 135
4.4.1.1.2 溶磷圈 136
4.4.1.1.3 溶三磷化鈣活性參數 137
4.4.1.2中溫溶磷細菌生長和菌落型態 137
4.4.1.2.1菌落 137
4.4.1.2.2 溶磷圈 138
4.4.1.2.3溶三磷化鈣活性參數 138
4.4.1.3 耐高溫溶磷放線菌生長和菌落型態 139
4.4.1.3.1 菌落 139
4.4.1.3.2 溶磷圈 139
4.4.1.3.3溶三磷化鈣活性參數 140
4.4.1.4 中溫溶磷放線菌生長和菌落型態 141
4.4.1.4.1 菌落 141
4.4.1.4.2 溶磷圈 141
4.4.1.4.3溶三磷化鈣活性參數 141
4.4.1.5 耐高溫溶磷黴菌生長和菌落型態 142
4.4.1.5.1 菌落 142
4.4.1.5.2 溶磷圈 143
4.4.1.5.3 溶三磷化鈣活性參數 143
4.4.1.6 中溫溶磷黴菌生長和菌落型態 144
4.4.1.6.1 菌落 144
4.4.1.6.2 溶磷圈 144
4.4.1.6.3溶三磷化鈣活性參數 144
4.4.2 耐高溫溶磷細菌分離株多樣性 145
4.4.3 耐高溫溶磷微生物的篩選 147
4.4.4 三種三磷化鈣培養基對於耐高溶磷微生物溶三磷化鈣活性影響 147
4.4.4.1 無菌對照實驗組 147
4.4.4.2 pH和溶三磷化鈣活性偵測 147
4.4.4.3 培養基和溫度對於耐高溫溶磷微生物溶三磷化鈣活性影響 148
4.4.4.4 pH與培養時間對耐高溫溶磷微生物溶三磷化鈣活性影響 152
4.4.4.4.1 耐高溫溶磷微生物溶三磷化鈣活性 152
4.4.4.4.2 PVK液態檢測法 153
4.4.4.4.3 SCP液態檢測法 154
4.4.4.4.4 NBRIP液態檢測法 155
4.4.4.4.5 耐高溫溶磷細菌分離株液態檢測法連續偵測 157
4.4.4.4.6 pH與耐高溫溶磷微生物溶三磷化鈣活性相關性 161
4.4.4.4.7 具高溶三磷化鈣活性耐高溫溶磷微生物之篩選 163
4.4.5 PVK、SCP及NBRIP平板檢測法與液態檢測法比較 166
4.4.5.1 耐高溫溶磷細菌之菌落型態與溶三磷化鈣活性相關性 166
4.4.5.2 耐高溫溶磷放線菌之菌落型態與溶三磷化鈣活性比較 169
4.4.5.3 耐高溫溶磷黴菌之菌落型態與溶三磷化鈣活性比較 170
4.5 結論 173

第五章 多功能微生物 274
5.1 摘要 274
5.2 前言 275
5.3 材料與方法 277
5.3.1 供試菌株 277
5.3.2 培養基 277
5.3.2.1 營養洋菜培養基 277
5.3.2.2 PDA培養基 277
5.3.2.3 三磷化鈣培養基 277
5.3.2.3.1 PVK培養基 277
5.3.2.3.2 SCP培養基 278
5.3.2.3.3 NBRIP培養基 278
5.3.2.3.4 磷酸鐵培養基 278
5.3.2.3.5 磷酸鋁培養基 278
5.3.2.3.6 Hydoxyapatite培養基 278
5.3.2.3.7 磷礦石培養基 278
5.3.2.5 可溶性澱粉-酵母抽取物培養基 279
5.3.2.6 Mendles-Reese medium培養基 279
5.3.2.7 幾丁質分解酵素分析培養基 279
5.3.2.8果膠質分解酵素分析培養基 279
5.3.2.8 蛋白質分解酵素分析培養基 279
5.3.2.9 脂質分解酵素分析培養基 280
5.3.2.10 Winogradsky’s nitrogen-free mineral培養基 280
5.3.2.11 Nitrogen-free mannitol培養基 280
5.3.2.12 抗菌活性分析培養基 280
5.3.3 培養條件 280
5.3.4 耐高溫溶磷微生物的活化 280
5.3.5 平板檢測法 280
5.3.6 液態檢測法 281
5.4 結果與討論 281
5.4.1 三磷化鈣、磷酸鐵、磷酸鋁、hydroxyapatite及磷礦石平板檢測法探討 281
5.4.2 不同磷酸鹽培養基和培養溫度對耐高溫溶磷微生物生長與菌落型態影響 281
5.4.2.1 耐高溫溶磷細菌生長與菌落型態 281
5.4.2.2 耐高溫溶磷放線菌生長與菌落型態 282
5.4.2.3 耐高溫溶磷黴菌生長與菌落型態 283
5.4.3 耐高溫溶磷微生物溶磷活性多樣性 284
5.4.3.1 功能計分設立 284
5.4.3.2 耐高溫溶磷微生物溶磷能力多樣性之功能計分評量 284
5.4.4. 磷酸鐵、磷酸鋁及磷礦石液態檢測法 285
5.4.4.4 培養基和溫度對於耐高溫溶磷細菌溶、磷酸鋁及磷礦石活性之影響 285
5.4.5 耐高溫溶磷微生物碳源之利用 287
5.4.5.1 澱粉質、纖維素、幾丁質以及果膠質分解酵素活性之偵測 287
5.4.5.2 不同單一碳源培養基和培養溫度對耐高溫溶磷微生物生長與菌落型態影響 287
5.4.5.2.1 耐高溫溶磷細菌生長與菌落型態 287
5.4.5.2.2 耐高溫溶磷放線菌生長與菌落型態 288
5.4.5.2.3 耐高溫溶磷黴菌生長與菌落型態 289
5.4.5.3 耐高溫溶磷微生物碳源利用能力多樣性探討 290
5.4.5.3.1耐高溫溶磷微生物碳源利用能力多樣性之功能計分評量 290
5.4.6 耐高溫溶磷微生物蛋白質和脂質分解酵素活性之探討 290
5.4.6.1. 耐高溫溶磷微生物蛋白質和脂質分解酵素活性偵測 291
5.4.6.2培養溫度對於耐高溫溶磷微生物在蛋白質和脂質分解酵素分析培養基平板檢測法的影響 291
5.4.6.2.1 耐高溫溶磷細菌生長與菌落型態的觀察 291
5.4.6.2.2耐高溫溶磷放線菌生長與菌落型態 292
5.4.6.2.3 耐高溫溶磷黴菌生長與菌落型態的觀察 292
5.4.7 耐高溫溶磷微生物固氮酵素活性探討 293
5.4.7.1耐高溫溶磷微生物固氮活性偵測 293
5.4.7.2培養溫度對於耐高溫溶磷微生物在固氮酵素分析培養基平板檢測法影響 293
5.4.7.2.1 耐高溫溶磷細菌生長與菌落型態 293
5.4.7.2.2耐高溫溶磷放線菌生長與菌落型態 294
5.4.7.2.3 耐高溫溶磷黴菌生長與菌落型態 294
5.4.8 耐高溫溶磷微生物抗菌活性探討 295
5.4.8.1 耐高溫溶磷微生物抗菌活性偵測 295
5.4.8.2培養溫度對於耐高溫溶磷微生物在抗菌活性培養基平板檢測法之影響 295
5.4.8.2.1耐高溫溶磷細菌生長與菌落型態 295
5.4.8.2.2耐高溫溶磷放線菌生長與菌落型態 295
5.4.8.2.3 耐高溫溶磷黴菌生長與菌落型態觀察 296
5.4.9 耐高溫溶磷微生物功能多樣性計分評量 296
5.4.10 多功能性耐高溫溶磷微生物之挑選和鑑定 296
5.5 結論 298

第六章 多功能生物肥料製作 336
6.1 摘要 336
6.2 前言 337
6.3 材料與方法 339
6.3.1 原料 339
6.3.1 供試菌株 341
6.3.2 培養基 341
6.3.2.1 營養洋菜培養基 341
6.3.2.2 PDA培養基 341
6.3.2.3 含鏈黴素和環己亞胺甘油酵母萃取物培養基 341
6.3.2.4 Rose Bengal培養基 341
6.3.2.5 PVK培養基 342
6.3.2.6 Mendles-Reese medium培養基 342
6.3.2.7 蛋白質分解酵素分析培養基 342
6.3.2.8 脂質分解酵素分析培養基 342
6.3.2.9 以三磷化鈣為唯一磷源含鏈黴素和環己亞胺甘油酵母萃取物培養基 342
6.3.2.10 以三磷化鈣為唯一磷源Rose Bengal培養基 342
6.3.3 培養條件 342
6.3.4 耐高溫溶磷微生物的活化 342
6.3.5 API ZYM試紙測試 343
6.3.6接種菌液製備 343
6.3.7 生物肥料製作 343
6.3.8 微生物相 344
6.3.9 性質分析 345
6.3.9.1 溫度 345
6.3.9.2 重量 345
6.3.9.3 顏色、黏性、顆粒大小及氣味 345
6.3.9.4 pH 345
6.3.9.5 水份含量 346
6.3.9.6 可溶性磷含量 346
6.3.9.7 發芽率 347
6.3.9.8 灰份含量 347
8.3.9.9 總有機碳含量 347
6.3.9.10 總氮含量 348
6.3.9.10 C/N 349
6.3.9.11 總磷含量 349
6.3.10 統計分析 350
6.4 結果與討論 350
6.4.1 供試耐高溫溶磷細菌分離株耐高溫酵素測試 350
6.4.2 堆肥性質分析 350
6.4.2.1 堆肥溫度 350
6.4.2.2 水份含量 352
6.4.2.3 pH 352
6.4.2.4 灰份含量 353
6.4.2.5 總有機碳 353
6.4.2.6 總氮量 353
6.4.2.7 碳氮比 354
6.4.2.8 發芽率 355
6.4.1.9 乾重消失比 356
6.4.1.10 氣味顏色顆粒飛蟲 356
6.4.3 磷變化 359
6.4.3.1 總磷含量 359
6.4.3.2 可溶性磷含量 360
6.4.3.3 溶磷效率 361
6.5 微生物相 363
6.5.1禽畜糞生物肥料(A) 363
6.5.2 禽畜糞生物肥料(B) 364
6.5.3 豬糞稻草生物肥料 367
6.6 結論 370

第七章 松樹素於禽畜糞生物肥料製作影響 396
7.1 摘要 396
7.2 前言 396
7.3 材料與方法 398
7.3.1 松樹素 398
7.3.2原料基質 398
7.3.2 培養基 399
7.3.2.1 營養洋菜培養基 399
7.3.2.2. PVK培養基 399
7.3.2.3 Mendles-Reese medium培養基 399
7.2.2.4以三磷化鈣為唯一磷源含鏈黴素和環己亞胺甘油酵母萃取物培養基 400
7.2.2.5以三磷化鈣為唯一磷源Rose Bengal培養基 400
7.2.2.6甘油酵母萃取物培養基 400
7.2.2.7以carboxymethylcellulose為唯一碳源Rose Bengal培養基 400
7.3.3肥料製作 400
7.3.4 微生物相偵測 401
7.3.5 性質分析 401
7.3.6 統計分析 401
7.4 結果與討論 401
7.4.1 原料基質和堆肥場 401
7.4.1.1 禽畜糞生物肥料原料基質 401
7.4.1.2. 生物肥料製作 405
7.4.1.2.1 性質分析 405
7.4.1.2.3 微生物相 406
7.4.1.2.4 磷含量 409
7.5 結論 410

參考文獻 421


表目錄
表2-1. 堆肥和生物肥料樣品特性 59
表2-2. 豬糞堆肥場Ch1豬糞堆肥A溶磷微生物相 60
表2-3. 豬糞堆肥場Ch1豬糞堆肥B溶磷微生物相 62
表2-4. 豬糞堆肥場Ch1豬糞堆肥E溶磷微生物相 64
表2-5. 生物肥料樣品C溶磷微生物相 65
表2-6. 生物肥料(接種土)樣品D溶磷微生物相 66
表2-7. 218株溶磷微生物分離株 66
表2-8. 豬糞堆肥堆積過程155株溶磷微生物分離株分佈 67
表2-9. 豬糞堆肥堆積過程中以National Botanical Research Institute’s phosphate growth medium (NBRIP)所分離之51溶磷微生物分離株分佈 67
表2-10. 豬糞堆肥堆積過程經由NBRIP傾倒培養基法分離之44株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 68
表2-11. 豬糞堆肥堆積過程經由NBRIP傾倒培養基法分離之44株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 69
表2-12. 豬糞堆肥堆積過程經由NBRIP傾倒培養基法分離之44株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 70
表2-13. 豬糞堆肥堆積過程經由NBRIP傾倒培養基法分離之44株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 71
表2-14. 豬糞堆肥堆積過程中以Pikovsakays’s medium (PVK)所分離之57溶磷微生物分離株分佈 72
表2-15. 豬糞堆肥堆積過程經由PVK傾倒培養基法分離之53株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 73
表2-16. 豬糞堆肥堆積過程經由PVK傾倒培養基法分離之53株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 74
表2-17. 豬糞堆肥堆積過程經由PVK傾倒培養基法分離之53株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 75
表2-18. 豬糞堆肥堆積過程經由PVK傾倒培養基法分離之53株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 76
表2-19. 豬糞堆積過程中以sucrose calcium phosphate medium (SCP)所分離之47溶磷微生物分離株分佈 77
表2-20. 豬糞堆肥堆積過程經由SCP傾倒培養基法分離之43株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 78
表2-21. 豬糞堆肥堆積過程經由SCP傾倒培養基法分離之43株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 79
表2-22. 豬糞堆肥堆積過程經由SCP傾倒培養基法分離之43株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 80
表2-23. 豬糞堆肥堆積過程經由SCP傾倒培養基法分離之43株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 81
表2-24. 豬糞堆肥堆積過程分離之140株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 82
表2-25. 生物肥料63株溶磷細菌分離株分佈 83
表2-26. 生物肥料61株耐高溫溶磷細菌分離株分佈 83
表2-27. 生物肥料由NBRIP傾倒培養基法分離之20株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 84
表2-28. 生物肥料由NBRIP傾倒培養基法分離之20株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 85
表2-29. 生物肥料由NBRIP傾倒培養基法分離之20株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 86
表2-30. 生物肥料由NBRIP傾倒培養基法分離之20株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 87
表2-31. 生物肥料由PVK傾倒培養基法分離之18株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 88
表2-32. 生物肥料由PVK傾倒培養基法分離之18株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 89
表2-33. 生物肥料由PVK傾倒培養基法分離之18株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 90
表2-34. 生物肥料由PVK傾倒培養基法分離之18株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 91
表2-35. 生物肥料由SCP傾倒培養基法分離之23株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落分佈 92
表2-36. 生物肥料由SCP傾倒培養基法分離之23株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶磷圈分佈 93
表2-37. 生物肥料由SCP傾倒培養基法分離之23株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天溶三鈣化磷活性參數(tri-calcium phosphate-solubilizing activity indices，TCPSAI)分佈 94
表2-38. 生物肥料由SCP傾倒培養基法分離之23株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 95
表2-39. 生物肥料分離之61株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 96
表2-40. 不同堆積時期豬糞堆肥和生物肥料分離之201株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 97
表3-1. 堆肥和生物肥料樣品特性 119
表3-2. 堆肥和生物肥料溫度 121
表3-3. 堆肥和生物肥料溶磷微生物相 122
表3-4. 堆肥和生物肥料堆積過程溶磷微生物相 124
表3-5. 利用傾倒培養基法初步分離溶磷微生物來源和數量 125
表3-6. 堆肥和生物肥料溶磷微生物分離株數量 126
表3-7. 不同堆肥和生物肥料堆積過程溶磷微生物分離株數量 127
表3-8. 豬糞堆肥堆積過程430株溶磷微生物分離株分佈 127
表3-9. 雞糞堆肥堆積過程343株溶磷微生物分離株分佈 128
表3-10. 牛糞堆肥堆積過程103株溶磷微生物分離株分佈 128
表3-11. 雞、牛及豬糞堆肥堆積過程876株溶磷微生物分離株分佈 129
表3-12. 生物肥料堆肥堆積過程101株溶磷微生物分離株分佈 129
表3-13. 禽畜糞堆肥和生物肥料所分離977株溶磷微生物分佈 130
表4-1. 621株耐高溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 174
表4-2. 621株耐高溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶磷圈分佈 175
表4-3. 621株耐高溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 176
表4-4. 202株耐中溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 177
表4-5. 202株耐中溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶磷圈分佈 178
表4-6. 202株耐中溫溶磷細菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 179
表4-7. 50株耐高溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 180
表4-8. 50株耐高溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶磷圈分佈 181
表4-9. 50株耐高溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 182
表4-10. 40株耐中溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 182
表4-11. 40株耐中溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶磷圈分佈 183
表4-12. 40株耐中溫溶磷放線菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 184
表4-13. 23株耐高溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 185
表4-14. 23株耐高溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶磷圈分佈 188
表4-15. 23株耐高溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 188
表4-16. 42株中溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養菌落分佈 189
表4-17. 42株中溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養 溶磷圈分佈 191
表4-18. 42株中溫溶磷黴菌分離株於PVK、SCP與NBRIP平板檢測法五天培養溶三磷化鈣參數分佈 192
表4-19. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於50°C培養菌落前25大之耐高溫溶磷細菌分離株 193
表4-20. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於25°C培養菌落前25大之耐高溫溶磷細菌分離株 194
表4-21. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於50°C培養溶磷圈前25大之耐高溫溶磷細菌分離株 195
表4-22. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於25°C培養溶磷圈前25大之耐高溫溶磷細菌分離株 196
表4-23. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於50°C培養溶三磷化鈣參數前25高之耐高溫溶磷細菌分離株 197
表4-24. 621株耐高溫溶磷細菌以PVK、SCP與NBRIP平板檢測法於25°C培養溶三磷化鈣參數前25高之耐高溫溶磷細菌分離株 198
表4-25. 50株耐高溫溶磷放線菌以PVK、SCP與NBRIP平板檢測法於50°C培養菌落前15大之耐高溫溶磷放線菌分離株 199
表4-26. 50株耐高溫溶磷放線菌以PVK、SCP與NBRIP平板檢測法於50°C培養溶磷圈前15大之耐高溫溶磷放線菌分離株 200
表4-27. 51株耐高溫溶磷放線菌以PVK、SCP與NBRIP平板檢測法於50°C培養培養溶三磷化鈣前15高之耐高溫溶磷放線菌分離株 201
表4-28. 977株溶磷微生物以PVK、SCP與NBRIP平板檢測法於25和50°C培養5天所挑選菌株特性 202
表4-29. 未接菌PVK、SCP以及NBRIP液態培養基於25和50°C連續培養10天測得之pH和可溶性磷含量 208
表4-30. 耐高溫溶磷細菌分離株於PVK、SCP及NBRIP平板和液態檢測法溶三磷化鈣活性比較 209
表4-31. 中溫溶磷細菌分離株於PVK、SCP及NBRIP平板和液態檢測法溶三磷化鈣活性比較 222
表4-32. 耐高溫溶磷放線菌分離株於PVK、SCP及NBRIP平板和液態檢測法溶三磷化鈣活性比較 223
表4-33. 耐高溫溶磷黴菌分離株於PVK、SCP及NBRIP平板和液態檢測法溶三磷化鈣活性比較 225
表4-34. 耐高溫溶磷分離株以PVK、SCP以及NBRIP液態培養基於25和50°C培養時所測得溶三磷磷化鈣能力之排列與計分 226
表4-35. 耐高溫溶磷微生物於PVK液態培養基25和50°C所得之最高溶三磷化鈣活性 228
表4-36. 耐高溫溶磷微生物於SCP液態培養基25和50°C時所得之最高溶三磷化鈣活性 230
表4-37. 耐高溫溶磷微生物於NBRIP液態培養基於25和50°C時所得之最高溶三磷化鈣活性 232
表4-38. 以NBRIP、PVK及SCP平板檢測法所得菌落、溶磷圈及溶三磷化鈣參數與以液態檢測法連續偵測10天所得pH和溶三磷化鈣活性 234
表4-39. 47株耐高溫溶磷細菌分離株以NBRIP、PVK及SCP液態檢測法所得pH分佈 261
表4-40. 47株耐高溫溶磷細菌分離株以NBRIP、PVK及SCP液態檢測法所得可溶性磷含量分佈 264
表4-41. 47株耐高溫溶磷細菌分離株以NBRIP、SCP及PVK液態檢測法於25和50°C培養10天可得最高可溶性磷含量數量比 266
表4-42. 47株耐高溫溶磷細菌分離株以NBRIP、SCP及PVK液態檢測法於25和50°C培養天數對其表現最高可溶性含量影響 266
表4-43. 47株耐高溫溶磷細菌分離株以NBRIP、SCP及PVK液態檢測法於25和50°C培養所得pH和可溶性磷含量相關性分析 267
表4-44. 耐高溫溶磷微生物分離株以液態檢測法所得之pH (X)與所得最高溶三磷化鈣活性(Y)之直線歸相關性分析 268
表4-45. 已發表之中溫溶磷菌和本研究所分離之具有較佳溶磷能力的耐高溫溶磷微生物在PVK和NBRIP液態培養下溶磷酸鈣活性比較 269
表4-46. 621株耐高溫溶磷細菌以NBRIP、PVK及SCP平板檢測法於25和50°C培養五天菌落、溶磷圈及溶三鈣化磷活性參數分佈 270
表4-47. 47株耐高溫溶磷細菌分離株以NBRIP、SCP及PVK平板和液態檢測法於25和50°C培養5天所得菌落型態參數和可溶性磷含量相關性分析 272
表4-48. 耐高溫溶磷微生物之平板檢測法(X)和液態檢測法中所得溶磷酸鈣能力(Y)之直線回歸相關性分析 273
表5-1. 不同磷酸鹽培養基對耐高溫溶磷細菌分離株生長和溶磷活性比較 300
表5-2. 不同磷酸鹽培養基對耐高溫溶磷放線菌分離株生長和溶磷活性比較 305
表5-3. 不同磷酸鹽培養基對耐高溫溶磷黴菌分離株生長和溶磷活性比較 306
表5-4. 耐高溫溶磷微生物分離株溶磷多樣性之功能計分評量 307
表5-5. 未接菌之磷酸鐵、磷酸鋁以及磷酸鈣液態培養基於25和50°C連續培養10天所得之可溶性磷含量和pH 309
表5-6. 具有較高溶三磷化鈣活性之耐高溫溶磷微生物分離株在磷酸鐵和磷酸鋁液態檢測法之pH和最高溶磷活性 310
表5-7. 不同碳源對於耐高溫溶磷細菌分離株生長和酵素活性影響 312
表5-8. 不同碳源對於耐高溫溶磷放線菌分離株生長和酵素活性影響 317
表5-9. 不同碳源對於耐高溫溶磷黴菌分離株生長和酵素活性影響 318
表5-10. 耐高溫溶磷微生物分離株之碳源利用多樣性功能計分評量 319
表5-11 耐高溫溶磷細菌分離株蛋白質和脂質分解酵素偵測 321
表5-12. 耐高溫溶磷放線菌分離株蛋白質和脂質分解酵素偵測 323
表5-13. 耐高溫溶磷黴菌分離株蛋白質和脂質分解酵素偵測 323
表5-14. 耐高溫溶磷細菌分離株固氮活性測試 324
表5-15. 耐高溫溶磷放線菌分離株固氮活性測試 326
表5-16. 耐高溫溶磷黴菌分離株固氮活性測試 326
表5-17. 耐高溫溶磷細菌分離株抗菌活性測試 327
表5-18. 耐高溫溶磷放線菌分離株抗菌活性測試 329
表5-19. 耐高溫溶磷黴菌分離株抗菌活性測試 329
表5-20. 耐高溫溶磷微生物的功能多樣性之功能計分評量 330
表5-21. 較多功能活性之耐高溫溶磷微生物 334
表5-22. 具高溶三磷化鈣耐溶磷微生物分離株之型態觀察 335
表6-1. 生物肥料製備供試耐高溫溶磷微生物菌株 371
表6-2. 禽畜糞生物肥料A原料基質重量 371
表6-3. 禽畜糞生物肥料B原料基質重量 372
表6-4. 稻草豬糞生物肥料原料組成 372
表6-5.以API ZYM試紙試驗測試耐高溫溶磷細菌供試菌株耐高溫酵素活性 373
表6-6. 禽畜糞生物肥料A堆積過程顆粒顏色氣味飛蟲變化 374
表6-7. 禽畜糞生物肥料B堆積過程顏色氣味變化 375
表6-8. 豬糞稻草生物肥料堆積過程顏色氣味變化 376
表7-1. 生物肥料B原料組成 411
表7-2. 生物肥料B堆積過程中性質變化 412
表7-3. 生物肥料B成品濕重和乾重重量損失比率 414
表7-4. 生物肥料B堆積過程中微生物菌相變化 415
表7-5. 生物肥料B堆積第35至56天之中溫放線菌和黴菌菌相變化 418
表7-6. 生物肥料B成品磷含量和溶磷效率 420

圖目錄
圖6-1. 禽畜糞生物肥料A堆肥溫度 377
圖6-2. 禽畜糞生物肥料B堆肥溫度 378
圖6-3. 豬糞稻草生物肥料堆肥溫度 379
圖6-4. 禽畜糞生物肥料A性質分析 380
圖6-5. 禽畜糞生物肥料B性質分析 381
圖6-6. 豬糞稻草生物肥料性質分析 382
圖6-7. 禽畜糞生物肥料A磷變化 383
圖6-8. 禽畜糞生物肥料B磷變化 384
圖6-9. 豬糞稻草生物肥料磷變化 385
圖6-10. 禽畜糞生物肥料A微生物相 386
圖6-11. 禽畜糞生物肥料A微生物比例 387
圖6-12. 禽畜糞生物肥料B微生物相 388
圖6-13. 禽畜糞生物肥料B放線菌和黴菌相 389
圖6-14. 禽畜糞生物肥料B微生物比例 390
圖6-15. 禽畜糞生物肥料B放線菌和黴菌比例 381
圖6-16. 豬糞稻草微生物肥料微生物相 392
圖6-17. 豬糞稻草生物肥料放線菌和黴菌相 393
圖6-18. 豬糞稻草生物肥料微生物比例 394
圖6-19. 豬糞稻草生物肥料放線菌和黴菌比例 395

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