臺灣博碩士論文加值系統

農業有機廢棄物可利用傳統的一般堆肥化 (composting process) 方式達到堆肥腐熟並做為植物生長所需肥料之目的，近年來新興的蚓糞堆肥化 (vermicomposting process) 更是一種降低處理成本之生物穩定化方式，在上述兩種堆肥化過程中，其內所包含的微生物均扮演重要角色，然而過去研究主要著重於一般堆肥化過程中微生物相的變化，鮮少有針對相同來源廢棄物，再經由兩種堆肥化處理後之微生物群落比較。本研究即在探討台灣大宗的農業廢棄物豬糞與養菇廢棄包混合物，經一般堆肥化與蚓糞堆肥化對微生物群落結構之影響。利用培養法與未經培養之基因選殖法，分析廢棄物原料、預堆產物、一般堆肥化成品與蚓糞堆肥化成品中微生物之組成，評估經兩種堆肥化處理後微生物群落結構之差異。
以平板塗抹之菌數分析法測定九類群微生物活菌數，結果可得知細菌、真菌、游離固氮菌、溶磷菌與螢光假單孢菌之數量，皆因預先堆肥化溫度超過70 ℃而呈現下降的趨勢。經由預堆高溫亦使豬糞與養菇廢棄包混合物原料中沙門氏菌數，由2.9×106cfu/g下降至1.9×104 cfu/g，但對大腸桿菌數則無明顯影響。經兩種堆肥化處理後其成品中皆可測得因預堆而減少之細菌、真菌、游離固氮菌與溶磷菌數皆於腐熟的堆肥中有增加之趨勢，兩種堆肥成品中細菌、真菌、放線菌、游離固氮菌、溶磷菌與幾丁質分解菌數差異不大，於蚓糞堆肥中游離固氮菌與溶磷菌數仍有105 cfu/g，幾丁質分解菌數則仍大於106 cfu/g。利用培養法與未經培養之基因選殖法分析樣品中之微生物群落結構，由好氧異營細菌分離株之16S rDNA分析結果得知全部樣品中可測得細菌族群共歸類至6個菌門中，一般堆肥中革蘭氏陽性菌比例較高，其中以Firmicutes菌門之菌群為優勢，蚓糞堆肥中革蘭氏陰性菌比例較高，其中以α-Proteobacteria與β-Proteobacteria菌群為優勢，由未經培養之16S rDNA基因選殖分析結果得知全部樣品中可測得細菌族群共歸類至16個菌門中，一般堆肥中以γ-Proteobacteria菌門之菌群為優勢，佔23 %，蚓糞堆肥中α-Proteobacteria與β-Proteobacteria菌群為優勢，分別為23 %與27%。相較於培養法之分析結果，未經培養之基因選殖法可分析出更多之微生物種類，其中一般堆肥中之菌群共歸類於8個菌門，而蚓糞堆肥中之菌群可歸類於11個菌門，且於兩種堆肥成品中之微生物種類有18個菌屬之差異，顯示蚓糞堆肥化處理會增加細菌種類多樣性，且相同原料經不同堆肥化後對於細菌群落結構影響甚為顯著。
此試驗結果可得知由豬糞作為主原料進行一般堆肥化下優勢細菌群落變化為由Bacteroidetes菌門，經由預堆高溫期後轉變為Firmicutes菌門，最後於腐熟階段一般堆肥以γ-Proteobacteria為優勢菌門，蚓糞堆肥中則以α-Proteobacteria與β-Proteobacteria菌群為優勢菌門。本研究成功建立一般堆肥與蚓糞堆肥廢棄物中細菌群落結構之分析方法，並探討兩種不同堆肥化處理對豬糞與養菇廢棄包混合物中細菌群落結構之影響，可望作為有機廢棄物堆肥化過程中微生物相研究之基礎。

Agricultural organic wastes can serve as fertilizers to promote plant growth after composting process. The emerging technology namely vermicomposting provides an alternative way to stabilize wastes meanwhile reduce the cost for waste treatment. Microorganisms within composting or vermicomposting process play major roles in accelerating the stabilization of organic materials. But study on the comparison of microbial community structure between compost and vermicompost processed from the same raw materials is lack. The present study was undertaken to explore dynamics of microbial community structure during composting and vermicomposting of the mixture of pig manure and mushroom waste. Enumeration of a total of nine microbial groups within raw organic wastes, 14 days pre-composting wastes, composts and vermicomposts was performed separately on selective agar plates. Besides, culture-dependent and culture-independent approaches were used to determine 16S rRNA gene sequences from pure cultures or clone libraries derived from these samples.
The results demonstrated that viable number of bacteria, fungi, free-living nitrogen fixers, phosphate solubilizers and fluorescent Pseudomonads declined after 14 days pre-composting treatment. Number of populations belonging to the genus Salmonella changed from 2.9 x 106 cfu/g to 1.9 x 104 cfu/g, but there was no significant decrease in number of coliform. Viable number of bacteria, fungi, actinomycetes, free-living nitrogen fixers, phosphate solubilizers and chitin degraders was similarin compost and vermicompost samples.Strains isolated from all samples can be assigned into six bacterial phyla, as revealed by their 16S rRNA gene sequence analyses. The percentage of Gram-positive bacteria was higher in compost samples, and these were dominated by the phylum Firmicutes. In contrast, vermicompost harbored higher percentage of Gram-negative bacteria, which were dominated by the phylum α- Proteobacteria and β- Proteobacteria. From 16S rRNA gene clone libraries the sequences derived from all the samples affiliated to 16 bacterial phyla. The γ-Proteobacteria was dominant (23%) in compost samples, while α- Proteobacteria and β- Proteobacteria were dominant (23% and 27%) in vermicompost samples. The culture-independent clone library construction and sequence analysis provided higher diversebacterial lineage information within samples, as compared to culture-dependent method. Microbial community in compost samples consisted of eight bacterial phyla, and there were 11 in vermicompost samples. A total of 18 distinct bacterial genera were recorded when compared with compost and vermicompost samples, and this demonstrated that the microbial community structure after composting and vermicomposting process was quite different.
Dynamics of microbial community structure during composting and vermicomposting of the mixture of pig manure and mushroom waste were explored in the present study. The results showed that bacteria belonging to the phylum Bacteroidetes dominated in raw organic wastes, whichthen changed to the phylum Firmicutes, and the phylum γ-Proteobacteria was dominant in compost samples while α- Proteobacteria and β- Proteobacteria were dominant in vermicompost samples. The methodologies adopted here were useful to monitor and differentiate microbial community structure during two different composting processes. This will provide bases in the clarification of microbes-mediated mineralization and stabilization of organic materials during composting.

摘要………………………………………................…………….................................Ⅰ
Abstract………………………………………………………………...........................III
目錄…………………………………....………………..………………........................V
表次……………………………………………………………………..........................X
圖次………………………………………………………………………………….....XI
附表次…………………………………………………………………......................XIII
附圖次………………………………………………………………………………..XIV

壹、前言……………………………………………………………………………..…...1
貳、前人研究…………………………………………………………………………….2
一、禽畜糞廢棄物資源化………………………………………………........................2
二、 有機廢棄物之堆肥化……………………………………..………………...….….3
(一) 般堆肥化作用……………………………………………………......................….3
(二) 蚓糞堆肥化作用………………………………………………………………..….5
(三) 預先堆肥化作用………………………………………………………………..….8
三、物群落結構分析方法……………..………………………………………….…..…9
(一) 環境樣品中微生物核酸萃取………………………………………………..…….9
(二) 聚合酶連鎖反應(Polymerase chain reaction, PCR)….….....................................10
(三) 分子指紋技術(Molecular fingerprint technique)…………….……......................13
1. 變性梯度膠體電泳 (DGGE)…………………………..….…………………….....13
2. 選殖株基因庫 (Clone libraries) 的構築…………….……….……………………14
四、環境樣品微生物親緣關係分析…………………………………….......................15
參、研究目的…………………………………………...................................................16
肆、材料與方法………………………………………………………………..……....17一、供試資材……………………………………………………..…………………....17
(一) 試驗資材……………………………..………………………………...................17
(二) 試驗蚯蚓…………………………………..…………………..…….....................17
(三) 試驗容器………………….………………………………..…...…………….......17
二、試驗方法及步驟…………………………………………………………………...17
(一) 試驗處理……………………………………………...…......................................17
1. 預先堆肥化………………………………………...…………………………….....17
2. 一般堆肥化與蚓糞堆肥化試驗………………………………...……………….....17
(1) 一般堆化…………………………………………..…...………………………......18
(2) 蚓糞堆肥化……………………………..………………...……..............................18
(3) 對照組………………………………..……………...........………………………..18
(二) 一般堆肥及蚓糞堆肥採樣法………………………...……..……..……………..19
1. 堆肥採樣方法……………………………………………………............................19
2. 蚓糞堆肥採樣方法………………………………………………............................19
三、微生物群落結構分析試驗…………………………..........……………..………...19
(一) 菌種培養…………………………...………………………………..……………21
(二) 好氧性異營細菌分離株純化及定序………….………………………................28
1. 好氧性異營細菌純化……………….………………………………………….......28
2. 反覆凍融試驗……………………………………….…….………..........................28
3. 擴增之16S rDNA進行電泳……………….…………….………………………....28
四、樣品中微生物多樣性與群落結構分析……………………………………......….30
(一) 樣品中微生物核酸萃取……………………………………..…...……………....31
(二) PCR-DGGE (聚合酶連鎖反應-變性梯度膠體電泳)分析……………..………...31
1. 菌株染色體16S rDNA之擴增…………………..………………………………...31
2. 擴增之16S rDNA進行電泳……………………………………...………………..31
3. 變性梯度膠體電泳步驟……………………………………...………………..…...33
(1) 組合水平式電泳膠片配件……………………………….…..…………..……......33
(2) 膠片染色、退染與照膠…………………………………………………………...34
(3) 切膠與DNA回溶……………………………………………………….…….…...34
(4) PCR反應…………………………………………………………….………….…..36
(三) 樣品中微生物選殖株基因庫構築………………………………….…….....…...38
1. 菌株染色體16S rDNA之擴增………………………………………………..…...38
2. 擴增之16S rDNA進行電泳……………………………………………..……..….38
3.　PCR (聚合酶連鎖反應)產物純化………………………...………………....……38
4. 菌種DNA濃縮………………………………………………………...….………...40
5. ligation (接合作用)……………………………………………….……………...….40
6. Transformation (轉形作用)……………………………………...………………......41
7. 繼代培養…………………………………………………………............................42
8. 質體DNA中嵌入之16S rDNA分析………………………...………………..….42
伍、結果與討論……………………………………………………………………….45
一、原料、預堆14天堆積物及兩種堆肥化處理下各微生物族群菌落數(CFUs)…... 45
(一) 原料與預堆14天堆積物之菌落數比較…………………………………….......45
1. 主要微生物群落比較 (細菌、真菌、放線菌)…………………………..…….….45
2. 植物有益微生物群落比較………………………………………………………....47
3. 病原菌群落比較 (大腸桿菌、沙門氏菌)……………………………………..….48
(二) 一般堆肥化與蚓糞堆肥化55天之菌落數比較……………………………......50
1. 主要微生物群落比較 (細菌、真菌、放線菌)……………………………….…..50
2. 植物有益微生物群落比較…………………………………….………………...…53
二、原料、預堆14天堆積物及兩種堆肥化處理下好氧異營細菌分離株16S rDNA分析…………………………………………………….……………………………....55

(一) 豬糞與養菇廢棄包混合原料(OW)、預堆14天堆積物(PCW)與各堆肥化處理下(BK55、COM55、VM55) 細菌分離株菌屬分類…………………..…….....................57
1. 豬糞與養菇廢棄包混合原料、預堆14天堆積物中細菌分離株菌屬分類與菌群比較…………………………………………………………………………………….....57
2. 各堆肥化處理下細菌分離株菌屬分類與預堆14天堆積物中菌群比較……………………………………………………………………………….……....58
3. 一般堆肥與蚓糞堆肥中菌群比較…………………………………………………59
三、原料、預堆14天堆積物及兩種堆肥化處理下經選殖株基因庫構築 (clone
libraries) 微生物基因型分析………..……………………………..………………….62
(一) 豬糞與養菇廢棄包混合原料(OW)、預堆14天堆積物(PCW)與各堆肥化處理下 (BK55、COM55、VM55) 整體細菌群落菌門 (phylum) 分類……………………62
1. 豬糞與養菇廢棄包混合原料(OW)、預堆14天堆積物 (PCW)中細菌群落菌門分類與比較……………………………………………………………………………….62
2. 各堆肥化處理下 (BK55、COM55、VM55) 細菌群落菌門分類與預堆14天堆積物中菌群比較………………………………………….………………………………63
3. 一般堆肥與蚓糞堆肥中菌群比較……………………………………………..…..65
(二) 豬糞與養菇廢棄包混合原料(OW)、預堆14天堆積物(PCW)與各堆肥化處理下 (BK55、COM55、VM55)整體細菌族群菌屬 (genus)分類…………………………65
1. 豬糞與養菇廢棄包混合原料 (OW)、預堆14天堆積物 (PCW) 中細菌群落菌屬分類與比較…………………………………………………………………………….67
2. 各堆肥化處理下細菌群落菌屬分類與預堆14天堆積物中菌群比……………..68
3. 一般堆肥與蚓糞堆肥中菌群比較………………………………………………....70
四、原料、預堆14天堆積物及兩種堆肥化處理下細菌族群16S rDNA進行
變性膠體梯度電泳(DGGE)分析………………………………………..….................75
陸、結論…………………………………………………………………………….....77
柒、參考文獻………………………………………………………………………….78
捌、別錄…………………………………………………………………………….…90

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