臺灣博碩士論文加值系統

黑翅土白蟻是臺灣唯一屬於大白蟻亞科的白蟻，屬真菌栽培白蟻，可建立大型菌落在地底下的白蟻巢穴，其內含有許多菌圃，對枯枝落葉的降解有很大的影響。菌圃或「真菌花園」是由黑翅土白蟻消化植物基質後的糞便所建構的通風結構，可供共生真菌雞肉絲菇的菌絲生長。先前的研究指出黑翅土白蟻和雞肉絲菇間具有共生關係，白蟻從雞肉絲菇的菌絲中獲取含氮化合物，並提供了適合這些真菌生長的棲地。
在缺少白蟻的條件下，迄今仍無法培養出雞肉絲菇的子實體，因而本研究主要意義為探討在體外條件下，黑翅土白蟻與菌圃間的共生菌之交互作用關係，以期增加雞肉絲菇的培養性。本實驗運用培養方法與非培養方法來分析在白蟻腸道及菌圃中的微生物菌相，藉以釐清在其中的微生物菌群生態結構與關係。首先利用變性梯度凝膠電泳（DGGE）及株落選殖等非培養方法來偵測細菌和真菌群落；接著利用不同的培養基來培養所分離純化的微生物群落。我們推測這些微生物群落能促進纖維素與半纖維素的水解、腸道發酵、生產養分以及菌圃老化，使得其共生真菌雞肉絲菇開始生長。
從微生物菌相生態實驗中，於白蟻腸道及菌圃中發現變形菌門、厚壁菌門等不同的細菌菌株，及些許無法培養的菌株，其中主要的細菌菌株屬厚壁菌門。而實驗所分離到的真菌主要是雞肉絲菇與一些小團體，如酵母菌。接著將這些分離純化到的細菌菌株與侵入性的microfungi Trichoderma harzianum進一步研究其相互作用關係。藉由這些研究得知腸道共生菌在調控菌圃侵入性microfungi過渡生長上扮演著重要的角色。實驗發現芽孢桿菌會抑制T. harzianum的生長因而促進雞肉絲菇的生長。因此，它被視為啟動的真菌生長的互惠者。隨後在分離純化到的菌株中分析其木質纖維素降解、固氮及生物制氫的功能。
此外，利用不同的碳基質來研究黑翅土白蟻腸道內的共生菌如可分解纖維素的芽孢桿菌和具發酵力的梭菌以模擬白蟻腸道產生氫氣作用。隨後以固態NMR檢測芽孢桿菌、梭菌和雞肉絲菇對菌圃老化及體外芒果樹基質之木質纖維素降解的協同作用。結果顯示，芽孢桿菌在此扮演著互惠的行為，透由其所產生之厭氧環境，使梭菌得以生長並產生氫氣進而增加雞肉絲菇對基質木素纖維素的降解。因此，這三種微生物間的協同作用可應用於白蟻衍生性的生物燃料處理技術上。最後，探討菌圃的化學組成及有機成分的降解模式，其老化結果與共同培養芽孢桿菌、梭菌和雞肉絲菇在芒果基質上的降解的趨勢是相似的。本實驗主要集中在白蟻腸道之微生物菌相，和黑翅土白蟻的菌圃系統，以及它們的功能方面和微生物仿生生物制氫的過程之探究。

The black winged subterranean termite Odontotermes formosanus is the only known macrotermitine termite in Taiwan. These termites construct large colonies with numerous fungus combs in subterranean nest and have great impact on the degradation of plant litter. The fecal material of O. formosanus containing the ingested plant material is made into a small ventilated structure, the fungus comb or the “fungus garden” to which the mycelium of the symbiotic fungi Termitomyces grows. Previous studies portray the symbiotic relationship between Odontotermes formosanus and Termitomyces species. By this relationship, the termite acquires nitrogenous compounds from the fungal hyphae of Termitomyces and in turn these fungi are provided with a suitable habitat for their growth and sustenance.
So far, the fruiting body of Termitomyces has not been cultivated in the laboratory in the absence of termites. This research has gained significance as gut symbionts are used to enhance the growth of Termitomyces mycelia by culturing. Therefore it is necessary to study the functioning of the O. formosanus termite-gut fungus comb symbionts under in vitro conditions. For this research, the microbial communities harboring in the gut and fungus comb of this termite are analyzed by both culture dependent and culture independent methods to better understand the ecology and community structure of their microflora. For the detection of bacterial and fungal communities by culture independent methods, denaturing gradient gel electrophoresis (DGGE) and clonal selection are performed; and for culture dependent methods, microbial communities are isolated using different culture mediums. These microbes are hypothesized to contribute to cellulose-hemicellulose hydrolysis, gut fermentation, nutrient production, fungus comb ageing and the initiation of the growth of the symbiotic fungus Termitomyces.
From the microbial ecological studies by culture dependent and independent methods in the termite gut and the fungus comb, the different bacterial strains are identified to belong to Proteobacteria, Firmicutes and other uncultured bacteria, of which Firmicutes are predominant. The dominant fungal isolate belong to Termitomyces species where as other minor groups like yeasts are also detected. The bacterial strains isolated by the cultural approach are further used for the interaction studies along with the invasive microfungi Trichoderma harzianum. These studies are performed to know if the gut symbionts contributed an important role in regulating the growth of other invasive microfungi overgrowing on the fungus comb. The Bacillus strains are detected to suppress the growth of T. harzianum and promote the growth of the dominant fungi Termitomyces. Therefore, they are considered as mutualists that initiate the growth of the fungi. The functional roles of the isolated strains are assessed for lignocellulosic degradation, nitrogen fixation and biohydrogen production.
In addition, the gut symbionts from the macrotermitine termite Odontotermes formosanus, the cellulolytic Bacillus and fermentative Clostridium are studied in batch experiments using different carbon substrates to biomimic the termite gut for hydrogen production. Their fungus comb ageing and the in vitro lignocellulosic degradation of the mango tree substrates by the synergistic interaction of Bacillus, Clostridium and Termitomyces are detected by Solid-state NMR. From the results, Bacillus species act as mutualists, by initiating an anaerobic environment for the growth of Clostridium for biohydrogen production and enhancing the lignocellulosic degradation of substrates in the presence of Termitomyces in vitro and in vivo. Thus, the synergistic collaboration of these three microbes can be used for termite-derived biofuel process technology. The chemical composition of the fungus comb is monitored and the degradation pattern of the organic components as a result of ageing is similar to the mango tree substrates that are incubated with cocultures of Bacillus, Clostridium and Termitomyces. The overall research is focused on studying the microbial community in the termite gut and fungus comb system of O. formosanus, their functional aspects and biomimicking their microbial process for biohydrogen production.

Acknowledgements i
Chinese abstract iii
English abstract iv
Table of contents vi
List of Tables x
List of Figures xi

Chapter 1: General introduction
1.1. Importance of termites 1
1.2. Types of termites and the functional role of the gut microbial consortium 1
1.3. Macrotermitine termites 2
1.4. Fungus comb and their function in the macrotermitine system 2
1.5. The model macrotermitine termite: Odontotermes formosanus (The black winged subterranean termite) 3
1.6. Digestome and microbial communities residing in Odontotermes formosanus 4
1.7. Fungus combs of Odontotermes species 5
1.8. Protection of fungus comb 6
1.9. Importance of Termitomyces 7
1.10. The bacterial and fungal symbionts 7
1.11. Main objective of this research 10
1.12. Strategy of the experiment 11

Chapter 2: Microbial community analysis in the termite gut and fungus comb of Odontotermes formosanus
2.1. Introduction 12
2.2. Materials and methods 13
2.2.1. Procuring the termites and the fungus comb from the site and their preservation 13
2.2.2. Culture dependent methods 14
2.2.2.1. Isolation of bacterial cultures from the termite gut and fungus comb 14
2.2.2.2. Culturing of Termitomyces and microfungi 14
2.2.3. Culture Independent methods 15
2.2.3.1. DNA extraction 15
2.2.3.2. Checking the concentration and purity of DNA 16
2.2.3.3. Detection of the bacterial species in the termite gut and fungus comb 16
2.2.3.4. Detection of the fungal species in the termite gut and fungus comb 17
2.2.3.5. Denaturing gradient gel electrophoresis and cloning 17
2.2.3.6. Clonal selection of bacterial cultures from the termite gut and fungus comb and rarefaction analysis 18
2.2.3.7. Phylogenetic analysis 18
2.3. Results 19
2.3.1. Bacterial community structure analysis by DGGE 19
2.3.2. Clonal selection 20
2.3.3. Rarefaction analysis of the bacterial clones 20
2.3.4. Culture dependent methods (Bacteria) 20
2.3.5. Phylogenetic analysis of the Bacillus strains 21
2.3.6. Fungal community analysis by culture independent methods 21
2.3.7. Isolation of microfungi 22
2.4. Discussion 23

Chapter 3: Microbial strains isolated from the termite gut and fungus comb of Odontotermes formosanus and their characterization
3.1. Introduction 28
3.2. Materials and methods 30
3.2.1. Enzyme assays of Bacillus species isolated from the termite gut and fungus comb 30
3.2.2. Functional analysis of Clostridium sp. 31
3.2.2.1. Batch experiments using Clostridium sp. 32
3.2.2.2. Hydrogen gas determination by Clostridium sp. 32
3.2.2.3. Hydrogenase gene targeted PCR and phylogenetic analysis 32
3.2.3. Characterization of microorganisms belonging to the phylum Proteobacteria 33
3.2.3.1. Functional aspects of Ochrobactrum species isolated from the termite gut of O. formosanus 33
3.2.4. Interaction assays in vitro 34
3.3. Results 35
3.3.1. Functional analysis of the Bacillus species 35
3.3.2. Functional analysis of the Clostridial species 35
3.3.3. Characterization of the Proteobacterial species 36
3.3.3.1. Functional aspect of the nitrogen fixing bacteria 37
3.3.4. Interaction studies 37
3.4. Discussion 38

Chapter 4: Development of biomimic process in the termite gut and the fungus comb
4.1. Introduction 44
4.2. Materials and methods 48
4.2.1. Detection of mineral elements and carbohydrates 48
4.2.2. NMR spectroscopy of the different layers of the fungus comb 49
4.2.3. Bacterial strains and growth conditions 49
4.2.4. Experimental conditions 50
4.2.4.1. Addition of the carbon sources 50
4.2.4.2. Hydrogen gas determination 51
4.2.4.3. NMR studies of the mango tree substrates with the gut symbionts in the presence and absence of Termitomyces 52
4.3. Result 52
4.3.1. Comparison of the chemical nature of fungus combs of Odontotermes formosanus from two different locations in Taiwan 52
4.3.2. Chemical composition of the fungus comb on the basis of their ageing (in vivo conditions) 53
4.3.3. Hydrogen production using various carbon substrates 54
4.3.4. Chemical composition of mango tree substrates under in vitro conditions 55
4.4. Discussion 57

Chapter 5: Conclusion and future work 63
Tables 65
Figures 74
Supplementary information 93
References 97
Appendices 117
Publications, posters and oral presentations related to this work from 2008 till 2012 141

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