臺灣博碩士論文加值系統

塔塔加高山生態系是台灣長期生態研究網 (Taiwan Ecological Research Network，TERN)研究站之一，本研究以雲杉、鐵杉及草原區這三處不同營養區，依土壤深度進行不同季節的塔塔加森林土壤微生物多樣性分析。其中微生物生質碳、生質氮、微生物族群、水分含量、總有機碳和總氮隨土壤深度增加而下降。雲杉區微生物生質碳和生質氮分別介於394-1323 µg/g 及 71-233 µg/g，在鐵杉區則分別介於430-1340 µg/g 和 56-261 µg/g，草原區則分別介於141-870 µg/g 和 46-161 µg/g，其中以草原區為最低。微生物生質碳和生質氮在春季較高，於冬季時最低。草原區因土壤中有機物質含量低，故微生物族群量最低，微生物族群中以細菌族群量最高。細菌多樣性分析先以16S rDNA進行聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR)，再以變性梯度膠凝電泳 (Denaturing Gradient Gel Electrophoresis, DGGE)分析增幅子(amplicons)，得知三採樣區主要電泳片段非常相似，僅在次要片段有差異，且由PCR-DGGE分析亦顯示土壤剖面不同層間的差異，顯示在不同深度土壤分層均有特定微生物，得知微生物族群的數量與多樣性受地形影響。本研究生物技術所使用之可培養細菌，是以功能性篩出並以16S rDNA鑑定分析，這些分離株具有有機物、纖維素及有芳香環(酚)分解能力與溶磷功能。分離菌株以纖維分解菌最多、其次為溶磷及酚降解菌。產纖維酵素分離菌包括Chryseobacterium、Janthinobacterium、Flexibacter及Pedobacter等屬；溶磷菌包含Burkholderia、Enterobacter、Pseudomonas及Serratia等屬，而分離菌株中Acinetobacter、Bacillus及Staphylococcus等屬具有高效的酚降解能力。
研究更進一步以塔塔加分離株進行微生物產氫研究，利用加熱前處理技術分離出的塔塔加功能性產氫微生物族群，進行不同基質濃度產氫評估。由PCR-DGGE分析及氫氣產量得知，當增加前處理溫度時，產氫的微生物種類亦隨之改變，在15-20g葡萄糖基質下，最大產氫速率、氫累積量及產氫量分別為1390-1576 ml/l/day、2966-3146 ml H2/l及1.83-1.93 mol H2/mol glucose。本研究顯示加熱前處理法如同以特殊溫度篩選功能性菌株般，可有效從土壤分離產氫菌Clostridium。本研究探討加熱前處理法處理之森林土壤，在以纖維素合成培養基增菌後纖維素基質產氫能力，且完成以分批醱酵觀察不同濃度纖維素基質產氫效果。

Tatachia Forest is one of the Taiwan Ecological Research Network (TERN) sites. The microbial diversity of Tatachia forest soils along soil depth in three different vegetation sites-spruce, hemlock and grassland was analyzed at different seasons during the study period. Microbial biomass carbon (Cmic), biomass nitrogen (Nmic), microbial population, moisture content, total organic carbon (Corg), and total nitrogen (Ntot) decreased with increasing soil depth. In spruce, the Cmic and Nmic ranged from 394 to 1323 µg/g and from 71 to 233 µg/g, respectively. In hemlock, the values of Cmic and Nmic ranged from 430 to 1340 µg/g and from 56 to 261 µg/g, respectively. In grassland, the values ranged from 141 to 870 µg/g and from 46 to 161 µg/g, respectively. The grassland had low microbial biomass among the tested sites. The Cmic and Nmic contents were higher in spring and the minimal values were obtained in winter season. The grassland site had the lowest microbial populations among the three tested sites due to the low organic matter content. Bacterial population was the highest among the microbial populations. Analysis using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA showed that the bacterial diversity of the three sites were very similar to each other in the major bands, and the variation was in the minor bands. Vegetation influenced the quantity and diversity of microbial populations. The culturable bacterial population for different biotechnological uses were isolated by functional screening and identified by 16S rDNA analysis. The isolated strains were able to degrade organic matter, cellulose and aromatic compounds such as phenol, and able to solubilize inorganic phosphates. Cellulolytic strains were predominantly isolated, followed by phosphate-solubilizing and phenol-degrading strains. Several new cellulase producing bacteria belonging to the genus Chryseobacterium, Janthinobacterium, Flexibacter and Pedobacter were isolated. The phosphate solubilizing strains belonged to the genus Burkholderia, Enterobacter, Pseudomonas and Serratia. Among the isolated isolates, strains belonging to the genus Acinetobacter, Bacillus and Staphylococcus were effective phenol-degrading strains.
The microbial diversity of Tatachia forest was further explored for biohydrogen production. A functional hydrogen producing consortium was isolated from Tatachia forest soil by heat pretreatment technique and hydrogen production at different substrate concentration was evaluated. As revealed by PCR-DGGE analysis and hydrogen yield, the hydrogen producing microbial community changed with increase in heat pretreatment temperatures. The maximum hydrogen production rate, cumulative hydrogen and hydrogen yield with 15-20 g glucose were 1390-1576 ml/l/day, 2966-3146 ml hydrogen/l and 1.83-1.93 mol hydrogen/mol glucose, respectively. The study showed that heat pretreatment approach is effective for isolating hydrogen producing natural Clostridium consortium from the soil as enumerations of the functional strains need specific temperature range to flourish. The performance of hydrogen production from cellulosic substrates by heat pretreated forest soil enriched in defined medium containing cellobiose was also investigated. Batch fermentation was carried out to observe the effects of different substrate concentrations on the hydrogen production from cellobiose.

CONTENTS
Page No.
ACKNOWLEDGEMENTS................................iii
ABSTRACT.........................................iv
ABSTRACT (Mandarin)...............................vi
CONTENTS........................................viii
LIST OF FIGURES....................................x
LIST OF TABLES....................................xiv

CHAPTER-I General Introduction................1
1.1 Importance of soil microorganisms................2
1.2 Methods to determine microbial diversity........3
1.3 Exploration of microbial diversity for biotechnology...10
1.3.1 Exploring microbial diversity for bioenergy........................11
1.3.2 Biological hydrogen production (or Biohydrogen)..............................13
1.3.3 Fermentative hydrogen producing microflora........................17
1.3.4 Fundamentals of fermentative hydrogen production ...................20
1.3.5 Factors affecting fermentative hydrogen production....................24
1.4 Research background..............................30
1.5 Research objectives....................................31
1.6 Organization of the dissertation...............................33
1.7 Contribution of the dissertation..............................34

CHAPTER-II Effect of vegetations and seasons on microbial biomass carbon and nitrogen in Tatachia forest soils.....................................35
2.1 Introduction........................................36
2.2 Materials and methods.........................39
2.3 Results............................................46
2.4 Discussion........................................71
2.5 Conclusion...........................................74

CHAPTER-III Diversity and biotechnological uses of culturable bacteria from Tatachia forest soils...........75
3.1 Introduction......................................76
3.2 Materials and methods................................82
3.3 Results........................................87
3.4 Discussion........................................103
3.5 Conclusion.........................................107

CHAPTER-IV Isolation of hydrogen producing functional consortium from soil: heat pretreatment approach...........................108
4.1 Introduction..............................109
4.2 Materials and methods...............................111
4.3 Results..............................................115
4.4 Discussion...........................................125
4.5 Conclusion.........................................128

CHAPTER-V Biohydrogen production using cellulosic substrates...........................129
5.1 Introduction.....................................130
5.2 Materials and methods...............................132
5.3 Results............................................135
5.4 Discussion..................................141
5.5 Conclusion.........................................144

REFERENCES.........................................145
APPENDIX A.....................................184
APPENDIX B.............................................185

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