MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Hongchen Jiang Candidate for the Degree Doctor of Philosophy ________________________________________________________ Dr. Hailiang Dong, Director ________________________________________________________ Dr. Chuanlun Zhang, Reader ________________________________________________________ Dr. Yildirim Dilek, Reader ________________________________________________________ Dr. Jonathan Levy, Reader ________________________________________________________ Dr. Q. Quinn Li, Graduate School Representative ABSTRACT GEOMICROBIOLOGICAL STUDIES OF SALINE LAKES ON THE TIBETAN PLATEAU, NW CHINA: LINKING GEOLOGICAL AND MICROBIAL PROCESSES By Hongchen Jiang Lakes constitute an important part of the global ecosystem as habitats in these environments play an important role in biogeochemical cycles of life-essential elements. The cycles of carbon, nitrogen and sulfur in these ecosystems are intimately linked to global phenomena such as climate change. Microorganisms are at the base of the food chain in these environments and drive the cycling of carbon and nitrogen in water columns and the sediments. Despite many studies on microbial ecology of lake ecosystems, significant gaps exist in our knowledge of how microbial and geological processes interact with each other. In this dissertation, I have studied the ecology and biogeochemistry of lakes on the Tibetan Plateau, NW China. The Tibetan lakes are pristine and stable with multiple environmental gradients (among which are salinity, pH, and ammonia concentration). These characteristics allow an assessment of mutual interactions of microorganisms and geochemical conditions in these lakes. Two lakes were chosen for this project: Lake Chaka and Qinghai Lake. These two lakes have contrasting salinity and pH: slightly saline (12 g/L) and alkaline (9.3) for Qinghai Lake and hypersaline (325 g/L) but neutral pH (7.4) for Chaka Lake. We have taken an integrated approach combining geochemistry, molecular phylogeny (both DNA and RNA based, both 16S rRNA and amoA gene), quantitative PCR (total Bacteria, Archaea, total crenarchaeota, AOA, and AOB), and cultivation and isolation. Both lake water and sediments have been analyzed. The results are divided into four chapters and they are summarized below. In addition, I also studied microbial communities and functions in sediments from South China Sea, a potential site for gas hydrate deposits. This work was done as extra add-on to the microbial ecology studies in Titeban lakes. GEOMICROBIOLOGICAL STUDIES OF SALINE LAKES ON THE TIBETAN PLATEAU, NW CHINA: LINKING GEOLOGICAL AND MICROBIAL PROCESSES A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Geology By Hongchen Jiang Miami University Oxford, Ohio 2007 Dissertation Director: Hailiang Dong TABLE OF CONTENTS Chapter 1: Introduction…………………………………….…………….………………………….1 References………………………………………………………………………...5 Chapter 2: Microbial Diversity in Water and Sediment of Lake Chaka: an athalassohaline lake in Northwestern China…………………………………10 Abstract…………………………………………………………………………..11 Body Text………………………………………………………………………..12 References………………………………………………………………………..32 Chapter 3: Microbial Response to Salinity Change in Lake Chaka, a Hypersaline Lake on Tibetan Plateau.………………………………………………………48 Summary.………………………………………………………………………...49 Body Text………………………………………………………………………...50 References………………………………………………………………………..74 Chapter 4: Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in the Sediments of Qinghai Lake, Northwestern China…………………….95 Abstract…………………………………………………………………………..96 Body Text………………………………………………………………………...97 References………………………………………………………………………107 Chapter 5: Dominance of Putative Marine Benthic Archaea in Sediments from Qinghai Lake, Northwestern China………………………………………….117 Abstract…………………………………………………………………………118 Body Text……………………………………………………………………….119 References………………………………………………………………………130 Chapter 6: Microbial Diversity in the Deep Marine Sediments from the Qiongdongnan Basin in South China Sea……………………………………143 Abstract…………………………………………………………………………144 Body Text……………………………………………………………………….145 References………………………………………………………………………158 Chapter 7: Summary………………………………………………………………….175 Appendix: Archaeal lipids and 16S rRNA genes characterizing gas hydrate- impacted sediments in the Gulf of Mexico…………………………………...179 ii LIST OF TABLES Chapter 2: Microbial Diversity in Water and Sediment of Lake Chaka: an athalassohaline lake in Northwestern China………………………………...10 1 – PLFA composition pf samples from Chaka Lake…………………………………..38 2 – Microbial isolates obtained from the lake water and sediment samples……………38 3 – Coverage and diversity index for the clone libraries for the lake water and sediments from Chaka Lake……………………………………………………………………38 Chapter 3: Microbial Response to Salinity Change in Lake Chaka, a Hypersaline Lake on Tibetan Plateau………………………………………………………48 1 – Distribution of cultured bacterial and archaeal isolates from Lake Chaka sediments…………………………………………………………………………….85 2 – Primers used in this study……………………………………………………………85 Chapter 4: Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in the Sediments of Qinghai Lake, Northwestern China…………………….95 1 – Primers used in this study…………………………………………………………..111 2 – Quantitative analyses of AOB and AOA by Q-PCR……………………………….111 3 – Phylogenetic affiliations of AOB and AOA amoA gene clones retrieved from the Qinghai Lake sediments…………………………………………………………….111 Chapter 5: Recovery and Dominance of Putative Marine Benthic Archaea in Sediments from an Inland Lake: Qinghai Lake, Northwestern China……117 1 – Carbon and oxygen isotopic compositions in the Qinghai Lake sediments………..137 2 – Quantitative analyses of cell abundance determined by Q-PCR…………………...137 3 – Phylogenetic affiliations and compositions of archaeal 16S rRNA gene clones retrieved from the Qinghai Lake sediments………………………………………...138 Chapter 6: Microbial Diversity in the Deep Marine Sediments from the Qiongdongnan Basin in South China Sea……………………………………143 1 – Pore water chemistry………………………………………………………………..166 iii LIST OF FIGURES Chapter 1: Introduction………………………………………………………………1 1 – Location and monsoon systems of the Tibetan Plateau…………………………….8 2 – Location of Lake Chaka, Qinghai Lake and South China Sea……………………...9 Chapter 2: Microbial Diversity in Water and Sediment of Lake Chaka: an athalassohaline lake in Northwestern China………………………………..10 1 – Location of Lake Chaka, northwestern China..…………………………………….41 2 – Depth distributions of geochemistry and microbial abundance in the Lake Chaka sediments……………………………………………………………………………41 3 – Physiological properties of selected isolates………………………………………..42 4 – Neighbor-joining tree showing the phylogenetic relationships of bacterial 16S rRNA gene sequences cloned from the Lake Chaka samples to closely related sequences from the GenBank……………………………………………………………….43-44 5 – Neighbor-joining tree showing phylogenetic relationships of archaeal 16S rRNA gene sequences cloned from the Lake Chaka samples to closely related sequences from the GenBank database…………………………………………………………………..45 6 – Frequencies of phylotypes affiliated with the major phylogenetic groups in the bacterial and archaeal clone libraries……………………………………………….46 7 – Clustering of the differenct bacterial clone libraries based upon ∆Cxy values deteremined from LIBSHUFF analysis……………………………………………..47 Chapter 3: Microbial Response to Salinity Change in Lake Chaka, a Hypersaline Lake on Tibetan Plateau………………………………………………………48 1 – A geographic map showing lakes on the Tibetan Plateau and the location of Lake Chaka in Northwestern China……………………………………………………….88 2 – Depth distributions of geochemistry and microbial abundance in the Lake Chaka sediments…………………………………………………………………………….88 3 – Depth profile of PLFA composition for 10 selected sediment subsamples………….89 4 – Neighbor-joining tree showing the phylogenetic relationships of bacterial 16S rRNA gene sequences cloned from the sediment subsamples of the 900-cm sediment core to closely related sequences from the GenBank database………………………………90 iv 5 – NaCl requirements of representative isolates from the core………………………...91 6 – Time-course production of Fe(II) from microbial reduction of Fe(III) in NAu-2 and the source sediment………………………………………………………………….92 7 – Neighbor-joining tree showing the phylogenetic relationships of archaeal 16S rRNA gene sequences cloned from the sediment subsamples of the 900-cm core to closely related sequences from the GenBank database……………………………………...93 8 – Schematic figure of the sediment core and frequencies of phylotypes affiliated with major phylogenetic groups in the bacterial and archaeal clone libraries……………94 Chapter 4: Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in the Sediments of Qinghai Lake, Northwestern China…………………….95 1 – A geographic map showing lakes on the Tibetan Plateau and the location of Qinghai Lake, NW China, and the drilling site in the lake………………………………….113 2 – Depth distribution of certain geochemical parameters in the pore water of the Qinghai Lake sediments……………………………………………………………………..113 3 – Neighbor-joining tree showing the phylogenetic relationships of bacterial amoA gene sequences cloned from four sediment subsamples of the 5-m core to closely related sequences from the GenBank database…………………………………………….114 4 – Neighbor-joining tree showing the