MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Gengxin Zhang Candidate for the Degree: Doctor of Philosophy _______________________________________ Hailiang Dong, Director(Advisor) ________________________________________ John Rakovan, Reader ________________________________________ Jonathan Levy, Reader ______________________________________ Yildirim Dilek, Reader ______________________________________ Matthew W. Fields, Graduate School Representative ABSTRACT Geomicrobial Processes and Diversity in Ultra-High Pressure Metamorphic Rocks and Deep Fluids from Chinese Continental Scientific Deep Drilling By Gengxin Zhang This dissertation investigates the microbial communities and microbe-mineral interactions in ultra-high pressure metamorphic rocks and deep fluids from the Chinese Continental Scientific Drilling (CCSD) project by using geochemical, mineralogical, cultivation and molecular microbiology methods. The drilling site is located in the eastern part of the Dabie-Sulu ultra high-pressure metamorphic (UHPM) orogenic belt at the convergent plate boundary between the Sino-Korean and Yangtze Plates. This integrated approach conclusively demonstrates that microbes can survive in the deep continental subsurface (down to 3350 m) and they play important roles in mineral transformations and elemental cycling. The first half of this study focuses on geochemical conditions and diversity and metabolic functions of microbial community. Characterization of SSU rRNA genes indicated that the bacterial clone sequences shifted form a Proteobacteria-dominated community to a Firmicutes-dominated one with increased depth. From the ground surface to 2030 m, most clone sequences were related to nitrate reducers, with a saline, alkaline, and cold habitat. From 2290 to 3350 m most sequences were closely related to anaerobic, thermophilic, halophilic or alkaliphilic bacteria. The archaeal diversity was low. Most archaeal sequences from the ground surface to 3350m were not related to known cultivated species, but to environmental clone sequences recovered from subsurface marine environments. An important contribution of this research is an enrichment of a thermophilic (optimal temperature of 68oC) organism from 2450m with an ability to reduce Fe(III) and oxidize Fe(II) under different conditions. This enriched organism was capable of reducing Fe(III) in aqueous form and in the structure of clay minerals and iron oxides at acidic pH. This organism was also capable of oxidizing Fe(II) in aqueous form and in the structure of pyrite and siderite. The second half of this dissertation focuses on microbe-mineral interactions by using enriched and isolated cultures to react with clay and iron oxide minerals. Mesophilic and thermophilic iron-reducing bacteria were incubated with lactate as the electron donor and structural Fe(III) in solid minerals as the sole electron acceptor. Extensive mineral reaction took place. One important such reaction was the smectite to illite reaction promoted by mesophilic and thermophilic metal reducing bacteria. This particular reaction highlights the significant role of iron-reducing bacteria in promoting the smectite to illite reaction at high temperature. Geomicrobial Processes and Diversity in Ultra-High Pressure Metamorphic Rocks and Deep Fluids from Chinese Continental Scientific Deep Drilling 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 Gengxin Zhang Miami University Oxford, Ohio 2006 Dissertation Director: Hailiang Dong, Ph.D. TABLE OF CONTENTS Chapter 1: Introduction 1 References 3 Chapter 2: Microbial Diversity in Ultra-High Pressure Rocks and Fluids 6 From the Chinese Continental Scientific Drilling in China Abstract 7 Body Text 8 References 31 Chapter 3: Unique Microbial Community in Drilling Fluids From 50 Chinese Continental Scientific Drilling Abstract 51 Body Text 52 References 71 Chapter 4: Evidence for Microbial-Mediated Iron Redox cycling 90 in the Deep Subsurface Abstract 91 Body Text 92 References 113 Chapter 5: Microbial Reduction of Structural Fe(III) in Nontronite 141 by Thermophilic Bacteria and Their Roles in Promoting the Smectite-Illite Reaction Abstract 142 Body Text 143 References 154 Chapter 6: Microbial Effects in Promoting the Smectite to Illite Reaction: 171 Role of Organic Matter Intercalated in the Interlayer Abstract 172 Body Text 173 References 187 Chapter 7: Summary 204 ii LIST OF TABLES Chapter 2: Microbial Diversity in Ultra-High Pressure Rocks and Fluids 6 From the Chinese Continental Scientific Drilling in China 1 - Summary of Anions, TOC and 13C isotope compositions for the 36 rocks and drilling fluids 2 - Chemical composition of the rock samples from CCSD 37 3 - Phylogenetic bacterial rDNA clone-type analysis 38 Chapter 3: Unique Microbial Community in Drilling Fluids From 50 Chinese Continental Scientific Drilling 1 - Anion and cation composition, pH, salinity and in-situ temperature 78 for the drilling fluid samples Chapter 4: Evidence for Microbial-Mediated Iron Redox Cycling 90 in the Deep Subsurface 1 - Composition of M1 and AG medium 122 2 - Anion and cation composition, pH, salinity and in-situ temperature for 123 the drilling fluid sample 3 - Experimental conditions and employed analysis methods 124 Chapter 5: Microbial Reduction of Structural Fe(III) in Nontronite 141 by Thermophilic Bacteria and Their Roles in Promoting the Smectite-Illite Reaction 1 - Experimental conditions used for nontronite reduction in bacterial 161 cultures and abiotic controls 2 - Change in pH and Eh as a result of Fe(III) bioreduction 162 Chapter 6: Microbial Effects in Promoting the Smectite to Illite 171 Reaction:Role of Organic Matter Intercalated in the Interlayer iii LIST OF FIGURES Chapter 2: Microbial Diversity in Ultra-High Pressure Rocks and Fluids 6 From the Chinese Continental Scientific Drilling in China 1 - A map showing general geology in the Dabie-Sulu orogen 43 of central-eastern China 2 - A back-scattered electron image showing fractures in garnet and pyroxene 44 3 - An optical micrograph showing various mineral/fluid inclusions in 45 the UHP rocks 4 - Neighbor-joining tree of nearly full-length sequences (~1400 bp) of 46 isolates and representative examples of bacterial clone sequences 5 - Typical electropherograms of bacterial T-RFLPs 47 6 - Phylogenetic relationships of representative phylotypes of bacterial 48 16S rRNA gene sequences 7 - Phylogenetic relationships of representative phylotypes of archaeal 49 16S rRNA gene sequences Chapter 3: Unique Microbial Community in Drilling Fluids From 50 Chinese Continental Scientific Drilling 1 - A map showing general geology in the Dabie-Sulu orogen 81 of central-eastern China 2 - PLFA profiles for the drilling fluid samples 82 3 - Neighbor-joining tree of nearly full-length sequences (~1400 bp) 83 of isolates and representative examples of bacterial clone sequences 4 - Ferrihydrite was reduced by CCSD_DF2290_FWA_60_ isolate1 84 5 – Nontronite was reduced by CCSD_DF2450_M1_68_ isolate6 85 6 - Phylogenetic relationships of representative phylotypes of bacterial 87 16S rRNA gene sequences 7 - Phylogenetic relationships of representative phylotypes of archaeal 88 16S rRNA gene sequences 8 - Stacked bar graph showing the contribution of each family of bacteria 89 iv in the clone libraries for the drilling fluid samples Chapter 4: Evidence for Microbially-Mediated Iron Redox 90 Cycling in the Deep Subsurface 1 - Change in 0.5 N HCl-extractable Fe(II) with time in the first 128 transfer from the enrichment culture in the M1 medium 2 - Changes of Fe(II) concentration, lactate, acetate Eh and pH 129 with time in the second transfer in AG medium 3 - Change in 0.5 M HCl-extractable Fe(II) with time in the 131 third transfer in AG medium with nontronite, ferric citrate or ferrihydrite 4 - Photographs of biological oxidized FeS and abiotic FeS control 133 5 - XRD patterns of bio-oxidized FeS, abiotic control and reference 134 6 – Secondary electron image showing that ferric citrate was reduced 135 to form vivianite and then partially oxidized in the third transfer in AG medium 7 – Secondary electron image showing ferrihydrite as an oxidation 136 products of FeS 8 – TEM micrographs of microbially oxidized FeS 137 9 – Mössbauer spectra of the bioreduced ferric citrate at different temperature 139 10 – Phylogenetic relationships of representative phylotypes of bacterial 140 16S rRNA gene sequences Chapter 5: Microbial Reduction of Structural Fe(III) in Nontronite 141 by Thermophilic Bacteria and Their Roles in Promoting the Smectite-Illite Reaction 1 - Production of biogenic Fe(II) from bioreduced nontronite samples 165 2 - XRD patterns for of nontronite samples 166 3 - Secondary electron images of bioreduced and nonreduced nontronite 167 4 - Secondary electron image showing evolution of illite crystal morphology 168 5 - TEM micrographs for bioreduced and nonreduced nontronite 169 v 6 - Histograms showing the distribution of layer spacings in bioreduced 170 and nonreduced nontronite Chapter 6: Microbial Effects in Promoting the Smectite to Illite 171 Reaction:Role of Organic Matter Intercalated in the Interlayer 1 - XRD patterns for oriented specimens of nontronite 194 2 - Comparison of FTIR spectra for nontronite, cysteine 195 and cysteine-NAu-2 complex 3 - Production of biogenic Fe(II) from bioreduced nontronite 196 4 - Secondary electron images