UNIVERSITY OF CALIFORNIA, SAN DIEGO Ecology of microbe/ basaltic glass interactions: Mechanisms and Diversity A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Marine Biology by Lisa A. Sudek Committee in charge: Professor Bradley M. Tebo, Chair Professor Hubert Staudigel, Co-Chair Professor Katherine A. Barbeau Professor Douglas H. Bartlett Professor Michael J. Sailor Professor Alexis S. Templeton 2011 Copyright Lisa A. Sudek, 2011 All rights reserved. The Dissertation of Lisa A. Sudek is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California San Diego 2011 iii This work is dedicated to my family: Both, overseas and here, including the little lady who will join us soon. I wouldn’t have accomplished this without your love and support. iv Table of Contents Signature page....................................................................................................... iii Dedication............................................................................................................. iv Table of Contents.................................................................................................. v List of Figures...................................................................................................... vi List of Tables....................................................................................................... xi Acknowledgements.............................................................................................. xii Vita and Publications........................................................................................... xix Fields of study..................................................................................................... xx Abstract................................................................................................................ xxi Chapter I Introduction.................................................................................. 1 Chapter II Microbial ecology of Fe (hydr)oxide mats and basaltic rock from Vailulu’u Seamount, American Samoa............................................................... 19 Chapter III Siderophore-mediated weathering of the ocean’s crust: Microbes and the role of free organic ligands................................................................................... 71 Chapter IV Exploring volcanic glass colonization by the deep-sea, heterotrophic Fe(II)oxidizing bacterium Pseudomonas stutzeri VS-10................... ………… 99 Chapter V Towards an understanding of Pseudomonas stutzeri VS-10 interaction with basaltic glass 1. Auxotrophic mutants of P. stutzeri VS-10 generated by Tn5 transposon mutagenesis: phenotypic, physiological and genetic characterization.... 155 2. Characterization of Amonabactin-metal complexes in Pseudomonas stutzeri VS-10 ........................................................................................ 182 3. Organic acid production by P. stutzeri VS-10 and their potential role in basalt dissolution.................................................................................... 193 Chapter VI Growth of Pseudomonas stutzeri VS-10 on basaltic glass and its effect on rock surface texture and chemistry...................................................... 231 v List of Figures Chapter II Figure 2.1 Bathymetric map of the summit region of Vailulu’u Seamount 50 Figure 2.2 Rarefaction curves for clone libraries from the Nafanua Summit Mat, the Nafanua Summit Rock and the Eastern Moat Mat…. 51 Figure 2.3 Phylogenetic composition of two mat samples and one rock sample from Vailulu’u……………………………………….. 51 Figure 2.4 Phylogenetic distribution of cultured strains of Fe(II)-oxidizing bacteria, Mn(II)-oxidizing bacteria and siderophore-producing bacteria…………………………………………..................... 52 Figure 2.5 Phylogenetic distribution of cultured strains………………... 53 Figure 2.6 Scanning electron micrographs of Fe (hydr)oxides in a mat sample from the summit of Nafanua, a microaerophilic enrichment culture of lithotrophic FeOB, an isolated culture from those enrichments and an abiotic control………………………….. 54 Chapter III Figure 3.1 Basic redox cycle of Fe……………………………………… 88 Figure 3.2 CAS plates from the 2003 cruise to Loihi Seamount, Hawaii……………………………………………………….. 88 Figure 3.3 Structure of Pyoverdin………………………………………. 89 Figure 3.4 Structure of Desferrioxamine B (DFO-B)………………….. 89 Figure 3.5 CAS plate after 1st transfer of colonies……………………… 90 Figure 3.6 Spectrum for basalt PVD and basalt DFO………………….. 90 Figure 3.7 The complexation of Fe from basalt by PVD and DFO over 16 days………………………………………………………. 91 vi Figure 3.8 Fe-PVD complexation in all three experimental set-ups compared to the negative controls……………………………………… 92 Figure 3.9 Basalt/DFO-B system under anaerobic conditions………….. 92 Figure 3.10 The complexation of Fe from basalt and rhyolite PVD and DFO…………………………………………………………. 93 Figure 3.11 Amount of Fe released from both basalt and rhyolite by DFO and PVD over 16 days normalized to their surface area………… 94 Chapter IV Figure 4.1 Growth of P. stutzeri VS-10 on chelexed minimal medium without additives or with the addition of either basalt, rhyolite, quartz or 100 μM FeCl2……………………………………………….. 136 Figure 4.2 Photograph of deep-sea bacteria grown on organic-rich F-plates and Prussian-Blue test………………………………………. 137 Figure 4.3 Liquid Chromatography Mass Spectrometry (LC-MS) results on the presence of siderophore in the supernatant of a P. stutzeri VS-10 culture………………………………………………... 138 Figure 4.4 Structures of Amonabactin P750 and P693…………………. 139 Figure 4.5 Diffusion chamber experiments demonstrating growth of P. stutzeri VS-10 on chelexed minimal glycerol medium in the presence of basalt………….………………………………… 139 Figure 4.6 Scanning electron microgrpahs (SEM) of P. stutzeri VS-10 biofilms on basaltic glass…………………………................ 140 Figure 4.7 Scanning electron micrographs of P. stutzeri biofilms on basalt and rhyolite…………………………………………………. 141 Figure 4.8 Power production by P. stutzeri VS-10 on minimal glycerol medium in a microbial fuel cell……………………………... 142 Figure 4.9 Microaerobic microbial fuel cell run………………………... 143 vii Figure 4.10 Scanning electron micrograph of the anode felt from the microbial fuel cell…………………………………………… 144 Figure 4.11 Polarization and power curves for the microbial fuel cell….. 145 Figure 4.12 Cyclic voltammograms of the microbial fuel cell…………… 146 Figure 4.13 Scanning electron micrograph of P. stutzeri on a silicon wafer chip…………………………………………………………. 146 Figure 4.14 Results from nanolithographical experiments………………. 147 Chapter V Figure 5.1 Typical set-up for a microbial fuel cell (MFC)…………….. 201 Figure 5.2 Growth curve of WT and mutants of strain VS-10 on minimal medium with and without basalt……………………………. 201 Figure 5.3 Growth of WT and mutants on LB and LBKan medium after 24 hrs at 37 °C………………………………………………. 202 Figure 5.4 Growth of mutants and WT on a regular LB plate and on a motility LB plate……………………………………………. 202 Figure 5.5 LC-ESI-MS results on the detection of siderophores in the supernatant of high cell densities WT and mutant cultures.... 203 Figure 5.6 Growth of WT and mutants (including VIII21 and XII5) on organic-rich F-plates………………………………………… 204 Figure 5.7 Gradient tubes inoculated with either WT or mutant cultures (2A and 9G)………………………………………………… 204 Figure 5.8 Scanning electron micrographs (SEMs) of mutant 2A and WT biofilms grown on LB medium with basalt……………. 205 Figure 5.9 SEMs of WT and mutants grown on minimal medium and basalt for 4 days…………………………………………….. 206 Figure 5.10 SEMs of mutants on basalt at high cell densities on minimal medium (close-ups of Figure 9)…………………………….. 207 viii Figure 5.11 Power curves for WT and two mutants (2A and 9G) collected over a time period of ~ 70 hours……………………………. 208 Figure 5.12 SEMs of anode graphite fibers from the fuel cell experiments 209 Figure 5.13 Genome region in P. stutzeri A1501……………………….. 210 Figure 5.14 Mutant rescue experiments including amendments with exogenous siderophores and homoserine lactones……………………… 211 Figure 5.15 Growth curves for WT and mutant 2A on minimal medium with addition of 8.7 mM of L-proline………………………. 212 Figure 5.16 Growth curves for WT and mutants on minimal medium after the addition of 0.5 % of Casamino acids (CAA)…………… 212 Figure 5.17 Scanning electron micrographs of mutants 2A, XII5 and 11G grown on minimal medium, basalt and 0.5 % of CAA for 3 days…………………………………………………………. 213 Figure 5.18 Presence of amonabactin P750 and P693 in the supernatant of WT and mutant 2A grown on minimal medium, rhyolite and 8.7 mM proline………………………………………………………. 214 Figure 5.19 Power and polarization curves for WT and mutant 2A in minimal medium fuel cells containing 8.7 mM of L-proline………… 215 Figure 5.20 Color and UV-visible spectrum of the Fe-siderophore complexes…………………………………………………… 216 Figure 5.21 pH characteristics of Fe-siderophore complex(es)…………. 217 Figure 5.22 Formation of different siderophore complexes with Fe (II + III) and Mn (II)………………………………………………….. 218 Figure 5.23 UV-Vis spectra of Fe (II) and Fe (III) complexes under aerobic and anaerobic conditions………………………………………… 219 Figure 5.24 Color of the Fe (II) and Fe (III) and Mn (III)-siderophore complexes under aerobic and anaerobic conditions……………………... 219 Figure 5.25 Growth curves and pH of P. stutzeri VS-10 cultures on minimal medium with and without basalt…………………………….. 220 ix Figure 5.26 Detection