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When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk UNIVERSITY OF SOUTHAMPTON FACULTY OF ENGINEERING , SCIENCE AND MATHEMATICS School of Ocean and Earth Science The role of microbial populations in the cycling of iron and manganese from marine aggregates by Sergio Balzano Thesis for the degree of Doctor of Phylosophy June 2009 BIOTRACS Bio-transformations of trace elements in aquatic systems THIS THESIS WAS COMPLETED USING FRAMEWORK 6 FUNDING FROM THE EUROPEAN UNION. RESEARCH DG HUMAN RESOURCES AND MOBILITY PROJECT NO: 514262 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF ENGINEERING, SCIENCE AND MATHEMATICS SCHOOL OF OCEAN AND EARTH SCIENCES Doctor of philosophy THE ROLE OF MICROBIAL PROCESSES IN MARINE AGGREGATES ON THE CYCLING OF IRON AND MANGANESE By Sergio Balzano Marine aggregates play an important role in the cycling of carbon, nutrients and trace metals. Within aggregates the oxygen depleted by aerobic microbial respiration may not be replaced rapidly, generating anoxic or suboxic microzones. Reduced compounds that are unstable in the oxygenated water column have been previously found associated with marine snow. Therefore, in the experiments described in this thesis, artificial aggregates were made in the laboratory from senescent phytoplankton material and incubated to investigate the role of the associated microbial populations to the biogeochemical redox cycling of iron and manganese and to the degradation of organic matter. The release of dissolved iron from artificial aggregates which did not contain any measurable (~10 µm) anoxic microzones, was demonstrated under dark conditions. The rate of release was controlled by the amount of reducible Fe(III) available, and appears to be limited by the competing oxidation of Fe(II). Moreover highly significant releases in reduced Mn were detected from aggregates incubated under a constant velocity shear, although the same aggregates did not affect the dissolution of iron. A possible reason is likely associated with the higher stability of Mn(II), compared to Fe(II) in aerobic environments. Molecular (16S rRNA gene) analyses showed the bacterial community associated with artificial aggregates to be similar to that found in natural aggregates and dominated by (predominantly uncultured) α- and γ-Proteobacteria, Bacteroidetes, - Planctomycetes and Cyanobacteria . It was possible to culture NO 3 -, Fe(III)- and Mn(IV)-reducing bacteria from the artificial aggregates, and marine particles incubated with Fe(III) under anaerobic conditions contained a range of γ- and δ-Proteobacteria known to respire Fe(III) and in most cases Mn(IV). Moreover several microorganisms belonging to γ-Proteobacteria were isolated from marine aggregates and strains affiliated to the genera Amphritea , Marinobacterium and Marinobacter, were demonstrated to grow through the reduction of Fe(III), with Marinobacter also capable of respiring Mn(IV). Whilst the precise mechanism of reduction is not clear, it is evident that marine aggregates can be a source of Fe(II) and dissolved Mn, in coastal waters and most probably other natural water systems. Fatty acid analyses revealed the prevalence of saturated over unsaturated fatty acids indicating that aggregates were already partially degraded when incubation started. Nonetheless, the lipids in the artificial aggregates were rapidly degraded further as indicated by a depletion in short chain (<20) saturated and monounsaturated fatty acids. In contrast, the concentrations of linear and branched, saturated long chain (>20) fatty acids fluctuated, suggesting that some of these lipids could have been produced in situ by marine microorganisms rather than deriving from I higher plant debris. In addition, a bacterial branched monounsaturated fatty acid (11- methyl-octadecenoic acid), which has not previously been found in marine particles was present in artificial aggregates. Roseobacter litoralis found among the aggregate- attached bacteria contains 11-methyl-octadecenoic acid, and other bacteria present in artificial aggregates have the potential to produce long-chain saturated and polyunsaturated fatty acids. Thus, the fatty acid assemblage appears to reflect both organic matter degradation, including selective preservation, but also changes in the microbial assemblage. A range of future studies are suggested to elucidate the mechanisms for Fe(III) and Mn(IV) reduction in aggregates. These include microscale analyses of dissolved species and evaluation of the presence of metal binding ligands associated with aggregates. Moreover it is important to assess the activity of the Fe(III)- and Mn(IV)- reducing bacteria present in aggregates in situ and the production of long chain fatty acids in degrading aggregates. II Graduate School of the National Oceanography Centre, Southampton This PhD thesis by Sergio Balzano has been produced under the supervision of the following persons Supervisors: Prof. Peter J Statham Dr Richard D Pancost Prof. Jonathan R Lloyd Chair of advisory panel: Dr Duncan Purdie III Table of contents Abstract………………………………………………………………………………….……….I Table of contents…………………………………………..…………………………...………IV List of Tables ……………………………………………………………………………...…...XI List of Figures ……………………………………………………………………..…………XIII Declaration of authorship…………………………………………………………………….XVI Acknowledgments………………………………………………………………………….XVII List of Abbreviations……………………………………………………………………….XVIII Chapter 1. Introduction ………………………………………………….…………...……...1 1.1 Importance of biogeochemical cycling in the water column …..…....................1 1.1.1 Iron and manganese…………………………………………………....…..2 1.1.2 Impact of sinking material on trace metal cycles…………….…………....2 1.2 Phytoplankton growth and aggregate formation …………………………....…2 1.2.1 Aggregation of particles in the water column…………….…………...…...2 1.2.2 Transparent exopolymer particles (TEP)………………………………..…4 1.2.3 Marine aggregates: composition and classification…………...…………...5 1.3 Artificial marine aggregates ……………………………………………...…....6 1.3.1 Sampling natural marine snow: difficulties and limitations…………….....6 1.3.2 Previous attempts to aggregate organic matter…………………………….7 1.4 Impact of sinking particles on nutrients and trace metal cycles ……………….7 1.4.1 Trace elements in seawater…………………………..……….…................7 1.4.2 Trace metal chemistry……………………….……………..……................8 1.4.3 Biogeochemistry of iron……………………………………..….................9 1.4.4 Biogeochemistry of manganese……………………………...…………...12 1.5 Microbial colonisation of marine aggregates ………………………...………13 1.5.1 Marine microbes……………………………………………….................13 1.5.2 Interactions of heterotrophic eukaryotes with marine particles……….….13 1.5.3 Prokaryotes attached to marine aggregates………………….…................14 1.5.4 Ecological successions in marine snow………………..............................15 1.6 Degradation of marine particles …………………………………………...…17 1.6.1 Freshness and lability of organic matter in aggregates……………..….…17 1.6.2 Indicators of diagenetic status of organic matter……………………...….17 1.6.3 Alteration of organic matter with depth…………………………………..18 1.6.4 Fate of phytodetritus on the seafloor…………………..............................18 IV 1.6.5 Degradation of lipids in marine particles……………………………....…19 1.6.6 The particle decomposition paradox…………………………………...…22 1.7 Boundary layers and anoxia in marine aggregates …………………………...22 1.7.1 Implications for microbial respiration in aggregates…………………..…23 1.7.2 Reduced species and anaerobic bacteria in aggregates……………...……24 1.8 Anaerobic degradation of organic matter ……………………………...……..25 1.8.1 Electron acceptors alternative to oxygen………………………...…….…25 1.8.2 Fermentation and its implications on trace metal cycling……...………...26 1.8.3 Microbial reduction of iron and manganese……………….......................27 1.9 Aims and objectives ……………………………………………..…………...28 1.9.1 The BIOTRACS project……………………………………..…………...28 1.9.2 Objectives……………………………………………...……………29 Chapter 2. Materials and Methods……………………………………………...…...30 2.1 Artificial aggregates as model particles for biochemical studies ……..….…..30 2.2 Formation of artificial marine aggregates ………………………………...….30 2.2.1 Cleaning procedures…………………………………………………........30 2.2.2 Method overview……………………………………….........……..…......31 2.2.3 Cultivation of phytoplankton strains…………………………..…..……...31 2.2.4 Cultivation of mixed phytoplankton assemblages……………..…..…..…32 2.2.5 Formation of artificial marine aggregates………………………..…...…..33 2.3 Release of reduced iron, fatty acids and microbial populations in aggregates…………..…………………………………………………..…….35 2.3.1 Aggregates from cultures of Thalassiosira and Alexandrium….................35 2.3.2 Aggregates from natural seawater samples…………..…………...............36 2.3.3 Incubation