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Environmental Processes
Sulfur isotope fractionation by sulfatereducing microbes can reflect past physiology Andre Pellerin, Christine Wenk, Itay Halevy, and Boswell Wing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05119 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018
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Sulfur isotope fractionation by sulfate-reducing microbes can
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reflect past physiology
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André Pellerin1*, Christine B. Wenk2, Itay Halevy2 and Boswell Wing3
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[1] Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny
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Munkegade 114, Aarhus C 8000, Denmark
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[2] Department of Earth and Planetary Sciences, Weizmann Institute of Science, Rehovot
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76100, Israel
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[3] Geological Sciences, University of Colorado Boulder UCB 399, Boulder, CO 80309-
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0399, USA
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* Correspondence to: André Pellerin (
[email protected])
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ABSTRACT
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Sulfur (S) isotope fractionation by sulfate-reducing microorganisms is a direct manifestation of
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their respiratory metabolism. This fractionation is apparent in the substrate (sulfate) and waste
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(sulfide) produced. The sulfate-reducing metabolism responds to variability in the local
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environment, with the response determined by the underlying genotype, resulting in the
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expression of an ‘isotope phenotype’. Sulfur isotope phenotypes have been used as a diagnostic
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tool for the metabolic activity of sulfate-reducing microorganisms in the environment. Our
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experiments with Desulfovibrio vulgaris Hildenborough (DvH) grown in batch culture suggest
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that the S isotope phenotype of sulfate respiring microbes may lag environmental changes on
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timescales that are longer than generational. When inocula from different phases of growth are
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assayed under the same environmental conditions, we observed that DvH exhibited different net
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apparent fractionations of up to -9‰. The magnitude of fractionation was weakly correlated with
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physiological parameters, but was strongly correlated to the age of the initial inoculum. The S
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isotope fractionation observed between sulfate and sulfide showed a positive correlation with
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respiration rate, contradicting the well-described negative dependence of fractionation on
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respiration rate. Quantitative modelling of S isotope fractionation shows that either a large
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increase (≈50X) in the abundance of sulfate adenylyl transferase (Sat), or a smaller increase in
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sulfate transport proteins (≈2X) is sufficient to account for the change in fractionation associated
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with past physiology. Temporal transcriptomic studies with DvH imply that expression of sulfate
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permeases doubles over the transition from early exponential to early stationary phase, lending
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support to the transport hypothesis proposed here. As it is apparently maintained for multiple
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generations (≈1-6) of subsequent growth in the assay environment, we suggest that this
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fractionation effect acts as a sort of isotopic ‘memory’ of a previous physiological and
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environmental state. Whatever its root cause, this physiological hysteresis effect can explain
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variations in fractionations observed in many environments. It may also enable new insights into
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life at energetic limits, especially if its historical footprint extends deeper than generational.
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INTRODUCTION
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Stable isotope analysis can monitor microbiologically-catalyzed reactions in the environment
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where the progressive enrichment of heavy isotopologues in metabolic substrates and their
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depletion in the corresponding wastes (known as ‘fractionation’) can be used to constrain
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degradation rates of contaminants by indigenous microbial populations1-8 . The progressive
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isotopic enrichments and depletions observed in the environment reflect cellular-level
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fractionations that depend on a microorganism’s physiological response to the local
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environment9-11 as well as the underlying genotype12,
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phenotypic trait14. While the sulfur isotope phenotype of sulfate-reducing microorganisms has
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been widely used to constrain microbial activity in modern and ancient marine sediments9, 15-18
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the ubiquity of aqueous sulfate in many continental aquifers19,
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fractionation is a critical tool for monitoring the functioning of microbial populations in these
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environments. For example, in aquifers that contain petroleum hydrocarbons, sulfate reduction
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contributes significantly to hydrocarbon decomposition and sulfur isotope fractionation can be
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used as a proxy to estimate rates of sulfate reduction, and hydrocarbon consumption in-situ3, 21-23
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, providing a tool for estimating the effectiveness of bioremediation in contaminated aquifers as
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well as the potential ‘souring’ of petroleum reserves. The underlying assumptions that enable
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sulfur isotope fractionation to be used as a probe of sub-surface microbial activity are: (1)
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genotype variations between environments and in time have no important impact on the S
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isotope phenotype, (2) the magnitude of isotope fractionation responds rigorously to
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environmental conditions and (3) microbial isotope fractionation responds rapidly to
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environmental changes.
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The first assumption has been the focus of studies investigating the standing variability of
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genotypes in nature and its influence on fractionation13, as well as studies focusing on the
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importance of evolution to fractionation24. The second assumption has been tested through
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repeated measurements in batch cultures24, chemostats25 and retentostats11 and are largely
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, and can thus be thought of as a
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means that S isotope
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confirmed in directionality and magnitude. Observations that lead to the generally held third
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assumption, that microbial isotope fractionation responds instantaneously to environmental
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change, are widespread. For example, pure cultures of Archaea that perform chemolithotrophic
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methanogenesis show isotopic fractionation magnitudes that differ between exponential and
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stationary phase26, suggesting that the transition time between the exponential and stationary
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phase is long enough for the isotope phenotype to reflect the changing conditions. Similar
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conclusions can be drawn for carbon and hydrogen isotope fractionation in pure and co-cultures
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of hydrogenotrophic methanogenic or acetogenic microbial cultures with H227,
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isotope fractionation due to experimentally controlled rate limitation by CO2 supply to
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cyanobacteria29, for carbon isotope fractionation by methanotrophs30, for S and oxygen isotope
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fractionation during pyrite oxidation by Acidithiobacillus ferrooxidans31 and for S isotope
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fractionation by sulfate reducing bacteria in batch culture and retentostats9, 11, 32-34.
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Despite indications for rapid organismal responses to changing environmental conditions,
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striking differences in physiology are evident throughout a batch cycle and may measurably
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affect the isotope phenotype. For instance, it has been observed, that the past short-term history
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of cells affects nitrogen isotope fractionation during denitrification35, suggesting that physiology
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of microorganisms may not respond instantaneously to changing environmental conditions.
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Fitness optimization and stress responses of microbes in fluctuating environments may be
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capable of driving such decoupling in physiology and environment36-38. Cellular metabolic
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systems with turnover times exceeding the lifetime of a cell will respond to environmental
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changes on timescales potentially longer than the microorganism’s generation time39, and would
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thus decouple the isotope phenotype from the one expected based on environmental state, at least
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temporarily. Such lags in the response of microbiological isotope fractionation could confound
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, for carbon
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laboratory experiments looking at the relationship between fractionation and environmental
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changes, unless they were run under conditions of balanced growth24, 40. Likewise, isotopic lags
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could also have profound implications for interpretations of the behavior of natural microbial
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populations from stable isotope fractionations. For example, repeated acetate amendments aimed
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at inducing uranium bioremediation in contaminated aquifers have shown hysteresis in sulfur
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isotope fractionation on a timescale of years41. While such a lag in S isotope fractionation has
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been attributed to the stimulation of microbial growth (and successful bioremediation), a
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decoupling of growth physiology from environmental change may also impart a lag in S isotope
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fractionation, leading to erroneous interpretations of the response times of natural microbial
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populations to environmental stimulation.
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Motivated by these concerns, we designed an experiment aimed at isolating the isotopic response
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of cells with differing physiology to new environmental conditions. Different physiological
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states were accessed by transferring cells from a pure culture of a sulfate-reducing
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microorganism (SRO), Desulfovibrio vulgaris Hildenborough (DvH) in various stages of batch-
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culture growth to fresh media. If the isotope phenotype responded instantaneously to a change in
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environmental conditions, all cultures should produce the same isotope phenotype in the assay
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conditions (the fresh media). However, if preexisting physiology plays a role in shaping the
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isotope phenotype, then cells with physiology adapted to an environment closely resembling the
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assay conditions (early to mid exponential phase) and cells adapted to significantly different
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environmental conditions from the assay conditions (late exponential to stationary phase) should
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fractionate isotopes differently. If the isotope phenotype is delayed because the physiology of the
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cells does not adjust instantaneously to the assay conditions, the isotopic discrimination should
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reflect, at least in part, the cell’s changing physiology, its past physiology and the environmental
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conditions under which it was previously growing. The experimental approach offers a simple
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measure of the speed at which the isotope phenotype reflects the new environmental conditions.
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Our results support the latter scenario, resolving what may be a short term decoupling of the S
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isotope phenotype from bacterial physiology, with implications for measurements of isotope
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fractionation in pure cultures and in nature.
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METHODS
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Choice of model organism
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DvH is commonly used as a model organism to investigate the evolutionary, physiological,
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enzymatic, genetic and growth characteristics of sulfate-reducing bacteria42-47. Experiments have
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shown that populations of DvH can express a wide range of S isotope fractionations that vary
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predictably with the rate of sulfate respiration25. Recent models of S isotope fractionation can
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reproduce the observed S isotope signatures produced by DvH14, and suggest that these
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signatures reflect intracellular metabolite levels, thereby providing a mechanistic framework for
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interpreting the resulting fractionations.
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Growth media
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All experiments were performed in a Tris-buffered chemically defined medium (MOLS4) that
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consists of 30 mM sodium sulfate, 60 mM sodium lactate, 8 mM MgCl2, 20 mM NH4Cl, 2 mM
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K2HPO4-NaH2PO4, 30 mM Tris-HCl, as well as solutions of trace elements, Thauer’s vitamins
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and rezasurin as an oxygen indicator48. The pH was adjusted to 7.2 with hydrochloric acid. 80
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mL of MOLS4 was placed into 120-mL serum bottles for the assays. Bottles were crimp sealed
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with chloro-butyl rubber stoppers, and the headspace was purged of oxygen by flushing with
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pure N2 gas. After purging, individual crimp-sealed medium bottles were sterilized in an
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autoclave.
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Characterization of sulfur isotope fractionation
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Five mL of culture was transferred into N2-purged and sterilized serum bottles containing 80 mL
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of sterile growth medium and a magnetic stir bar. The assay bottles were vigorously stirred while
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being simultaneously purged with pure N2 gas for two hours to remove any sulfide that was
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carried over with the inoculum. Repeated tests showed that the sulfide blank in the assay medium
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after purging was
