Microbial sulfur cycling in chemosynthetic biofilms, deep sea sediments, cyanobacterial mats, and phototrophic aggregates

Current lab members: Kat Dawson (postdoctoral researcher) and Min Sub Sim (postdoctoral Researcher) and Hank Yu (ESE graduate student)

Former lab members on ‘team sulfur’: Postdocs, David Fike, Derek Smith, Lizzy Wilbanks and student Crystal Gammon

33S isotope tracers and single cell isotopic phenotyping within sulfur metabolizing chemosynthetic biofilms at White Point

The objective of this project is to develop techniques for the application of 33S and 34S labeled sulfate and elemental sulfur in probing biological sulfur cycling. Here we describe experiments to examine the intracellular transfer of biologically derived sulfide in environmental microbial communities with triple stable isotope labeling experiments: 13C, 15N and 33S or 34S. Silicon wafers colonized by microbial mats in situ, were incubated with isotopically labeled substrates and subsequently analyzed by fluorescent in situ hybridization (FISH) coupled to nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowing the simultaneous measurement of 13C/12C, 15N/14N, 33S/32S and 34S/32S of individual cells.

Image (right): Illustration of the workflow from multiple-isotope SIP incubations through to the identification of stable isotope phenotypes and their properties via cluster analysis of NanoSIMS data. From Dawson, Katherine S. and Scheller, Silvan and Dillon, Jesse G. et al. (2016) Stable Isotope Phenotyping via Cluster Analysis of NanoSIMS Data As a Method for Characterizing Distinct Microbial Ecophysiologies and Sulfur-Cycling in the Environment. Frontiers in Microbiology, 7 . Art. No. 774.

After colonization with microbial biomass, NanoSIMS compatible Si-wafers were incubated with multiple stable isotope labeled substrates. Microscopy, using FISH probes, identified regions that were mapped for subsequent NanoSIMS analysis of up to seven parallel masses. NanoSIMS data was processed using Look@NanoSIMS to define single cell regions of interest (ROIs) and extract associated isotope and elemental composition ratios. Several cluster analysis algorithms were evaluated to determine the best method, which was then applied to partition the NanoSIMS ratio data. The properties of the stable isotope phenotype clusters were examined and compared to independent FISH images to investigate label uptake in a multispecies metabolic network.

Fluroescent in situ hybridization coupled to nanometer-scale secondary ion mass spectrometry (FISH-NanoSIMS)

FISH micrographs of Deltaproteobacteria associated with Gammaproteobacteria in incubations with a) 13C-acetate, 15NH4+ and ~10 at.% 34SO42- or b) 13C-acetate, 15NH4+ and ~15 at.% 33SO42-. The bottom row shows the corresponding nanoSIMS ratio image for c) 34S/32S or d) 33S/32S.


Dawson, K. S., Scheller, S., Dillon, J. G., & Orphan, V. J. (2016). Stable Isotope Phenotyping via Cluster Analysis of NanoSIMS Data As a Method for Characterizing Distinct Microbial Ecophysiologies and Sulfur-Cycling in the Environment. Frontiers in Microbiology, 7, 774. http://doi.org/10.3389/fmicb.2016.00774

Identification and analysis of S intermediates in environmental samples using UPLC-ToF-MS

(Postdoc Derek Smith, collaborator Dr. Alex Sessions)

The intermediate redox species of sulfur can be responsible for the diagenetic alterations of sulfur, carbon, and iron, control the solubility of trace metals, and are the substrates for the microbial oxidation, reduction, and disproportionation of sulfur. However, most of these intermediate redox species have very rapid rates of chemical and biologic reactions leading to the concept of the ‘cryptic sulfur cycle’. With the growing understanding of the complexity of the sulfur cycle, there is a greater need to characterize these sulfur intermediates, but there has been a deficiency in methodologies to do so.

We are developing field compatible methods that enable the preservation, separation, and analysis of compound specific sulfur isotope analysis (CSSIA) on intermediate redox states of sulfur (e.g. thiosulfate, sulfite, methanethiol, bisulfide). This method relies on the use of bromobimane to derivatize and stabilize sulfur species from environmental samples in the field followed by analysis on a UPLC-ToF-MS system.

Intracellular sulfur metabolites in the dissimilatory sulfate reduction pathway

(Postdoc Min Sub Sim, collaborators Drs. Alex Sessions and Jess Adkins)

Sulfur isotope fractionation between sulfate and sulfide has been widely used to diagnose the activity of sulfate-reducing microbes in modern and ancient ecosystems, but a sub-cellular mechanism that shapes the patterns of sulfur isotope fractionation remains largely as a black box. Such limitations have hampered the quantitative use of naturally-occurring sulfur isotope fractionation as proxies for specific environmental or physiological parameters, but a recent advance in sulfur isotope analysis by MC-ICP-MS lowers the detection limit down to a few nmol S, an essential capability for measuring trace intracellular metabolites.

Figure 1. Schematic diagram showing the dissimilatory sulfate reduction pathway. Sulfate-reducing microbes utilize sulfate as a substrate and release sulfide as a waste product, which is not a single-step process at an intracellular scale. Note that sulfate is transported into the cell, activated into APS, and stepwisely reduced to sulfide. Theoretical models have provided a basis for linking isotopic fractionation to those internal cellular processes, but remain largely untested.

In collaboration with A. Sessions and J. Adkins, we are developing new analytical approaches for determining the concentrations and isotopic compositions of intracellular sulfur metabolites in sulfate-reducing microbes, which bridging a critical gap between theoretical models and experimental observations.

Previous work:

Two-dimensional mapping of sulfur cycling in microbial mats using Secondary ion mass spectrometry (SIMS) and CARD-FISH.

The metabolic activities of microbial mats have likely regulated biogeochemical cycling over most of Earth's history. However, the relationship between metabolic activity and the establishment of isotopic geochemical gradients in these mats remains poorly constrained. Here we present a parallel microgeochemical and microbiological study of micron-scale sulfur cycling within hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico. Dissolved sulfide within the mats was captured on silver discs and analyzed for its abundance and d34S isotopic composition using high-resolution secondary ion mass spectrometry (nanoSIMS). These results were compared to sulfide and oxygen microelectrode profiles. Two-dimensional microgeochemical mapping revealed well-defined laminations in sulfide concentration (on scales from 1 to 200 um), trending toward increased sulfide concentrations at depth. Sulfide d34S decreased from 10% to -20% in the uppermost 3mm and oscillated repeatedly between -10% and -30% down to a depth of 8mm. These variations are attributed to spatially variable bacterial-sulfate reduction within the mat. A parallel examination of the spatial distribution of known sulfate-reducing bacteria within the family Desulfobacteraceae was conducted using catalyzed reporter deposition fluorescence in situ hybridization. Significant concentrations of Desulfobacteraceae were observed in both oxic and anoxic zones of the mat and occurred in several distinct layers, in large aggregates and heterogeneously dispersed as single cells throughout. The spatial distribution of these microorganisms is consistent with the variation in sulfide concentration and isotopic composition we observed. The parallel application of the methodologies developed here can shed light on micron-scale sulfur cycling within microbially dominated sedimentary environments.

Follow up studies of mat-associated sulfide using the silver sulfide capture methodology using the 7F Geo SIMS for analysis of sulfide abundance and isotopic composition focused on changes in sulfide isotopes over a diel cycle, comparing full light conditions that maximize photosynthetic activity in the uppermost layers of mats and at night. By tracking profiles of oxygen and sulfide on the sub millimeter scale, we are characterizing daily changes in sulfur isotope profiles in response to this redox forcing. Through collaboration with members of the Exobiology group at the NASA Ames Research Center, we also characterized changes in sulfide isotope fractionation that arise from differing amounts of sulfate in the mats, using in situ Microcoleus mats from Guerrero Negro, Baja California Sur, Mexico (80 mM sulfate) and mat incubations maintained in the NASA Ames Greenhouse facility at 80 mM, 1 mM, and 0.2 mM sulfate concentrations. Efforts to understand the microscale changes in d34S-sulfide and effect of sulfate concentrations on isotope fractionations within these early earth analog ecosystems provides important constraints on our interpretation of sulfur biosignatures in the geologic record.


Fike, D. A., C. L. Gammon, W. Ziebis, and V. J. Orphan (2008) Micron-scale mapping of sulfur cycling across the oxycline of a cyanobacterial mat: A paired nanoSIMS and CARD-FISH approach. ISME Journal 2: 749-759

Fike, D.A., N. Finke, G. Blake, J. Zha, T.M. Hoehler, V.J. Orphan (2009) The effect of sulfate concentration on (sub)millimeter-scale sulfide δ34S in hypersaline cyanobacterial mats over the diurnal cycle. Geochem. et Cosmochem. Acta. 73: 6187-6204

Sulfur cycling in anoxygenic purple sulfur ‘berries’

(Excerpted from: Wilbanks, E. G., Jaekel, U., Salman, V., Humphrey, P. T., Eisen, J. A., Facciotti, M. T., Buckley, D. H., Zinder, S. H., Druschel, G. K., Fike, D. A. and Orphan, V. J. (2014), Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh. Environ Microbiol, 16: 3398–3415. doi:10.1111/1462-2920.12388)

Microbial metabolism is the engine that drives global biogeochemical cycles, yet many key transformations are carried out by microbial consortia over short spatiotemporal scales that elude detection by traditional analytical approaches. We investigate syntrophic sulfur cycling in the ‘pink berry’ consortia of the Sippewissett Salt Marsh through an integrative study at the microbial scale. The pink berries are macroscopic, photosynthetic microbial aggregates composed primarily of two closely associated species: sulfide-oxidizing purple sulfur bacteria (PB-PSB1) and sulfate-reducing bacteria (PB-SRB1). Using metagenomic sequencing and 34S-enriched sulfate stable isotope probing coupled with nanoSIMS, we demonstrate interspecies transfer of reduced sulfur metabolites from PB-SRB1 to PB-PSB1. The pink berries catalyse net sulfide oxidation and maintain internal sulfide concentrations of 0–500 μm. Sulfide within the berries, captured on silver wires and analysed using secondary ion mass spectrometer, increased in abundance towards the berry interior, while δ34S-sulfide decreased from 6‰ to −31‰ from the exterior to interior of the berry. These values correspond to sulfate–sulfide isotopic fractionations (15–53‰) consistent with either sulfate reduction or a mixture of reductive and oxidative metabolisms. Together this combined metagenomic and high-resolution isotopic analysis demonstrates active sulfur cycling at the microscale within well-structured macroscopic consortia consisting of sulfide-oxidizing anoxygenic phototrophs and sulfate-reducing bacteria.

(above) A. Intertidal pools in Little Sippewissett Salt Marsh form dense stands of pink berry aggregates at the sediment–water interface. B. Large aggregates can reach nearly a centimeter in size. C. Pink berries in sediment (0–5 cm) collected from an intertidal pool in Little Sippewissett. D. Berries can be easily washed free of marsh sediment and manipulated in the lab. E. Cross-section of a berry reveals pink tubules encased in a clear exopolymer matrix, scale bar is 0.5 mm. F. Higher magnification view of pink berry tubules, scale bar is 200 μm. Figure 1 from Wilbanks et al. (2014)

(right) Model of sulfur cycling in the pink berry consortium. PB-SRB1 (green rods) reduce sulfate to sulfide, oxidizing a variety of electron donors from either exogenous sediment sources or from locally supplied photosynthate produced by PB-PSB1. PB-PSB1 (pink cocci) consume syntrophic sulfide, oxidizing sulfide to sulfate and intracellular stores of elemental sulfur (S0, pale yellow circles). Should PB-PSB1 cells lyse, intracellular sulfur might be reduced and/or disproportionated by PB-SRB1 (grey dashed arrows). Some electron donors for PB-PSB1 (HS-) and PB-SRB1 (H2 or fatty acids) are also likely provided exogenously by compounds effluxed from the sediment (squiggly lines). During the day, the phototrophic PB-PSB1 fixes CO2 into biomass, while at night it may derive maintenance energy by respiring elemental sulfur and intracellular carbohydrate reserves and producing sulfide. Though the PB-SRB1 genome suggests the genetic potential to fix CO2, results from our stable and radiocarbon experiments suggest PB-SRB1 does not contribute significantly to carbon fixation in the berries under the conditions of our incubations. Figure 9 from Wilbanks et al. (2014)