Sulfur biogeochemistry 8 e- between stable redox states Polymerizes, cyclizes Reduced, intermediate, and oxidized solid forms Thousands of organic sulfur forms (organosulfur compounds; thiols have an –SH group, thioethers –C-S-C, thioesters and sulfonates are oxidized S forms, sulfoxides/sulfones RS(=O)R’, RS(=O)2R, thioketones, thioamides, sulfonium ylides less common)

Sulfur Cycle

Early earth ocean-atmosphere and S

Assimilatory vs. Dissimilatory S is an essential nutrient (key to amino acids cysteine and methionine) and many other cellular molecules, so all organisms need an assimilatory pathway Many dissimilatory reactions due to complicated intermedaite pathways involving S redox chemistry- leads to idea that S-utilizing organisms are the most diverse group of microbes which metabolize a single element

1 piece of sulfur oxidation pathways

Assimilatory pathways APS pathway – uptake of SO42- to APS (Adenosine phosphosulfate) using an ATP APS then goes thorugh 1 of 2 paths: –Forms PAPS (phosphoadenosine phosphosulfate) –Or forms organic thiosulfate derivative (G-S-S- O3) These are furthur reduced to HS- to form cysteine or other useful sulfur forms All of this COSTS ENERGY!

Dissimilatory SO 4 2- reduction Biological Sulfate Reduction (BSR) and Thermochemical Sulfate Reduction (TSR) At temperatures < ºC the reduction of SO 4 2- by reduced organics is VERY slow (though thermodynamically favorable) – formation of sulfide at low T is thus MICROBIAL ‘Mineralization’ process because H2S and metals strongly interact – form sulfide minerals – very low solubility!

Measuring rates of BSR Profiles and flux rates from gradients Culture-based incubations Radiolabeling using 35 S-labeld sulfate –Done quickly in sediments (reduce chance of re-oxidation) –Recovery of H 2 S produced can be difficult (if it quickly goes into pyrite for example it is harder to recover) –However, this is the most accurate and common technique

BSR and Carbon mineralization Carbon compound degradation to CO 2 through BSR –AT high sedimentation rates, BSR can account for significant fraction of this –At lower sedimentation rates, BSR is less important –WHY THE DIFFERENCE?? –In lake sediments this can be very different than in marine sediments, WHY?

Where do sulfate-reducing bacteria (SRB) hang out? Need anaerobic/microaerophilic environment, enough SO 4 2-, organics/ H 2 Reduced sediments Hydrothermal springs (deep sea, terrestrial) Cyanobacterial mats (where in the mats do you think??) SRB inhabit widest range of conditions – T , 0-28% NaCl

SRB Phylogeny Deep-branching, widely distributed across tree of life (both archaeal and bacterial), thermophilic Bacteria – mostly in  -proteobacteria, also spore-formers, gram+, in nitrospira group Archaea – Archeoglobus T max=92ºC LGT of dissimilatory sulfur reductase (DSR) gene supported across archaea, different bacterial species

SRB Metabolism pathway SO 4 2- import – costs energy, coupled to transport of H+ of Na+ ‘Activated’ by ATP sulfurylase  forms APS, which is then reduced to sulfite which is reduced to sulfide by the DSR enzyme (a reductase) H 2 S is highly toxic (interacts strongly with organics and metals)  rapidly excreted from the cell

DSR substrate limitations Require smaller, less recalcitrant substrates (anaerobes do not make radicals needed to degrade bigger molecules into something useable) Grow best on simple substrates like acetate, but can grow on a wide range of substrates, including some xenobiotics and even PO 3 3- Some are complete oxidizers, many incomplete – (incomplete ones grow faster) H 2 as an e - source, most are chemolithoheterotrophic, a few known chemolithoautotrophs…

SRB Diversity Over 100 different species known IN one study, 20 different species were identified from a single sediment sample! For the same metabolism – what other factors may play into which one(s) are predominant at any point in time or space??

Elemental sulfur S 8 a product of sulfide oxidation, some organisms store it intracellualry, also forms abiotically on interaction of H2S with metals, organics Elemental sulfur respiration coupled with H 2 or organic carbon oxidation (complete and incomplete) found in many organisms Several identified species of the  - proteobacterial clade that primarily metabolize S 8, Widespread archaeal metabolism – Crenarcheota, Sulfolobus, Acidianus, othrs

Sulfide oxidation Abiotic pathways – sulfide reaction with FeOOH or MnOOH is fast, reaction with O2 slower, with NO3- slow too… Plenty of differences in the intermediates of H 2 S oxidation depending on specific chemistry and availability of oxidants too

Black Sea

Green Lake, NY Voltammetric evidence for significant role of polysulfides in sulfide oxidation and elemental sulfur reactions

Sulfide Oxidizing Organisms Chemolithoautotrophs (and heterotrophs) exist that can oxidize H2S and other intermediates –Many can also reduce elemental sulfur… Use O 2 or NO 3 - as electron acceptor Most obligate or facultative aerobes, but some are obligately microaerophilic (can’t handle above a few tens of uM)

Intracellular S 8 Several S-oxidizers can store S 8 in vacuoles Noteably Beggiatoa and Thiothrix spp.

Cave formation and stratified analogues in central Italy Influx of sulfide-rich water accelerates cave formation: H 2 S + 2 O 2  SO H + CaCO 3 + H +  Ca 2+ + HCO 3 - S8S8 S x n- HS - S 2 O 3 2- HSO 3 - S 4 O 6 2-

Microbial ecology and sulfur speciation Different microbial communities found in different places --- related to BIG changes in S speciation! 3 different predominant mat types Potential (V vs. Ag/AgCl) Current (  A) White:  -proteobacterial mat Red: thiovulum mat Green: beggiotoa mat S8S8 S x n- HS - S 2 O 3 2- HSO 3 -

‘  -proteobacterial’ mats Potential (V vs. Ag/AgCl) Current (  A) Scans into white mat material S x n- Potential (V vs. Ag/AgCl) Current (  A) Scans above (green and into biofilm, red) Potential (V vs. Ag/AgCl) Current (  A) Above (green) and into biofilm (others) beggiatoa mats thiovulum mats S8S8 S8S8 S8S8 Sxn-Sxn- HS - S 2 O 3 2-

‘thiovulum’ mats, Pozzo di Cristale, Frassassi caves

Thiovulum mat profile data ~ 50  m thick biofilm Potential (V vs. Ag/AgCl) Current (  A) above

Snottite electrochemistry Potential (V) vs Ag/AgCl Current (  A) pH varies 1-3 in these snottite streamers

S 8 in biofilms at Frasassi Images courtesy Jenn Macalady, Penn State

Courtesy Macalady lab, Penn State 16s library of the biofilms in Frassassi New results looking at metagenomic data has identified a gene regulating elemental sulfur ‘docking’

Phototrophic S-oxidation Anoxygenic phototrophy using H 2 S, S 8, S 2 O 3 2- as electron donors Organisms are common, in 5 major groups: –Purple sulfur bacteria –Purple nonsulfur bacteria –Green sulfur bacteria –Green nonsulfur bacteria –Heliobacteria These archaic groupings derived from ‘sulfur’ groups depositing visible S8, nonsulfur ones did not – mistakenly thought they did not use reduced sulfur as a result, and we still use the names…

Phototrophic Mats - Cyanos Anoxygenic photosynthetic organisms oxidizing H 2 S across a VERY sharp gradient!! Electrode tip stuck bottom

Phototrophic mats - PSB Purple sulfur bacteria mats –Respond to light level changes in minutes  position in sediment and water column can vary significantly!

Light Manipulation experiments Jacket onJacket offHat on Hat off

S-oxidizer phylogeny Anoxygenic photosynthesis development before oxygenic photosynthesis? –Geochemical record of the earth’s oceans? –Photosystem less complicated –Anoxygenic organisms more deeply branching Others argued based on pigment biosynthesis pathways oxygenic photosynthesis is first Subsequent genetic analysis using genes related to pigment biosynthesis showed anoxygenic photosynthesis first (specifically, PSB) – but here are some complications involving possible LGT…

Disproportionation Sulfur’s equivalence to fermentation – intermediate oxidation state sulfur species (elemental sulfur, thiosulfate, sulfite) split into one more and one less oxidized forms, ex: –S 2 O H 2 O  H 2 S + SO 4 2-

S stable isotopes 4 stable isotopes of sulfur: 32 S (95.04%), 33 S (0.749%), 34 S (4.20%), 36 S (0.0156%) Thermodynamic equilibrium for the fractionation of S isotopes rarely obtained – observed fractionations largely kinetic SRB fractionations (cultures) 3-46‰ –Rates, species/enzymes, substrates affect this S-disproportionation also results in large fractionation (up to 37‰) SRB fractionations in nature up to >100+‰ S-oxidation (biotic or abiotic) does not produce much fractionation at all!