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 SO42- reduction

• Biological Sulfate Reduction (BSR) and Thermochemical Sulfate Reduction (TSR)

• At temperatures <150-200ºC the reduction of SO4

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 35S-labeld sulfate– Done quickly in sediments (reduce chance of

re-oxidation)

– Recovery of H2S 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 CO2 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 SO4

2-, organics/ H2

• Reduced sediments• Hydrothermal springs (deep sea, terrestrial)• Cyanobacterial mats (where in the mats do

you think??)

• SRB inhabit widest range of conditions – T 0-127, 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

• SO42- 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)

• H2S 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 PO3

3-

• Some are complete oxidizers, many incomplete – (incomplete ones grow faster)

• H2 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

• S8 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 H2 or organic carbon oxidation (complete and incomplete) found in many organisms

• Several identified species of the -proteobacterial clade that primarily metabolize S8,

• 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 H2S 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

Green Lake Voltammetric Profile

0

5

10

15

20

25

0 0.1 0.2 0.3Peak Current (A)

Dep

th(m

)

Oxygen(dissolved)

HydrogenSulfide

Polysulfide

ElementalSulfur

Sulfide Oxidizing Organisms• Chemolithoautotrophs (and heterotrophs)

exist that can oxidize H2S and other intermediates– Many can also reduce elemental sulfur…

• Use O2 or NO3- as electron acceptor

• Most obligate or facultative aerobes, but some are obligately microaerophilic (can’t handle above a few tens of uM)

Intracellular S8

• Several S-oxidizers can store S8 in vacuoles

• Noteably Beggiatoa and Thiothrix spp.

Cave formation and stratified analogues in central Italy

• Influx of sulfide-rich water accelerates cave formation: H2S + 2 O2 SO4

2- + 2 H+

CaCO3 + H+ Ca2+ + HCO3-

1.375

-0.230

0.000

0.200

0.400

0.600

0.800

1.000

1.200

-0.050-1.800 -1.500 -1.250 -1.000 -0.750 -0.500 -0.250

S8

Sxn- HS-

S2O32-

HSO3-

S4O62-

Microbial ecology and sulfur speciation• Different microbial communities found in different

places --- related to BIG changes in S speciation!

3 different predominant mat types0.871

-0.265

-0.200

-0.100

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

-0.050-1.800 -1.500 -1.250 -1.000 -0.750 -0.500 -0.250

Potential (V vs. Ag/AgCl)

Cur

rent

(A

)

White: -proteobacterial matRed: thiovulum matGreen: beggiotoa mat

S8

Sxn-

HS-

S2O32-HSO3

-

‘-proteobacterial’ mats 0.997

-0.331

-0.200

-0.100

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

-0.050-1.800 -1.500 -1.250 -1.000 -0.750 -0.500 -0.250

Potential (V vs. Ag/AgCl)

Cur

rent

(A

)

Scans into white mat material

Sxn-

Potential (V vs. Ag/AgCl)

Cur

rent

(A

)

1.275

-0.294

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

-0.050-1.800 -1.600 -1.400 -1.200 -1.000 -0.800 -0.600 -0.400 -0.200

Scans above (green and into biofilm, red)

Potential (V vs. Ag/AgCl)

Cur

rent

(A

)

0.420

-0.107

-0.050

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

-0.050-1.800 -1.500 -1.250 -1.000 -0.750 -0.500 -0.250

Above (green) and into biofilm (others)

beggiatoamats

thiovulummats

S8

S8

S8

Sxn-

HS-

HS-

HS-

S2O32-

‘thiovulum’ mats, Pozzo di Cristale, Frassassi caves

Thiovulum mat profile data

~ 50 m thick biofilm

Thiovulum Mat Profile Pozzo di Cristale

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60

Current (nA)

De

pth

fro

m b

otto

m (

um

)

elemental sulfur

sulfite

sulfide (uM)

Potential (V vs. Ag/AgCl)

Cur

rent

(A

)

0.420

-0.107

-0.050

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

-0.050-1.800 -1.500 -1.250 -1.000 -0.750 -0.500 -0.250

above

Snottite electrochemistry

Potential (V) vs Ag/AgCl

Cu

rren

t (

A)

0.278

-0.108

-0.075

-0.050

-0.025

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

-0.050-1.300 -1.000 -0.800 -0.600 -0.400 -0.200

Sulfide peak location on Au/Hg microelectrode in Mini-Primrose water over a range of pH values on HMDE

y = -0.0702x - 0.2255

R2 = 0.9837

-0.7

-0.65

-0.6

-0.55

-0.5

-0.45

-0.4

-0.35

-0.3

1 2 3 4 5 6 7

pH

Po

ten

tial

(V

vs.

Ag

/Ag

Cl)

pH varies 1-3 in these snottite streamers

S8 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 H2S, S8, S2O3

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 - CyanosPhototrophic Mat outside fracture

spring - Frassassi

-350

-300

-250

-200

-150

-100

-50

0

0 20 40 60

Conc (nA)

Dep

th (

m)

sulfide (uM)

elementalsulfur

oxygen

approximatetop of mat

Anoxygenic photosynthetic organisms oxidizing H2S 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!

Purple sulfur bacteria mats

-800

-700

-600

-500

-400

-300

-200

-100

0

0 500 1000 1500 2000

H2S(aq) Concentration (M)

Dep

th (

mic

ron

s)

Light Manipulation experimentsCyanobacteria Light Manipulation Experiment

0

50

100

150

200

250

300

350

400

450

-80 -60 -40 -20 0 20 40 60 80 100 120

time (seconds)

nA

H2S

Jacket on Jacket off Hat 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:– S2O3

2- + H2O H2S + SO42-

S stable isotopes• 4 stable isotopes of sulfur: 32S (95.04%), 33S

(0.749%), 34S (4.20%), 36S (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!