Investigating Pathways of Sulfidogenesis in
Thiosulfate-Reducing Halanaerobium Species
Annette De Capite
Department of Microbiology, University of Oklahoma
Sulfidogenic Bacteria
Sulfate-reducing bacteria
• Sulfate (SO42-)
• Sulfite (SO32-)
Sulfur-reducing bacteria
• Elemental sulfur (S8)
Thiosulfate-reducing bacteria
• Thiosulfate (S2O32-)
Sulfide
HS- (aq) / H2S (g)
Observations
1.5-4 M
Cl ,
o
49 C
Halanaerobium!
SRB?
Anaerobic fermenters
common to high salt, high
temperature oil production
waters.
Middle East Field 16S Results (OU Biocorrosion Center)
Materials
Sample
[Cl-] (Molar)
Temperature
Angola production water
1.5
49oC
Middle East Field production water
2.5
49oC
European Oil Reservoir
2.5
37oC
High Temperature, High Salt Production Waters
Enrichments established under fermentative and thiosulfate-reducing conditions.
Genus Halanaerobium
23 sulfidogenic
isolates
obtained!
Novel Genus?
Family Halanaerobiaceae
Halanaerobiaceae Isolation Results
TR denotes species confirmed for thiosulfate reduction
Bold strains represent species used in this study
Genus Halanaerobium
Firmicutes
Obligate Anaerobes
Obligate halophiles
Isolated from saline production waters, brine
lakes, and microbial mats
1 µm
Fermentative metabolism
Do not reduce sulfate!
4 species produce sulfide from the
reduction of thiosulfate and sulfur.
Halanaerobium kushneri
Sources of Sulfate and Thiosulfate
Sulfate
• Production waters
• Seawater
• Interconversion to
other sulfur anions
Thiosulfate
• Production waters
• Seawater
• Oxidation of
sulfides
HS
S 2O 3
2SO4
(Mineral)
2SO4
(Seawater)
2-
SO4
2-
2S2O3
S2O3
2-
HS
Molecular Detection of Sulfidogenic Bacteria
16S rRNA gene
Functional genes
Applications
Caveats
Assess if microbial
community has the
genetic potential for
sulfidogenesis.
Does not confirm viability of
bacteria or activity of
sulfidogenic enzymes.
Cultivation and physiology!
Gene Targets for SRB
Amplification ≠ Enzymatic Activity
dsrABD
aprBA
2SO4
Sulfate
APS
*
SO32Sulfite
APS
Reductase
HSSulfide
Dissimilatory
Sulfite Reductase
Gene Targets for TRB
Rhodanese*
S2O32-
Thiocyanate
S2O32aprBA
sat
S2O32phs
rdl
SCN
Thiosulfate
disproportionation
Thiosulfate
Reductase
Molybdopterin
protein
SO32Sulfite
* Proposed to be involved in
thiosulfate-reduction of
Halanaerobium species.
SO32-
Sulfite
HS-
HS-
Sulfide
SO42-
Sulfide
Sulfate
Sulfite Reductases
HS- dsrABD or asrABC
Sulfide
Investigating Enzymatic Pathways
1. Assess if other sulfur anions are reduced
2. Evaluate rhodanese activity
3. Identify end products of thiosulfate reduction
4. Determine sulfide/thiosulfate ratio
5. Genomic analysis
ZB2A: Sulfidogenesis
from Sulfur Anions
Sulfidogenesis
in H. vreelandii
*
*
Sulfide produced
from reduction of
S2O32- and S8
Thiosulfate
reduction also
confirmed in H.
congolense and
OKU7
*
Significantly different compared to fermentative, Student’s t-test, p-value < 0.05.
Growth of H. vreelandii in the Presence of Sulfur Anions
Similar results observed for H.congolense and OKU7
The Effect of Sulfite on Growth of H. vreelandii
Growth is improved as sulfite concentration decreases
Investigating Enzymatic Pathways
1. Assess if other sulfur anions are reduced
• Elemental sulfur and thiosulfate are reduced to
sulfide.
• Sulfite inhibits growth
2. Evaluate rhodanese activity
Rhodanese Activity: Quantification of Thiocyanate
Iron-thiocyanate complex
Thiocyanate
Ferric nitrate
reagent
Thiocyanate can only be
produced from rhodanese!
Rhodanese activity observed
in all Halanaerobium strains
tested!
Measure absorbance of
supernatant at 470 nm
Rhodanese Activity in H. vreelandii
S2O32CN
SO32-
(Inhibitory)
SCN
*
*Thiocyanate is only detected when culture is amended with cyanide.
Investigating Enzymatic Pathways
2. Evaluate rhodanese activity
• Both sulfidogenic and non-sulfidogenic species of
Halanaerobium possess rhodanese activity.
• Only a fraction of the predicted sulfite is detected
in the rhodanese assay.
 Where does the sulfite go?
3. Identify end products of thiosulfate reduction
Fate of Sulfur Anions
CN
S2O32Thiosulfate
• Sulfate was not
Thiosulfate
Thiosulfate
SCN Rhodanese
disproportionation
disproportionation
Thiocyanate
produced.
rdl
• Sulfide was generated
SO42Thiosulfate reductase
Sulfate
as an end product of
phs
thiosulfate reduction
Thiosulfate disproportionation
Thiosulfate
disproportionation
• Thiocyanate was
2or
SO
produced when cyanide
3
Sulfite
Thiosulfate reductase
was present.
(Inhibitory)
phs
• Sulfite was not detected Sulfite Reductases
in vivo, but detected in
dsr or ‘asr’
HSlow quantities in the
Sulfide
rhodanese assay.
Investigating Enzymatic Pathways
3. Identify end products of thiosulfate reduction
• Thiosulfate disproportionation can be ruled out as
an operable pathway.
• Rhodanese and thiosulfate reductase cannot not
be excluded.
• Sulfite must be metabolized to prevent growth
inhibition
4. Determine sulfide/thiosulfate ratio
Thiosulfate: Sulfide Ratio
1 S2O32Thiosulfate
SCN Rhodanese
• For every molecule of
Thiocyanate
rdl
thiosulfate reduced, two
molecules of sulfide are
Thiosulfate reductase
phs
generated (2:1).
Thiosulfate reductase
phs
• Suggests that both
SO32thiosulfate and sulfite
Sulfite
reductases are operable.
Sulfite
 Sulfite as an intermediate.
Reductases
dsr or ‘asr’
• Questions the applicability of
the rhodanese reaction.
2 HSSulfide
Investigating Enzymatic Pathways
4. Determine sulfide/thiosulfate ratio
• 2:1 Both sulfur atoms of thiosulfate are reduced
to sulfide
• Strongly suggests the activity of a sulfite
reductase
5. Genomic analysis
Genome Results
Rhodanese
Rhodanese
Anaerobic
sulfite
reductase
Rhodanese
H. congolense
Molybdopterin
protein
Rhodanese
Anaerobic
sulfite
reductase
H. vreelandii
Molybdopterin
protein
Rhodanese
H. kushneri
Rhodanese
Genes for
dissimilatory
sulfite reductase
were not
retrieved.
Molybdopterin
protein could
potentially
function as
thiosulfate
reductase.
Compiled Results
H. congolense H. vreelandii H. kushneri H. salsuginis
Thiosulfate-reduction
Yes
Yes
Yes?
No
Rhodanese Activity
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Sulfide: Thiosulfate Ratio
2 to 1
2 to 1
1 to 1
NA
Predicted Genes
Rhodanese,
sulfite
reductase
Rhodanese,
thiosulfate Rhodanese,
reductase,
thiosulfate
sulfite
reductase
reductase
NT
Investigating Enzymatic Pathways
1 S2O325. Genome Analysis
• Initial thiosulfate cleavage
remains unclear.
 Can a rhodanese
cleave thiosulfate into
sulfite and sulfide?
• Suggests that an ‘anaerobic
sulfite reductase’ is involved
in the metabolism of sulfite.
Thiosulfate
Rhodanese
rdl
S-X*
?
Thiosulfate
reductase
phs
SO32Sulfite
2 HSSulfide
Anaerobic
Sulfite
Reductase
‘asr’
* Sulfur accepting molecule
(traditionally CN in rhodanese)
‘Anaerobic sulfite reductase’
• Also observed in
Thermoanaerobacter, Clostridia and
Salmonella species
• May be involved in reducing sulfite
to prevent growth inhibition.
• Is not amplifiable using gene
targets for dissimilatory sulfite
reductase from SRB.
SO32-(Inhibitory)
Sulfite
asrABC
HSSulfide
Rethinking TRB Gene Targets
Rhodanese-like proteins are not practical
gene targets for sulfidogenic
Halanaerobium species.
Anaerobic sulfite reductase is prime target
• Directly responsible for the production
of sulfide.
• Inhibition of this enzyme be an effective
means of control for sulfidogenic
Halanaerobium species.
Take Home Messages
• Sulfidogenesis from
various sulfur anions and
alternative enzymatic
pathways must be
considered.
• Anaerobic sulfite
reductase may be a more
appropriate gene target
for sulfidogenic
Halanaerobium species.
1 S2O32Thiosulfate
Rhodanese
rdl
SO32-
Thiosulfate
reductase
phs
Sulfite
Anaerobic
Sulfite
Reductase
‘asr’
2 HSSulfide
Acknowledgements
Thesis Committee:
Dr. Kathleen Duncan
Dr. Ralph Tanner
Dr. Joseph Suflita
Dr. Michael McInerney
Thank you for your time.
I welcome your questions!