This document summarizes research on non-sulfate reducing bacteria (SRB) that produce sulfide through thiosulfate reduction in high salinity oil production facilities. Molecular detection of these thiosulfate-reducing bacteria (TRB) is challenging as they possess different enzymatic pathways than SRB. The researchers isolated novel Halanaerobiaceae from brine production waters that reduce thiosulfate and elemental sulfur to sulfide. Physiological experiments showed a rhodanese-like protein and possibly an uncharacterized sulfite reductase are involved in thiosulfate reduction. Genomic analysis identified anaerobic sulfite reductase subunits but SRB primers did not detect them, suggesting the

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Non-SRB Sulfidogenic Bacteria from High Brine Oil Production Facilities: Enzymatic Basis of Sulfide Production and Molecular Detection
A. T. De Capite, K. E. Duncan, R. S. Tanner
University of Oklahoma, Department of Microbiology and Plant Biology
Conventional molecular methods for assessing
microbially induced corrosion (MIC) from
sulfide production target the SRB (sulfate-
reducing bacteria) population through the
amplification of specific genes involved in the
dissimilatory sulfate reduction (DSR) pathway
[1]. Some members of the MIC community
can generate sulfide through the reduction
of other sulfur anions, such as thiosulfate.
However, thiosulfate reduction (TR) pathways
are more numerous and less characterized
than SRB enzyme systems. In extremely saline
environments, such as brine production waters,
TRB can out number SRB (Fig. 1).
• The enzymatic pathway
for sulfidogenesis in
Halanaerobiaceae isolates
will be elucidated using a
combination of genomic and
physiological approaches.
• After the enzymatic pathway is
determined, appropriate gene
probes can be developed to
specifically enumerate TRB
from environmental samples.
Fig. 1: 16S rRNA gene survey of brine production
waters from a site experiencing severe corrosion
indicate that Halanaerobiaceae (pink) comprise a
dominant portion of the microbial community. Not
all species of Halanaerobium produce sulfide from
the reduction of sulfur species [2]. Specific detection
of TRB can be achieved by employing probes that
target genes directly involved in sulfidogenesis. This
requires confirmation of the sulfidogenic pathway.
Activity (Labeled Pathway) Reactants Products
Ratio of sulfide
produced per
thiosulfate reduced
Rhodanese-like Protein (A) Thiosulfate, cyanide Sulfite, thiocyanate 0 to 1
Thiosulfate Reductase (B) Thiosulfate Sulfite, sulfide 1 to 1
Thiosulfate Disporportionation
(C)
Thiosulfate Sulfate, sulfide 1 to 1
Dissimilartory Sulfite reductase
(D,E)
Sulfite Sulfide 2 to 1
SO4
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SO3
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SCN
(A)
(A,B)
(B,C)
(D,E)
(C)
Fig. 2: Rhodanese-like proteins (rdlA) have been proposed as the enzymatic mechanism
for thiosulfate reduction in Halanaerobium spp. [2].
Enrichment and Isolation
Production water samples were received from crude oil
production sites and enriched in heterotrophic media amended
with thiosulfate. Isolates were obtained roll-tubes. All microbial
cultivation was under strictly anaerobic conditions [5].
Molecular Analyses
DNA was extracted and sequenced using the Illumina MiSeq
platform and analyzed with MG-RAST. Full 16S rRNA sequences
were obtained using PCR and rP2/fD1 primers [6]. PCR
amplification of various sulfur cycling functional genes was
attempted using genomic DNA as template [2,7,8]
Physiological Characterization
A terminal electron acceptor assay was performed to assess if
isolates reduced thiosulfate, sulfate, sulfite and elemental sulfur
to sulfide. An iodate-iodine assay was employed to quantify
thiosulfate, sulfite and sulfide [9]. Total sulfide produced was
determined by increasing pH of incubations to 10 and using a
DMPD sulfide assay [10]. Ion chromatography (Dionex 3000,
AS4A column) was employed to assess sulfate production.
Rhodanese activity was confirmed by quantifying the production
of thiocyanate [2].
Halanaerobium praevalens (T) DSM 2228 TR
Halanaerobium saccharolyticum subsp saccharolyticum (T) DSM 6643 TR
Halanaerobium kushneri (T) ATCC 700103
Halanaerobium acetethylicum EIGI (T) DSM 3532
Halanaerobium congolense (T) DSM 11287 TR
Halanaerobium hydrogeniformans ATCC PTA-10410
Halanaerobium salsuginis (T) ATCC 51327
ZB2A (Middle East Field Isolate) TR Novel species
Halanaerobium sp. L21-Ace-D5 (Hypersaline microbial mat isolate)
Halocella cellulosilytica (T) DSM 7362
Halothermothrix orenii (T) DSM 9562
OKU7 (Angola Field Isolate) TR
Halanaerobiales bacterium Ag-C55 (Juan de Fuca Ridge Isolate)
Halarsenatibacter silvermanii (T) DSM 21684
Halobacteroides halobius (T) DSM 5150
74
88
99
82
100
100
100
100
82
86
0.02
Novel species
Isolation of Novel Halanaerobiaceae
Materials and Methods
Fig. 3: Tree constructed using aligned full
16S rRNA sequences [14]. Bold letters “TR”
denote that a type strain or isolate reduced
thiosulfate to sulfide. Isolate ZB2A shares 97%
16S rRNA sequence identity to the type strain
Halanaerobium salsuginis. The most closely
related type strain to OKU7, Halothermothrix
orenii, shares only a 93% 16S rRNA sequence
identity.
Halanaerobium
kushneri [11]
Halanaerobium
salsuginis [12]
Halanaerobium
congolense [13]
Novel
Halanaerobium
Species
Novel
Halanaerobiaceae
Genus
Strain Designation VS-751 VS-752 SEBR-4224 ZB2A OKU7
Sulfate (10 mM) - - - - -
Thiosulfate (10 mM) - - + + +
Sulfite (5 mM) - - - - -
Elemental Sulfur (1%) - - + + +
Table 1: Not all species of Halanaerobium reduce thiosulfate; therefore, simply detecting Halanaerobium in a
16S survey will not differentiate the sulfidogenic population. By identifying the enzyme system responsible for
sulfidogenesis, appropriate gene probes can be designed to specifically detect these microorganisms.
0.0
0.1
0.2
0.3
0.4
0.5
0 2 4 6 8 10
Abs660(nm)
Incubation Period (Days)
Fermentative
Thiosulfate (5mM)
Sulfite (4mM)
Sulfite (1mM)
Sulfite (4mM) / Thiosulfate
(5mM)
Sulfite (1mM) / Thiosulfate
(5mM)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10
Abs660(nm)
Incubation Period (Days)
Fermentative
Thiosulfate (10mM)
Sulfite (4mM)
Sulfite (1mM)
Sulfite (4mM) /
Thiosulfate (10mM)
Sulfite (1mM) /
Thiosulfate (10mM)
Fig. 4: When isolate ZB2A is grown under sulfite-reducing conditions amended
with 5 mM thiosulfate, inhibition from sulfite significantly hinders cell growth (Fig.
4A); however, sulfite inhibition can be overcome by increasing the concentration
of thiosulfate to 10 mM (Fig. 4B).
Thiosulfate Amendment Can Overcome
Sulfite Growth Inhibition
For Every Molecule of Thiosulfate Reduced,
Two Molecules of Sulfide are Generated
Fig 5: Results indicate that both sulfur atoms of thiosulfate are
reduced to sulfide. This suggests that the sulfur anion produced from
initial cleavage of thiosulfate is subsequently reduced to sulfide.
Sulfate was not detected. Sulfite was detected in low levels the
rhodanese assay, but not in active cultures, suggesting that cells
rapidly metabolize sulfite before it accumulates to inhibitory levels.
SRB Primer Sets Are Not Appropriate for the
Detection of Halanaerobiaceae
Table 2: Anaerobic sulfite reductase subunits A-C were annotated in the
genomes of isolate OKU7 and H. congolense, yet DSR primers did not
amplify these sequences. Results suggest that rdl could be employed for the
specific detection of Halanaerobiaceae.
Gene Primer Set
Specific
Amplification
Annotated in
Genome
aps, αβ subunits AprB1F/AprA5R None None
dsr, αβ subunits Dsr1FK/Dsr4RK None OKU, H.congolense
rdl, partial 1513F/1515R
OKU7, H. congolense,
H. kusheri
All
Fig. 7:
• Thiosulfate disproportionation can be ruled
out because sulfate is not detected.
• A rhodanese-like protein must be
considered, as results demonstrate that
isolates have rhodanese activity (pathway A).
• A thiosulfate reductase enzyme must also
be considered, as the end product of this
reaction would be labile sulfite (pathway B).
• Isolates could be metabolizing sulfite
using an uncharacterized anaerobic sulfite
reductase system (pathway E).
Isolates Can Reduce Thiosulfate and Elemental
Sulfur to Sulfide, but Not Sulfate or Sulfite
Enzymatic Basis of Sulfidogenesis from Thiosulfate
Many Pathways, Many Enzymes!
Discussion
What Pathways are Operating?
There are two potential pathways responsible for the initial
cleavage of thiosulfate, and both produce sulfite as an end
product.
1. A molybdopterin thiosulfate reductase
2. A rhodanese-like protein
Results suggests that an uncharacterized ‘anaerobic
sulfite reductase’ (asr) is involved in the reduction of sulfite
generated from the cleavage of thiosulfate.
Fate of Sulfite?
• Sulfite has an inhibitory effect on growth; however, cells
can overcome sulfite inhibition via thiosulfate reduction.
• The amount of sulfite quantified is a fraction of the
expected sulfite in the rhodanese assay, suggesting
that sulfite has a rapid turnover rate.
Good Gene Targets for TRB?
• The rhodanese-like protein gene (rdl) shows potential
as a gene target for Halanaerobiaceae; however,
theoretically the rhodanese enzyme system is not
directly responsible for sulfide production.
• Thiosulfate reductase (phs gene) still must be
investigated!
• This asr gene may be a more appropriate gene target
because it’s enzymatic activity would directly produce
sulfide.
Research Funded by
OU Biocorrosion Center
References
[1] Muyzer, G., Stams, A.J.M. (2008). Nature Reviews: Microbiol. 6: 441-454.
[2] Ravot, G., Casalot, L., Ollivier, B., Loison, G., Magot, M. (2005). Research in Microbiol. 156:
1031-1038
[3] Burns, J. L. and DiChristina, T. J. (2009). Appl. Environ. Microbiol. 75: 5209–5217.
[4] Krämer, M. and Cypionka, H. (1989). Arch. Microbiol. 151: 232-237.
[5] Balch, W. E. and Wolfe, R. S. (1976). Appl. Environ. Microbiol. 32: 781-791.
[6] Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J. (1991). J Bacteriol. 173(2): 697-703.
[7] Meyer, B. and J. Kuever. (2007). Microbiol. 53: 2026-2044
[8] Klein M, Friedrich M, Roger AJ, Hugenholtz P, Fishbain S, Abicht H, Blackall LL, Stahl DA,
Wagner M. (2001). J Bacteriol. 183: 6028-6035.
[9] Koh, T. and Miura, Y. (1987). Analytical Sciences. 3: 543-547.
[10] Tanner, R. S. (1989). J. Microbiol. Meth. 10: 83-90
[11]Bhupathiraju, V. K., McInerney, M. J., Woese, C. R., Tanner, R. S. (1999).. Int. J. Syst.
Bacteriol. 49: 953–960
[12] Bhupathiraju, V. K., Oren, A., Sharma, P. K., Tanner, R. S., Woese, C. R., McInerney, M. J.
(1994). Int. J. Syst. Bacteriol. 44: 565–572.
[13] Ravot, G., Magot, M., Ollivier, B. Patel, B. K. C., Ageron, E., Grimont, P. A. D., Thomas, P.
Garcia, J. L. (1997). FEMS Microbiology Letters. 147: 81-88
[14] Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J. R. (2007). Appl. Environ. Microbio. 73(16):
5261-5267.
Fig. 6: Rhodanese activity was confirmed, as thiocyanate is
generated when culture is amended with thiosulfate and cyanide. The
amount of sulfite quantified in this assay is significantly lower than
the amount of expected, suggesting that sulfite is rapidly converted to
prevent accumulation of this inhibitory end product.
0
5
10
15
20
25
0 10 20 30 40 50 60 70 80
Concentraiton(mM)
Incubation Period (Minutes)
Thiocyanate
Thiosulfate
Sulfite (experimental)
Sulfite (theoretical)
Both Isolates Possess Active Rhodanese Activity
Sulfide Anions Produced from Thiosulfate
Reduction Can Elucidate Enzymatic Pathway
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(A) (A,B)
(B)
(E)
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Acknowledgements
(inhibitory)
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A. Rhodanese-like protein [2]
B. Thiosulfate reductase (molydopterin) [3]
C. Thiosulfate disproportionation [4]
D. Dissimilatory sulfite reductase [1]
E. Anaerobic sulfite reductase [1]
A. Rhodanese-like protein [2]
B. Thiosulfate reductase (molydopterin) [3]
E. Anaerobic sulfite reductase [1]
Introduction
Halanaerobiaceae Dominate Microbial Community of
Corroded Production Facility
4A
4B
This research would not have been possible without guidance from Dr. Joseph Suflita, Dr. Michael McInerney, Neil Wofford, Chris Marks and Chad Wiens
Images 1&2: Phase
contrast micrographs
of isolate ZB2A (1)
and OKU7 (2).
1
2