Activity of Sulfate-Reducing Bacteria
Under Simulated Reservoir Conditions
J.T. Roanea, SPE, Arne Graue, SPE, and Torlelv Lien, SPE, U. of Bergen
Summary. Sulfate-reducing bacteria (SRB) have been isolated from hot oilfield
Experiments with these bacteria in a reservoir simulator indicate th~t SRB may ~allltain the~r activity III the conditions found III most
North Sea reservoirs and, if precautions are not taken, may contribute to souring of the 011 and gas.
Introduction
Waterflooded hydrocarbon reservoirs may offer good conditions
for growth of the anaerobic SRB that produce toxic and reactive
H 2S. This is particularly the case for offshore oil fields where
oxygen-scavenged seawater with high sulfate concentration (28 mM)
is injected and mixed with the in-situ reservoir pore water contain-
ing different kinds of short-chained organic acids. For continuing
microbial activity, however, both chemical and physical require-
ments must be met. Hence, the SRB must be able to grow and gener-
ate H 2S at the in-situ pressures and temperatures in the reservoir.
Reservoir conditions were simulated with a flow rig and inves-
tigated with respect to SRB growth and activity. This flow rig may
simulate conditions in a reservoir down to 15,000 ft [4.6 Ian], cor-
responding to a geostatic pressure of 15,000 psi [100 MPa] and
a temperature up to 248°F [120°C]. Spore-forming thermophilic
SRB of the genus Desulfotomaculum were isolated from hot pro-
duced water on different North Sea oil platforms. The bacteria were
injected into brine-saturated sandstone cores inside the rig's pres-
sure vessel. Thereafter, the cores with bacteria were gradually ex-
posed to increasing temperature and pressure, and the SRB activity
at the various combinations of high temperature and pressure was
measured as sulfate reduction rate. Results showed that the bacter-
ia were active and produced H 2 S to a temperature of 176°F [80°C]
and a pressure of 4,500 psi [30 MPa]. Electron micrographs re-
vealed bacterial growth on mineral surfaces. Slimy extracellular
material was observed in connection with the settlement of the
bacteria.
Seawater injection often is used in the recovery of hydrocarbons
from subsea oil reservoirs. To obtain a successful recovery, the
microbiological aspects of the waterflood must also be considered.
Failure to do so may result in severe production problems. Bac-
terial activity downhole and in the reservoir formation results in
formation damage by loss of reservoir production performance, by
a reduction in oil quality, by souring of the oil, and by the devel-
opment of major corrosion problems, in both injection and pro-
duction wells.! H 2 S also is poisonous if inhaled and may present
a health hazard for platform personnel. Natural seawater contains
several types of slime-forming and fIlamentous bacteria, among
them SRB and iron-oxidizing bacteria. Under favorable conditions,
it is likely that these organisms will colonize the huge surface of
the reservoir matrix. 2 The availability of nutrients is important in
the development of a microbial community. The best nutrient con-
ditions for SRB are expected to occur in the mixing zone between
the injected and formation waters. This mixing zone contains a high
concentration of sulfate from seawater and soluble organic com-
pounds from the formation water. If chemical and physical require-
ments are met in such an anaerobic environment, the bacteria will
be active and produce H 2 S.
Although SRB activity has been known for decades, little is known
about their ability to be active under the extreme pressure and tem-
perature conditions found in oil reservoirs. In North Sea reservoirs,
pressure commonly ranges from 3,000 to 7,500 psi [20 to 50 MPa]
and temperatures from 140 to 212°F [60 to 100°C]. The highest
temperature at which biological sulfate reduction has been observed
so far is 199°F [93°C] by the bacteria Archaeoglobus jitlgidus. 3
Other thermophilic SRB are Thermodesulfobacterium mobile 4 and
Copyright 1991 Society of Petroleum Engineers
SPE Production Engineering, May 1991
~at~rs fr~m su?~ea ?il reservoi.r~ in the N0r:th Sea.
Thermodesulfobacterium commune, 5 with a maximum temperature
of 180°F [85°C], and Desulfotomaculum nigrijicans, with a maxi-
mum temperature of 158°F [70°C].6
In this study, we simulated an oil reservoir in a laboratory flow
rig, using realistic temperatures and pressures. Representative ther-
mophilic SRB, isolated from hot produced water on North Sea oil
platforms, are used to study the effects of these bacteria on the
reservoir.
Technical Procedure
Core Preparation. The cores used in this study were drilled from
a matrix block of the eolic Hopeman sandstone. The block was ob-
tained from an outcrop at Elgin in Scotland, the Clashach quarry.
Petrographic and mineralogical studies of the sandstone indicate
a rather pure (91 %) quartz composition and only trace amounts of
clay (muscovite). Grain sizes are on the order of 0.25 mm, with
only small variations. The average porosity is 18%, and the per-
meability ranges from 700 to 800 md. Average pore diameter (26
/-tm) was measured by mercury injection. 7 Cylindrical cores 2.0
in. [5.1 cm] in diameter were drilled with lengths from 4 to 30 in.
[10 to 80 cm]. All cores were air dried at 176°F [80°C] for 24
hours before cooling and weighing. End caps were mounted and
the cores epoxy-coated. The cores were flushed with oxygen-free
nitrogen and then evacuated. This procedure was repeated several
times to remove oxygen from the micropores. Several PV's of
anaerobic brine containing nutrients for the bacteria were flushed
through the cores before they were mounted in the flow rig. To
establish a systematical approach, only brine was used to saturate
the core in these experiments. In later experiments, both oil and
water were used.
Flow Rig. Fig. 1 illustrates the experimental flow rig. The main
component of the rig is a 3-ft [l-m] -long cylindrical steel pressure
vessel with an ID of 2.8 in. [7.1 cm] and a O.4-in. [l.O-cm] wall
thickness. A core prepared as described was pressurized in trans-
former oil inside the vessel. The rig can simulate pressure condi-
tions in a reservoir down to 15,000 ft [4 Ian], corresponding to
a geostatic pressure of 15,000 psi [100 MPa]. Pressure transducers
monitored the simulated overburden pressure, water injection pres-
sure, and the differential pressure across the core. Pumping the fluid
against a backpressure regulator gave pore pressures up to 10,500
psi [70 MPa]. Experimental flow rates ranged from 0.01 to 9.99
mL/min. A heating cable heated the pressure vessel and a thermostat
regulated the temperature. Three standard thermocouple K-elements
were placed at the inlet, in the middle, and at the outlet end of the
rig. Temperatures up to 248°F [120°C] could be maintained with
an accuracy of ± 1.8°F [± 1.0°C].
Porosity and Permeability Measurements. Average porosity was
measured with a Boyle's law porosimeter and checked by meas-
uring the volume of the fluid saturating the core.
Dual-piston pumps operating with pulseless constant flow rate
were used to inject fluids from a piston-type accumulator cell. A
programmable liquid sampler was attached to the outlet end of the
rig. The differential pressure across the core, flow-rate measure-
ments, and fluid and core characteristics were used in Darcy's equa-
tion to calculate permeability.
217
 Co_at
p.......re
pump
PRY Outlet
V
V
V
PT Pranre traDldueer
TP T_perahare probe
BPV Back pressure YIIl..
PRY SarelJ YIIl..
V Sbut-oll valve
Fig. 1-Schematlc of flow rig.
Bacteria and Their Growth Conditions. Anaerobic SRB were iso-
lated from hot produced water at 15soF [70°C] on an oil platform
in the Norwegian sector of the North Sea. An anaerobic synthetic
seawater mediumS containing spore elements,9 vitamins,lO and
fatty acids or alcohols was used for enrichment and cultivation.
The pH was adjusted to 7.0 to 7.2, the optimal range for SRB
growth. A 5-mL produced-water sample was added to the 50-mL
anaerobic growth medium. Then the carbon source was added to
obtain a final concentration of 20 to 40 mM. Pure cultures were
maintained in 50-mL serum bottles on an anaerobic liquid mineral
medium at 140°F [60°C].
Microscopic Investigations. The isolated thermophilic SRB, Strain
T93B, was grown on a liquid medium at 140°F [60°C] and at-
mospheric conditions. The morphology was examined with a phase-
contrast microscope. The bacteria were injected into brine-saturated
sandstone cores inside the pressure vessel of the rig. Thereafter,
the cores with bacteria were gradually exposed to increasing tem-
perature and pressure, and the SRB activity at various combina-
tions of high temperature and pressure was measured as a rate of
sulfate reduction. At the end of the experiments, the core was sec-
tioned and the mineral surfaces were examined by scanning elec-
tron microscopy.
Total count of bacteria and selective enumeration of thermophil-
ic SRB, Strain T93B, in the core effluent were carried out with
an epifluorescence microscope, after the cells were stained with
fluorechrome-Iabeled antibodies. Samples from the effluent were
filtered on a 0.22-I'm polycarbonate filter prestained with irgalan
black. The filter was stained with a lO-I'g/mL solution of 4,6-
diarnidino-2-phenylindole. Additional staining was performed with
fluorescine-isothiocyanate-labeled antibodies, which selectively bind
to the SRB membranes. Because we switched between two differ-
ent optical filter packages in the microscope, only one sample was
needed to determine both total bacterial and specific SRB counts. 11
In the determination of the SRB level, the advantages of the ap-
plied method, which was based on the use of antibodies, over con-
ventional methods are that no cultivation is necessary and the results
are obtained in hours. The technique detects SO to 90% of the SRB
present in the water sample. However, a prerequisite for using this
immunological method, of course, is that antibodies against the bac-
teria are available. As a part of this work, we made antibodies
against various types of SRB isolated from oil platforms in the North
Sea.
ChemkaI Detenninatiom. Sulfate was determined photometrically
after precipitation with barium chloride. 12 H 2 S was determined by
the methylene blue method 13 or by the copper sulfate method. 14
218
6.0-r-------------------,
..
CD
.a
E 5.0
::s
c
CD
()
.
C)
4.0
0
..J
3.0
1.0 2.0 3.0
Produced fluid [PV]
Fig. 2-Numbers of SRB In core effluent.
Bacterial activity was measured with a radiotracer technique as
the amount of sulfate converted to sulfide. 35S-labeled sulfate was
pumped into the core to a final concentration of 1.0 Ci/mL [37
kBq/mL] pore water. Then an incubation period (usually 24 hours)
was applied at the specified temperature and pressure. At the end
of the incubation period, several PV's of the synthetic seawater
medium were pumped through the core. Effluent samples were ex-
amined for H 2S content by adding a Zn-acetate solution and there-
by precipitating the 35S-labeled sulfide as Zn35S. A modified
radiorespirometric assay was used to determine the sulfate reduc-
tion rate. 15
Results and Discussion
Thermophilic SRB in Produced Water. Thermophilic SRB were
enriched and isolated from different sampling points on the water
separator system for different North Sea reservoirs. Strains of SRB
were found in most of the examined samples, indicating that ther-
mophilic SRB are widespread in the produced water from water-
flooded North Sea reservoirs.
A characteristic strain, Strain T93B, was spore-forming Gram-
positive rods with a growth temperature range of 109 to 172°F [43
to 7S°C]. The bacteria belong to the genus Desulfotomaculum.
Spores of the bacteria (1.0 I'm in diameter) germinated readily af-
ter being exposed to 266°F [l30°C] for 20 minutes at 30 psi [207
kPa] in an autoclave. The bacteria therefore are able to survive large
temperature fluctuations.
SRB Penetration Rates in Core. Earlier work on microbial mo-
tility through porous sandstone cores focused on random movement
of motile microorganisms and diffusive growth of nonmotile
microorganisms. 16,17 Bacterial penetration rates ranging from 0.06
to 0.47 cmlh for motile bacteria have been reported in stagnant
nutrient media. 16 The penetration rates of naturally occurring bac-
teria in a reservoir, however, will be dependent on the water injec-
tion rate. In oil reservoirs, water fronts typically advance 1 ftlD
[0.3 mid]. This seawater flow rate is three times faster than the
velocity of motile bacteria. According to the literature, continuous
seawater injection will be the main transport mechanism for the
bacteria when the average permeability exceeds 100 md and the
mean pore-throat distribution exceeds 6.0 I'm. 16
In this study, Strain T93B was injected into the core at concen-
trations lower than 5.0x 107 SRB/mL to avoid plugging at the in-
let. 17 By immunofluorescent labeling techniques, we detected the
SRB in the effluent after O.S PV of water had been injected, giving
information on early water breakthrough owing to dispersion. Earli-
er miscible displacements conducted in similar cores, where radio-
SPE Production Engineering, May 1991
 TABLE 1-SULFATE-REDUCTION RATES-PRODUCED
35S2- AT DIFFERENT TEMPERATURES AND PRESSURES

Produced 35S2-
Temperature Pressure Per Cubic Meter of Rock
(oF) (psi) (mol/D)
145 15 8.8x10- 2
145 2,900 3.7x 10- 2
167 2,900 5.6x10- 1
176 4,500 1.3x 10- 1
185 4,500 0.0

TABLE 2-ESTIMATED NUMBERS OF ACTIVE SRB

PER CUBIC METER OF RESERVOIR ROCK AT
DIFFERENT TEMPERATURES AND PRESSURES

Number of
Temperature Pressure SRB/m 3
(oF) (psi) of rock"
145 15 8.8x10 '4
145 2,900 3.7x 10 '4
167 2,900 5.6x 10 '5
176 4,500 1.3x10 '5
185 4,500 0.0
Assuming lhat one SRB reduces 1.0 x 10 -16 mol sulfate/D. 18 Fig. 3-Growth of SRB In reservoir pores (bar: 10 I'm).

labeled 22Na brine displaced inactive brine, confirmed these ob-
servations. 7 The numbers of bacteria in the first samples after
breakthrough were lower than in the injected seawater medium-
1.1 X 10 3 SRB/mL compared with an injected concentration of
5.1 X 10 6 SRB/mL (Fig. 2). This chromatographic effect on the
bacteria is caused by dispersion in porous media and by attractive
forces acting between bacteria and sand grains. Furthermore, small
and narrow pores may trap some bacteria.
Activity Measurements. The bacterial activities at different tem-
peratures and pressures were measured as sulfate reduction rates
per cubic meter 3 of reservoir rock (Table 1). We expect that the
majority of the active bacteria are located in the bioftlm covering
the surface of the sand grains, producing daughter cells into the
water stream. Typical numbers of SRB in the effluent after non-
flow periods were LOx 10 5 to 4.0x 10 5 SRB/mL. Therefore,
reported values from the literature on the activity per SRB (Table
2) are used to estimate the numbers of active SRB in the core be-
cause measuring specific sulfate reduction in the core has not been
possible.
Permeability Reduction. The permeability in a brine-saturated core
at 140°F [60°C] was measured to be 770 md. A 50-mL batch of
thermophilic SRB, Strain T93B, containing 2.0x 10 6 SRB/mL was
then dispersed in the core. A stagnant incubation period followed.
The permeability was measured after 5 and 11 days, and a reduc-
tion in permeability caused by microbial growth was found to be
16 and 33%, respectively.
Electron micrographs revealed that the thermophilic SRB grew
in colonies connected to extracellular material (Fig. 3). After ini-
tial colonization of available surfaces, aquatic organisms tend to
form a confluent bioftlm within which the sessile bacterial cells are
enmeshed in a matrix of exopolysaccharide glycocalyses. 19 H 2S
may precipitate metal ions as metal sulfides. This precipitation,
together with produced microbial biomass, can form particles of
sizes comparable to the pore-throat sizes and may lead to plugging
and reduction of permeability.
The supply of nutrients in the injection water is important for
the development of bioftlms and production of organic material.
Although these studies are carried out with an excess of inorganic
nutrients and suitable organic carbon source, results show that SRB
have a potential for growth and H 2S production, even under the
high pressure and temperature conditions found in North Sea oil
reservoirs.
Conclusions
Thermophilic sulfate-reducing bacteria were present in hot produced
water from several different waterflooded reservoirs in the North
Sea.
Spore-forming SRB can survive, proliferate, and stay active un-
der most of the physical conditions in North Sea oil reservoirs.
In reservoirs with moderate and high porosities, SRB will be trans-
ported and spread at the same velocity as the front of the injected
water.
SRB may result in loss of permeability resulting from precipita-
tion of metal sulfides associated with produced bacterial biomass.
Acknowledgments
We are indebted to the Norwegian Research Council for Science
and the Humanities and to Statoil for supporting this research
program.
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SPE Production Engineering, May 1991 219


Authors
Graue Rosnes Lien
..an Thoma. Ro.nes Is a scientist In the Dept. of Microbi-
ology and Plant Physiology at the U. of Bergen In Norway.
His primary resellrch Interests Include thermophilic bacteria
In porous reservoir cores, with special emphaSis on sulfate-
reducing bacteria. He holds an MS degree In general microbi-
ology from the U. of Bergen. Ame Graue, a scientist In the
Dept. of Physics at the U. of Bergen, has primary research
Interests In fundamental studies of reservoir parameters
focusing on EOR research. For the last 5 years, he has been
coordinator of the reservoir research cooperation at the U.
of Bergen. Graue has been a visiting scientist at Mas-
sachusetts Inst. of Technology and Phillips Petroleum Co. He
holds BS degrees In mathematics and physics and an MS
degree In nuclear physics, all from the U. of Bergen. Torlelv
Uen Is a professor In general microbiology In the Dept. of
Microbiology and Plant Physiology at the U. of Bergen. He
began as an assistant professor In 1971 and became an as-
sociate professor In 1977 and a professor In 1981. He holds
a PhD degree In microbiology from the U. of Bergen.
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51 Metric Conversion Factor.
OF (OF - 32)/1.8 °C
md x 9.869 233 E-04 #Lm2
psi x 6.894 757 E+OO kPa
SPEPE
Original SPE manuscript received for review Feb. 22, 1990. Paper accepted for publica-
tion Oct. 19, 1990. Revised manuscript received Aug. 20, 1990. Paper (SPE 19429) first
presented at the 1990 SPE Formation Damage Control Sym~lum heid in Lafayette LA
Feb. 22-23. -' ,
SPE Production Engineering, May 1991