Microbial Profile Modification With Spores
J.H. Bae, SPE, K.T. Chambers, SPE, and H.O. Lee, Chevron Petroleum Technology Co.
Summary
To overcome the shortcomings of conventional, near-wellbore profile
modification methods, a microbial profile modification (MPM) meth-
od with spores was investigated. A halotolerant, spore-forming meso-
phile was isolated and characterized. These biopolymer-producing
spores propagate easily in Berea cores with permeabilities more than
about 500 md. With a specifically formulated nutrient package, they are
readily germinated and produce biofilm, which reduces the permeabil-
ity of the rock. The depth of penetration and the degree of permeability
reduction can be controlled by varying injection schemes.
Introduction
The use of microbes in EOR has been discussed for more than 40
years.1 The idea behind microbial EOR (MEOR) is that microbes
can produce most of the agents used in EOR. These include surfac-
tants, polymers, solvents (such as ethanol and acetone), acids, and
gases (such as CO2 and methane). Thus, it has been purported that
these chemicals and gases produced in situ, alone or in combination,
improve the oil-displacement efficiency.
A flood of papers have been published in recent years on the labo-
ratory investigations as well as field trials of MEOR, both in the U.S.
and abroad. Many technical meetings devoted exclusively to
MEOR have been held, and the proceedings have been published.2
Unfortunately, many MEOR studies in the literature have been
poorly designed, and the mechanisms of oil recovery have not been
clearly delineated. Some were poorly documented and reported
with many questionable claims. As a consequence, MEOR is re-
ceived with much skepticism in the oil industry.
At the outset, we felt that, while MEOR is technically feasible
(that is, microbes produce chemicals in situ), the chance of produc-
ing the right chemicals in the right amount to effect the desired re-
sults is rather slim. On the other hand, MPM depends on in-situ bio-
film formation. Biofilm is the multilayer growth of cells and support
material on solid surfaces, consisting of biopolymer and biomass
produced by the microorganisms. Thus, this process does not de-
pend on delicate chemical systems produced by the microbes, and
it is much easier to achieve the desired results.
As we conceptualized, this process could be used for in-depth treat-
ments of high-permeability or thief zones. This method overcomes
the shortcomings of the conventional treatments, such as polymer
gels, squeeze cementing, and selective perforation, whose effects are
negated by crossflow in the reservoir. This process involves injection
of biopolymer-producing bacteria in the form of spores and nutrient
package. This process relies on injected microorganisms and nutri-
ents to reduce the permeabilities of watered-out thief zones or high-
permeability streaks by forming a resilient biofilm in the pore space
of the reservoir rock. Thus, MPM appeared to have the best chance
of technical and economic success. The objective of this paper is to
present our laboratory data on MPM with spores.
Desirable Characteristics of Microbes
for Profile Modification
Several characteristics are essential or highly desirable in microbes to
be used in MPM. To begin with, they should be able to grow and pro-
duce the biofilm in reservoir environments. This means that the mi-
crobes should thrive in anaerobic conditions, tolerate the reservoir
temperature and salinity, and be compatible with reservoir oil. Thus,
MPM requires the use of halotolerant anaerobes. Broadly speaking,
anaerobes can be divided into two categories: obligate anaerobes, for
which molecular oxygen is toxic, and facultative anaerobes, which
Copyright 1996 Society of Petroleum Engineers
Original SPE manuscript received for review Oct. 10, 1994. Revised manuscript received
May 2, 1996. Paper peer approved May 23, 1996. Paper (SPE 28617) first presented at the
1994 SPE Annual Technical Conference and Exhibition held in New Orleans, LA, Sept. 25–28.
SPE Reservoir Engineering, August 1996
can grow either in the presence or absence of atmospheric oxygen.
For ease of handling during storage and injection operations, faculta-
tive anaerobes are more suitable for MPM processes.
Anaerobes cannot use hydrocarbons as the carbon and energy
source; thus a nutrient medium should be provided. This medium
must contain a suitable carbon substrate and other elements required
for growth, such as nitrogen and phosphorous. This necessitates that
they have nonfastidious nutrient requirements.
For wide applications, the microbes should tolerate as wide a tem-
perature range as reasonably can be expected in the reservoir. Meso-
philes, which are generally suitable in the temperature range of 20
to 55°C, are more suitable than psychrophiles with a temperature
range of up to about 20°C. In high-temperature reservoirs, thermo-
philes with high-temperature optima will be required.
Additionally, because the microbes should be able to penetrate far
in the reservoir, they must be small in size. Bacterial spores, pro-
duced within some microbial cell, are small and are much more re-
sistant to adverse environmental conditions. They can survive in the
dormant state for many years. In fact, the use of spores in MEOR
appeared in the literature.3 When conditions are right, spores germi-
nate into vegetative cells, which will produce biofilm. Spores are
small, and their wall is inert; thus, they are easier to propagate in res-
ervoir rocks than vegetative cells. The spores should easily germi-
nate and grow when stimulated with a nutrient. The production of
polymers facilitates biofilm formation and enhances the stability of
the biofilm. Thus, bacteria that produce copious amounts of exo-
polymers are desirable. For obvious health reasons, they should be
nonpathogenic and must not produce animal or plant toxins.
Characteristics of SaltonĆ1
After a considerable effort, a culture was isolated from a saline sedi-
ment that appeared to have the desired characteristics.4 This isolate
was named Salton-1 and is used exclusively in this investigation.
The transmission electron microscope picture of the vegetative cells
and spores is shown in Fig. 1.
Biochemical and morphological tests identified Salton-1 as
closely resembling the species Bacillus licheniformis, as described
in Bergey’s Manual,5 the standard source for the classification of
bacteria. Salton-1 is a Gram-positive, rod-shaped bacterium about
0.2 to 0.3 mm wide and 0.5 to 1.0 mm long. The cells of Salton-1 are
somewhat smaller than the dimensions given in Ref. 5. They are fac-
ultative anaerobes and halotolerant or moderately halophilic, hav-
ing motility with peritrichous or polar flagella. As expected of genus
Bacillus, they are single-endospore-forming bacteria.
They produce an extracellular water-soluble polymer during the ear-
ly stationary phase of their growth cycle. Spectroscopic tests, acid hy-
drolysis, and chemical analysis determined the polymer to be primarily
polyglutamic acid. The molecular weight of the polymer was deter-
mined by gel permeation chromatography with polyacrylamide stan-
dards. The values obtained for two samples were 2.1 and 2.8 million.
Nutrient Formulations
As indicated above, spores of Salton-1 have several advantages over
vegetative cells for use in MPM. Vegetative cells are much larger
than the endospores, and they tend to aggregate together in short
chains or small masses. They are also coated with polymeric materi-
al and are thus difficult to propagate in porous media. Furthermore,
they tend to lyse rapidly when stored and lose viability.
Studies were conducted to find an optimal medium formulation
for rapid spore germination and growth and maximum polymer pro-
duction.6 The base formulation adopted, called PM-2, included
100 g NaCl, 1 g NaNO3, 0.7 g NH4NO3, 0.25 g MgSO4, 5 g
KH2PO4, 10 g sucrose, 1.3 g citric acid, and 0.5 g yeast extract/L
water, adjusted to pH 8.
The effects of components in the nutrient formulation PM-2 on
spore germination at room temperature are shown in Table 1. Each
163
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
Fig. 4—Change of medium pH with time. Fig. 5—Effect of NaCl on germination and growth.

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
TABLE 2—SUCROSE CONSUMPTION AND BIOPOLYMER
PRODUCTION

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
Incubation Sucrose Viscosity Biopolymer Dry Cell

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
(hours) (g/L) (Pa@s) (mg/L) (mg/L) Cells/mL

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
0 10.7 — — — 1.9 x 104
20.5 8.9 0.00104 70 174 2.1 x 108

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
26 — 0.00112 48 184 1.2 x 108

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
35 8.3 0.00145 135 — 1.4 x 108
44 5.3 0.00236 232 180 4.0 x 107

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
49 4.8 0.00264 318 188 1.6 x 107

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
57 4.5 0.0026 340 — —

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
68 2.2 0.00254 350 — —
75 2.2 0.00254 — 182 6.0 x 104

In Table 2, the kinetic data for sucrose consumption and biopolym-

Fig. 6—Effect of temperature on germination and growth.
er production are shown. The viscosity was measured with a Brook-
field LVT viscometer with ultraviolet-light adapter. The number of
cells reaches a plateau within a day. However, sucrose consumption
continues for many hours and so does the biopolymer production.

Core Tests
After the merger of Chevron and Gulf, a microbiology laboratory
was contracted to do some work for us. As preliminary tests, they
conducted constant-pressure core tests in 800-md Berea cores that
were saturated with North Ward Estes (NWE) water at room temper-
ature. This NWE brine contained 59.7 g NaCl, 13.2 g CaCl2, 38.5
g MgCl2, 3.3 g Na2SO4, and 0.43 g Na2CO3 in 1 liter of water. The
core was 10 cm in diameter by 5 cm long, and the injection pressure
was maintained at 3.5 psi. A 10-PV spore suspension of 106 cfu/mL
was injected and followed by a 100-PV of NWE brine, which was
followed by a 40-PV nutrient injection. The core was incubated for Fig. 7—Constant pressure core test.
24 hours and then continuously swept by brine until 100% perme-
ability reduction was achieved. In Fig. 7, the permeability reduction
ranged from 22% to 25%. After the 2-PV fluid injection, we did not
is shown for two pH values. Here the permeability ratio is the final
detect any spores in the effluent in 100-, 300-, 380-md cores. In a
permeability divided by the original permeability. During the injec-
660-md core, we detected a trace of spores. As shown in Fig. 8, spore
tion of spores, some permeability reduction was observed. During
the brine injection, the permeability was continuously reduced and breakthrough was prominent in 710- and 1350-md cores. In the high-
eventually the core was completely plugged. Even though this was er-permeability core, the effluent concentration reached 104 cfu/mL.
an unrealistic experiment, it showed the potential of the process. With a higher spore concentration of 109 cfu/mL, we had a signifi-
When we set up our own laboratory, we changed the experimental cant spore breakthrough in the effluent in a 650-md core, as shown in
procedure to be realistic. We fired all Berea cores used because they Fig. 9. On the other hand, with a 105-cfu/mL suspension, we did not
harbor indigenous bacteria that can be resuscitated by nutrient injec- detect any spores in a 1550-md core, indicating complete retention. As
tion. This procedure eliminated any permeability reduction caused expected, these results show that the transport of spores in Berea cores
by indigenous bacteria in the core. To remove any spurious effect of depends on the amount of the spores injected and the permeability of
gas produced by microbial activities on permeability reduction, we the rock, or, more precisely, the pore-size distribution. At this time, the
used a backpressure of 10 343 kPa. exact mechanisms of spore transport in porous media are not known.
However, spores may act more like fine particles and they may be
Spore Transport. Several core tests were run to investigate the trans- slowly retained in the rock by means of deep bed filtration.
port of spores in Berea cores. A 1-PV spore suspension of 107 cfu/mL
was injected into cores of different permeabilities, which was fol- Permeability Reduction. In Table 3, the experimental conditions
lowed by another PV of chase brine. The porosity of these cores for permeability reduction tests are listed. The spore concentration

SPE Reservoir Engineering, August 1996 165

 Fig. 8—Effluent spore concentration (injection concentration is Fig. 9—Effluent spore concentration (injection concentration is

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
107 cfu/mL). 109 cfu/mL).

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁ
ÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
TABLE 3—CORE TEST CONDITIONS*

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
Absolute
Permeability

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
Run (md) Porosity Injection Scheme Comments

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
1 1350 0.26 1S + 1N + I + 1B S and B at 100, N at 3 mL/h
2 1820 0.26 1S + 1B + 1N S and B at 100, N at 3 mL/h, some N through pressure tap

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
3 1610 0.26 1S + 1B + (5 + 2)N

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
4 1270 0.27 0.6S + 0.6N + I + 1B + 1N
5 1150 0.27 0.3S + 0.3N + I + 1B + 1N

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
6 1250 0.27 0.1S + 0.1N + I + 1B + 1N

ÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁÁ
*Numbers in injection scheme denote PV and abbreviations are as follows: S+spore suspension N+nutrient I+incubation B+brine.

used was 108 cfu/mL except Run 1 where 107 cfu/mL was used. The
permeability reduction data obtained at the end of the fluid injection
for the first three runs are shown in Fig. 10. In Run 1, after the nutri-
ent injection, the core was incubated for 3 weeks. In the next two
runs, the fluid injection rates were changed as noted in the table.
In the first two runs, the amount of spores and of nutrient used was
the same. Yet, the permeability reduction behavior was different. With
incubation, the reduction was about 50% along the core, while, with
higher injection rates in Run 2, permeability reduction was almost
100% in the first section and about 50% in the remainder of the core.
In Run 3, 5 PV of nutrient was injected through the last pressure
tap into the third section of the core to simulate the situation where
the nutrient flows through the tighter zone and crossflows into the
thief. In Fig. 11, the permeability reduction is shown as a function
of fluid injection. After about 2 PV injection, permeability was near
zero. The changes in permeability ratio during a 2-PV nutrient injec-
tion from the inlet are shown in Fig. 12. The ratio for the third sec-
tion is interesting in that it initially increased and then declined to
about 0.1, which is much higher than before the injection from the
inlet. We attribute this to the movement of biofilm in the core; this
phenomenon has been seen often. With larger amount of nutrient
and with a specific stimulation of the last section, the reduction was
almost complete all along the core.
The next three tests were conducted to see the amount of perme-
ability reduction with smaller volumes of spores and nutrient. In
each test, the same number of PV’s of spore suspension and nutrient
were injected, and the core then was incubated for 4 weeks. Then,
the permeability was measured by use of brine. In all cases, we did
not see any permeability reduction. Therefore, 1 PV of nutrient was
introduced to restimulate the microbes, and the results are shown in
Fig. 13. In general, about 20% to 30% reduction was attained. It ap-
pears that we may need a larger amount of spores and nutrient for
complete plugging than used in these runs. Thus, these tests indicate
that the permeability reduction depends on many variables, includ-
ing spore concentration and nutrient amount. On the other hand, this
also implies that the degree of permeability reduction could be con-
trolled by manipulating these variables.
Discussion
We have to keep in mind that we are dealing with living organisms
and many variables affect them during their life cycle. This is to say
that variability of experimental results involving microbes is much
greater than that we are accustomed to in a customary experiment

1
2
3

Fig. 10—Sectional permeability reduction. Fig. 11—Permeability reduction in third section.

166 SPE Reservoir Engineering, August 1996


Fig. 12—Permeability reduction during restimulation.
involving oil recovery. For instance, the size of Salton-1, both
spores and vegetative cells, varies somewhat depending on sporula-
tion and germination media, which, in turn, affects the transport in
the core and the degree of permeability reduction. Also, the process
is highly complex. From our experiments, it appears that the kinetics
of germination and growth in the bioreactor is quite different from
that observed in the core, and the relationship between biopolymer
produced and core plugging efficiency is not clear.
The results of core tests show that a small amount of spores and
nutrient might not be effective for permeability reduction. This can
be a major advantage in field applications. In a real situation, most
of the injected spores and nutrient will go into the high-permeability
zone. Fortunately, even if a small amount of spores penetrates the
low-permeability zones, those zones might not be plugged. Ob-
viously, the higher the permeability contrast among layers, the bet-
ter the process will work.
To be practical, we limited the slug size of spore suspension and
nutrient used in our experiments to 1 PV in most cases. Even this may
appear to be prohibitively large. However, in a real case, the volume
will be based on the thief-zone PV and the depth of treatment desired.
Thus, the amount will be a fraction of the total PV of the reservoir.
As shown in our experiment, spores, with their small size and inert
wall, can be transported easily in thief zones; thus, the depth of pe-
netration can be controlled by the amount and/or concentration of
spore suspension injected. However, if the permeability of the thief
zone is low (e.g., around 100 md), spore transport will be limited.
During the nutrient injection, when the near-wellbore region is
plugged, the nutrient will be diverted into other zones. When the nu-
trient crossflows into the thief zone where spores are retained beyond
the plugged zone, plugging will take place again. Hence, the problem
of crossflow can be used to advantage in this process. This process
can go on to any depth desired within the economic limitations.
Conclusions
1. Salton-1 has most of the desirable properties for MPM.
2. Salton-1 is mesophilic and haloterant with an optimum temper-
ature range of about 40 to 55°C and a salinity range of about 2 to 8
wt% NaCl.
3. With a 1-PV slug size, spores were detected in the effluent in
Berea cores having a permeability of more than about 500 md, indi-
cating easy propagation in these cores.
4. The spores can effectively reduce permeability of cores. The
depth of penetration and the degree of permeability reduction de-
pend on many variables.
5. Crossflow can be used for in-depth permeability reduction.
6. Overall, MPM is technically a viable process.
Acknowledgments
Original microbiological work was done by R.S. Silver, P.M. Bun-
ting, W.G. Moon, and W.A. Acheson of the former Gulf R&D Co.
We would also like to thank L.E. Henry who did most of the microbi-
ological work reported here.
SPE Reservoir Engineering, August 1996
4
5
6
Fig. 13—Sectional permeability reduction for spore slugs.
References
1. ZoBell, C.E.: “Bacterial Release of Oil from Oil-Bearing Materials,”
World Oil (1947) 125, 36.
2. Microbial Enhancement of Oil Recovery—Recent Advances, E. Premuzic
and A. Woodhead (eds.), Elsevier, Amsterdam (1993).
3. Jack, T.R. and Thompson, B.G.: Microbial Enhanced Oil Recovery, J.E.
Zajic et al. (eds.), PennWell Publishing Co., Tulsa, OK (1983).
4. Silver, R.S. et al.: “Bacteria and Its Use in a Microbial Profile Modifica-
tion Process,” U.S. Patent No. 4,799,545 (1989).
5. Bergey’s Manual of Systematic Bacteriology, J.G. Holt (ed.), Williams &
Wilkins, Baltimore (1984).
6. Silver, R.S. and Bunting, P.M.: “Phosphate Compound That is Used in a
Microbial Profile Modification,” U.S. Patent No. 4,947,932 (1990).
SI Metric Conversion Factors
cp 1.0* E*03 +Pa@s
°F (°F*32)/1.8 +°C
in. 2.54* E)00 +cm
md 9.869 233 E*04 +mm2
psi 6.894 757 E)00 +kPa
*Conversion factor is exact. SPERE
J.H. Bae is a research consultant at Chevron Petroleum TechnolĆ
ogy Co. in La Habra, CA. Previously, he was a section supervisor
with Chevron Oil Field Research Co. and a section director at
Gulf R&D Co. in Pittsburgh, PA. He has worked in all areas of EOR.
Bae holds a BS degree from Seoul National U. and MS and PhD
degrees from the U. of Florida, all in chemical engineering. K.T.
Chambers is a petroleum engineer with Chevron Overseas PeĆ
troleum Inc. Previously, he was a research engineer at Chevron
Oil Field Research Co. He holds a BS degree in chemical and peĆ
troleum refining from Colorado School of Mines and an MS deĆ
gree in chemical engineering from the U. of California, Berkeley.
Photograph is unavailable. H.O. Lee is a lead research scientist
at Chevron Petroleum Technology Co. Before joining Chevron,
she served as an assistant professor with the Dept. of Chemical
& Biochemical Engineering at Rutgers U. and worked as a postĆ
doctoral research assistant at the New Mexico Petroleum ReĆ
covery Research Center in Socorro. She holds a PhD degree in
chemical engineering from the U. of Illinois.
167