SPE 110134

A Pore Level Study of MIOR Displacement Mechanisms in Glass

Micromodels Using Rhodococcus sp. 094
Christian Crescente, SPE, StatoilHydro ASA; Andreas Rekdal, SPE, StatoilHydro ASA; Akram Abraiz, SPE,
Schlumberger; Ole Torsaeter, SPE, Lisbeth Hultmann, Arne Stroem, Kjetil Rasmussen, Norwegian University of
Science and Technology; and Espen Kowalewski, SPE, StatoilHydro ASA

Copyright 2008, Society of Petroleum Engineers

This paper was prepared for presentation at the 2008 SPE/DOE Improved Oil Recovery Symposium held in Tulsa, Oklahoma, U.S.A., 1923April2008.

This paper was selected for presentation by an SPE program committee following review of information contained in an abstract submitted by the author(s). Contents of the paper have not been
reviewed by the Society of Petroleum Engineers and are subject to correction by the author(s). The material does not necessarily reflect any position of the Society of Petroleum Engineers, its
officers, or members. Electronic reproduction, distribution, or storage of any part of this paper without the written consent of the Society of Petroleum Engineers is prohibited. Permission to
reproduce in print is restricted to an abstract of not more than 300 words; illustrations may not be copied. The abstract must contain conspicuous acknowledgment of SPE copyright.


Abstract
Micromodel experiments have been executed in order to have better insight into the displacement mechanisms allowing
Rhodococcus sp. 094 to increase oil recovery. Changes caused by the bacteria in the fluid interfaces and pore walls have been
recorded and are presented. The previously suspected mechanisms are further confirmed by the results, but a much better
insight into the details of how the process occurs has been obtained and a theory for this process is developed.

Introduction
Previous publications by the authors
1, 2
have already stated the effectiveness of Rhodococcus sp. 094 to increase oil
recoveries from Berea cores with pure hydrocarbons (dodecane) consistently with an additional 4 %
1
and at the most
favorable conditions with up to 9%
2
. The mechanisms suspected for the additional recovery have been: Selective plugging,
interfacial tension (IFT) reduction
3
, and changes in wettability. In those publications, the work has focused on standard
reservoir lab measurements, especially Berea corefloodings and interfacial tension and wettability measurements.
Glass micro model experiments can complement very well these experiments, by providing a look into the process as it
unfolds in the pore space. Even though the glass micromodels used have limitations from the larger pore size deriving from
the fabrication process limitations, and from the homogenous and two dimensional geometry of the flow, they can still be an
invaluable tool for understanding complex mechanisms, testing hypotheses and qualitatively provide a guide of the changes
occurring in the pore space with different EOR methods. It was also possible to estimate the saturation by using image
analysis software, which has made it possible to plot the enhanced oil recovery vs. the capillary desaturation curve (CDC).
A total of ten experiments where brine and brine with either the surfactant producing or the non-surfactant producing
variant of the Rhodococcus sp. 094 bacterium are presented. The results obtained in the micromodel flooding are analyzed in
conjunction with the results from coreflooding experiments, and in this way a much better understanding of the mechanisms
emerges, and a hypothesis can be proposed. By designing new experiments to test this hypothesis, it can be confirmed,
improved or disproved.

Theory review
In situ Microbial Improved Oil Recovery (MIOR) processes are processes in which bacterial activity is stimulated in the
porous space so that bacteria and their bioproducts can help mobilize additional oil from the reservoir.
Although these processes are usually inexpensive, and simple to apply logistically, the bacteria require for their growth
that enough of the following chemicals are readily available in the places in the reservoir where growth is to occur:
A carbon source, which is generally either oil or molasses
A source of nitrogen (like ammonium or nitrate)
Phosphate
An external electron acceptor for respiration (nitrite, nitrate and to a certain extent dissolved oxygen)
In previous publications by Crescente et al.
1,2
it has been established that there are three main mechanisms through which
Rhodococcus sp. 094 increases oil recovery:
Interfacial tension (IFT) reduction
4
: For Rhodococcus sp. 094 to grow on hydrophobic components, it needs to
produce a hydrophobic cell surface component, which allows the bacteria to attach to the oil drops, where the
2 SPE 110134
cells can reproduce in exponential growth first and linear growth later. These components are capable of forming
very stable emulsions of oil in water
3
, and Kowalewski et al.
5
reported IFT reductions from 38 to 0,006 mN/m
over a two weeks period for a different bacteria species, and additional field
6
and laboratory
7,8,9
investigations
report IFT reductions by bacteria. This IFT reduction will help release captured drops of oil, as shown in Figure
1 and by the Laplace equation.
Wettability changes
4
: Wettability is a complex phenomenon which is not yet fully understood. However, it is
known that weakly water-wet conditions are the most favorable for oil production under waterflooding.
Wettability changes both towards more water wet
10
and towards more oil wet
11
have been reported as a
consequence of bacterial applications. Changes in wettability will change the drainage/ imbibition processes. The
idealized pore system in Figure 2 shows how the wetting phase is connected with continuity throughout the
whole system, whereas the non wetting phase can leave disconnected drops (1) that will not move unless they are
reconnected like the drops in the continuous non-wetting fluid (2). A local wettability change might reconnect
the drops. On the other hand, a local wettability change on a surface, will release some of the previous wetting
phase enabling it for flow. The underlying mechanisms are complex and dependent on characteristics of the rock
and fluids, but in general, a wettability change can be positive and increase oil recovery regardless of the
direction of the change. Reported changes both towards more water wet
10
and towards more oil wet
11
conditions
can have a positive effect on recovery.
Changes in flow pattern
4
: Injected brine tends to follow established flow channels from the injection point to the
production point following the path of least resistance, which means that most of the flow will go through the
largest pores, leaving bypassed oil behind in areas which have been poorly swept. By changing these flow paths,
this bypassed oil can be reached and produced. Changes in the flow pattern can be caused by bacterial plugging
and by polymers or biofilm produced by bacteria
12, 13
. This effect has been widely reported
14-18
, even by field
tracer tests
19
. Two different kinds of pore blocking can occur, the first one is a gradual effective flow area
reduction by bacterial mass on the pore walls. As shown in Figure 3-a, initially bacteria and nutrients (in the
brine phase) will flow predominantly through the largest pore channels. Therefore, the rate of increase of
bacterial mass will be directly proportional to the flow rate through a given pore channel. Bacterial adhesion and
growth will reduce the effective flow area of the pore channel, deviating the flow through other flow paths,
improving the sweep efficiency and making possible the displacement of bypassed oil. And the second one is an
abrupt blocking caused by a large particle suddenly blocking a channel on a narrow point as seen in figure 3-b in
a similar fashion to polymers.

Capillary Number
The capillary number (N
c
) is a dimensionless ratio of viscous to capillary forces.
20
For a particular geometry and wetting
condition, the capillary number could be increased by either increasing the viscous force or by lowering the capillary force.
With the capillary number, the capillary desaturation curve (CDC) can be constructed by plotting the saturation of the non
wetting (S
nwr
) or wetting (S
wr
) phase on the y axis against the capillary number on a logarithmic x axis.
The critical capillary number (N
c
)
c
, is the point at which S
nwr
and S
wr
cease being constant, and start decreasing until
complete desaturation, which occurs at the total desaturation capillary number (N
c
)
t
. The capillary number relation used in
this work was:

=
c
N
Where
is the fluid viscosity,
is the fluid velocity and
is the interfacial tension between the fluids

Experimental Method
The present work expands on previous coreflooding experiments with Rhodococcus sp. 094 published by Crescente et al.
1, 2
,
and the same fluids are used. The experiments, though, are on a separate rig suited for investigations with micromodels.

Materials
Glass Micromodels
The glass micromodels used in this work are described in Figure 4 and Table 1. The micromodels consist of a very thin
pore structure etched on the surface of a glass plate. The pore structure is symmetrical with identical pores connected with
each other through four pore throats each. On top of this pore structure a second glass plate is placed, to cover the etched
pattern, creating a closed system. This second glass plate has an inlet and outlet hole at each end, allowing the displacement
of fluids through the pore network.
The pore structure is only one pore deep, which limits the flow to two dimensions, but allows observing the flow of the
different phases through the pore network.
SPE 110134 3
Two different micromodels have been used:
Micromodel 1: Is the smallest of the two, has an Aspect Ratio (ratio of the diameter of the pore body and the
pore throat) close to 1, as the pore throats are basically of the same size as the pores,
Micromodel 2: is the largest of the two micromodels, and has a higher Aspect Ratio (2.4)

Experimental Setup
The experimental setup can be seen in Figure 5, and it can be divided in two main systems according to their functions: a
system for flooding through the micromodel, and a system for capturing the images. A description of the main components
follows:
Fluid Reservoir and Burette
To the extreme left the fluid reservoir is seen on top of a magnetic stirrer/heater. When the fluid to inject is bacteria it
comes in a sterile sealed flask with a cork that has a silicone tubing on one connector and an air filter on another connector.
This allows extracting fluid without contamination from the environment. The pump inlet is connected to the sterile
connection hose of the flask, so that the fluid can be injected with the use of the pump. A burette is also seen and it is used to
inject by gravity at very low rates that the pump cannot achieve.
Pump
The pump used to inject the fluids was capable of a lower rate of 1 ml/hr. When the injected rates needed to be lower than
this, and when injecting toluene which is harmful for the pump, the burette was used, and by adjusting its height, the flow
rate could be controlled.
Hosing system
The main hosing system connecting the pump and burette to the micromodel is built with 1/16 tubing. T valves and stop
valves make it possible to easily switch between the pump and the burette, and also allow bypassing the micromodel through
a loop. The hosing connects to the inlet of the micromodel through a metallic port that has a screw that needs to be carefully
adjusted so that it is tight enough to hold the hose in place, but not too tight to prevent circulation, or to break the
micromodel. An identical port is connected to the outlet of the micromodel, and from here a hose comes out into a fluid
collection tube.
Microscope
The microscope allows taking a closer look at the surveillance points in the micromodel. It has exchangeable objective
lenses, and on its objective table (atop of which the micromodel is fixed) it has a coordinate system that was used to be able
to survey many points of the micromodel and easily return to each one of them repeatedly.
Video Camera
A video camera is placed on a special connector on the microscope, and is connected to a monitor and a PC, allowing
viewing and recording the images from the survey points.
Monitor
A monitor was connected to the camera. This allowed watching in real time the video signal from the camera. This
allowed easy surveillance of the points, and adjusting the positioning of the surveillance points, as the signal was in real time.
Computer
A computer with a TV tuner card was used to store and process the images. The TV tuner has an analog to digital signal
processing chip, and was therefore useful for capturing the signal from the camera.
Software
The software accompanying the video capturing card, could take pictures upon command, and could also be programmed
to take pictures at time intervals, which allowed surveillance of one point during prolonged periods without human
supervision. An open source image processing program was used to process the images and calculate the saturation. A
commercial photo editing tool was used to increase the contrast so that the image processing program could differentiate
between the phases.

Fluids
The fluids used in this experimental work have been the same fluids used in the coreflooding experiments published by
Crescente et al.
1, 2
.
Brine
The brine used to saturate and to waterflood the micromodel had the same composition as the dodecane medium used to
cultivate the bacteria, without the dodecane (Table 2). This was done to ensure that any improvement on the bacterial
floodings is due to the bacteria and not to the chemicals in the growth medium.
Hydrocarbon
n-Dodecane (C
12
) was the hydrocarbon used to saturate the micromodel.
Bacterial Suspensions
The alkane oxidizing Rhodococcus sp. 094, has been the species used in the present work, as in the previous publications
by Crescente et al. This bacterium was isolated from seawater from the fjord of Trondheim, and was found to be able to form
very stable crude oil in water emulsions. It was therefore thoroughly characterized by Bredholt et al.
3
, for its potential for
cleaning oil from contaminated sands. A useful feature of this bacterium is that its surfactant producing ability can be
4 SPE 110134
activated/deactivated depending on the carbon source used during the cultivation period (after using the pre-culture shown on
Table 2 to increase the number of initial cells in the culture), producing two different variants of the bacteria:
Cells of Surfactant-Producing Bacteria (SPB): By cultivating the bacteria in a medium where dodecane is the
carbon source, the bacteria are enabled to produce surfactants. The composition of the growth medium for this
variant can be seen in Table 2.
Cells of Non-Surfactant-Producing Bacteria (NSPB): When the cultivation medium has acetate as carbon source,
the bacteria will not produce surfactants. The composition of the growth medium for this variant can be seen in
Table 2.

Experimental Procedures
Cleaning
In biotechnology, experimental conditions are made strictly sterile. The realities of a reservoir engineering lab and in
particular the traits of flooding of micromodels makes this standard almost impossible to achieve. However, the circulating
system and micromodel were rigorously cleaned between experiments. The whole circulating system was flushed at high
rates with at least three consecutive cycles of methanol and distilled water. The micromodel though, required additional
caretaking:
Cleaning of Micromodels
After finishing the experiments the micromodels had often sediments and solid residues that were very difficult to extract.
A good cleaning method was therefore of extreme importance to have similar initial conditions in all the experiments: First,
about five flooding cycles of alternating distilled water/methanol are performed, in some of them injection is made through
the outlet of the micromodel. This removes a good part of the sediments. The micromodel is then flushed under hot tap water
through the inlet, then air was blown through the inlet. The cycle is repeated as many times as necessary, changing also the
direction of the cleaning. After the micromodel is clean, at least two more alternating cycles of methanol and distilled water
are injected with the pump.

Saturation with brine
The micromodel is initially saturated 100% with brine (Table 2), by flooding at rates up to 3 ml/min for about 10 minutes.
Primary drainage
Dodecane is injected into the micromodel by using the pump at rates of up to 0,5 ml/min, reaching Sw
i
and oil saturations
between 0,7 and 0,9.
Capillary desaturation curve
Two experiments were performed initially to create a capillary desaturation curve. Injection rates were the variable factor
in these experiments, and the rate was increased in steps until the residual oil saturation was zero. For the micromodel 2 the
injection rates were varied between 0,08 4 ml/min which was enough to extract all the oil from the micromodel. On
micromodel 1 the oil could not be extracted completely due to constraints in the inlet making the injection pressure too high.
Flooding experiments
Different sets of experiments were carried out to test the effectiveness of bacteria to reduce oil saturation in the
micromodels beyond what is possible with waterflooding, and surveying for observable changes that might give an indication
of the mechanisms in action during the bacterial floodings. Since there were slight differences between the different
experiments, the characteristics of each one are described on the results. During these experiments eight surveillance points
were selected, by cycling continuously between the points, the process could be followed at many points in the micromodel
at the same time. The coordinates system in the microscope stage allowed to continuously cycle between the surveillance
points and easily returning to each one of them.
Oil saturation determination
Determining oil saturation from images can be a difficult and time consuming task. To simplify this work, an open source
program with the ability to recognize different tones of light, and reporting how many pixels in a picture are occupied by a
given color was used. However, there was not a good enough tone contrast between the dodecane phase and the water phase.
Adding a colorant to the water phase was discarded because of the effect it might have on the bacteria, and adding Sudan Red
to the dodecane did not improve the tone contrast, so a commercial photo handling software was used to apply filters that
could improve the contrast through the application of a magnetic lasso filter which made the menisci clearer, allowing to
use the infill function to fill the dodecane drops with a color which gave a good contrast so the pictures could be finally
processed in the open source program. Before the oil saturation could be determined, the total pore space had to be
determined. This was done in a similar fashion, by filling the grains, and calculating the remaining area of the picture. Then
by dividing the oil area by the whole picture minus the grains area, the saturation was calculated (Figure 6).

Results and Discussion
CDC Determination
Experiment 1 was carried out to build the CDC for Micromodel 1. Eight surveillance points were selected from all the
areas of the micromodel, by selecting two X and four Y values from the coordinate system on the microscope stage. After the
establishment of irreducible water saturation, water injection started, initially by using the burette increasing the rate in steps
SPE 110134 5
until it was not possible to increase the rate with the burette any further. Then the pump was connected to the system.
However, when the rate was 0,787 ml/min a leakage occurred which meant the experiment had to be ended.
Experiment 3 was a new experiment carried out to build the CDC for Micromodel 1. The 8 surveillance points were
placed in the area close to the entrance of the micromodel. The resulting saturations, and the process for calculating the CDC
for this experiment are shown on Figure 7. The CDC curve for this experiment can be seen in Figure 8-a. It was not possible
to reduce the saturation of the surveillance points to zero, as this would require working at pressures beyond the pressure
limit, and risking to break the micromodel.
Experiment 5 was carried out to establish CDC for Micromodel 2. Eight surveillance points were established close to the
entrance of the micromodel by In this micromodel it was possible to increase the rates significantly beyond what was
possible with Micromodel 1. For Micromodel 2 it was possible to displace all the oil from the surveillance points by
increasing the rate, so a complete CDC was created (Figure 8-b). There is a flattening and slight increase in the mid section of
this curve which is due to some residual oil in the capillaries at the entrance of the micromodel pore network before these oil
drops moved into the model as the rate increased.

Waterflooding followed by bacterial flooding
Experiment 2 was a waterflooding followed by SPB flooding performed on Micromodel 1. From this experiment onwards
the surveillance points were fixed so that they would be the same for subsequent experiments with this micromodel. After
establishing Sw
i
brine was injected until there was no more oil production. At this point injection of bacterial suspension
could start. 15 pore volumes were injected before stopping the injection for 19 hours to allow the bacteria to start growing in
the micromodel. After reestablishing circulation the rates were increased stepwise while continuously injecting bacteria. It
was not possible to inject at as high rates as with the CDC experiment, as the pressure was higher during the bacterial
injection. The CDC for this experiment can be seen in Figure 8-a.
Experiment 4 was another waterflooding followed by bacterial flooding on Micromodel 1. CDC was calculated with
variation of IFT for both this experiment and Experiment 2 (Figure 8-a). Even though the oil saturation at the beginning of
this experiment was significantly higher than on Experiment 2, the endpoints were very close. It was apparent then that a
slight decrease in IFT was able to decrease the oil saturation in the surveillance points of this micromodel.
Experiment 6 was a waterflooding followed by bacterial flooding on Micromodel 2. After establishing So
r
through
waterflooding, 15 pore volumes of bacterial suspension were injected. Then the system was closed for a period of 24 hours.
After this, the rate was increased in steps through the whole experiment. However there were no major changes with respect
to the CDC from experiment 5 (Figure 8-b). However, the flooding period after the 24 hours shut-in lasted only 14 hours,
which might not be enough for the bacteria to have observable effects.

Bacterial flooding with SPB over a prolonged period
Experiment 7 was carried out with a different approach, with the goal to observe the changes in the system during a
bacterial flooding over a long period. After establishing Sw
i
, waterflooding is carried out until there is no more oil
production, then bacterial suspension is injected. The whole experiment is carried out at a constant rate of 0.08 ml/min, which
corresponds to a speed of 3.2 cm/min. Pictures of the surveillance point before the bacteria were introduced, and during the
bacterial flooding were taken throughout the whole experiment. During this experiment there were suspicion of possible
contamination with other bacteria, so fluid samples were sent to analysis, confirming that contamination had occurred and
that Rhodococcus sp. 094 was not the only bacteria present. But the fact that similar results are observed whenever
Rhodococcus sp. 094 is injected for long enough times in the micromodels makes it very unlikely that the results are the
result of one of the contaminant organisms.
During the first two days of bacterial injection few changes were observed (Figure 9-a and 9-b). Then on the interval from
71 to 89 hours drastic changes start to occur (Figure 9-c and 9-d). From Figure 9-f changes on the oil drops are clearly
observable. A film of dark color started growing around many drops, eventually changing their appearance completely into a
prune like, or corroded looking surface. The drops were no longer round in shape. This took place over a period of 18 hours,
which is consistent with bacterial exponential growth and with the period of most intensive emulsification (between 16 and
22 hours), as reported by Bredholt, et al.
3
As bacteria get in contact with the dodecane and have favorable conditions for
reproduction they start growing exponentially until population equilibrium is reached or one of the required chemicals are
depleted.
It was also observed that the growth appears to occur following the flow paths, and not another discernible characteristic
like drop size, drop placement, or local wettability. This is consistent with selective blocking mechanisms. The growth occurs
faster where there is more flow of brine, and therefore more flow of bacteria and the nutrients and oxygen needed for this to
grow. When the bacteria reach a drop of dodecane, then all the necessary conditions for its growth are met, and growth will
start earlier, and occur faster at these points in comparison with other areas with less flow.
In some places these black drops seem to have become solid, and stop moving altogether. The most likely explanation is
that bacteria are growing around the droplets, forming biofilm, which in some cases will attach to the surfaces of the
micromodel. This might appear to be counterproductive, but these oil drops were not moving in the first place, and plenty
others are being mobilized, as the flow pattern changes. Furthermore, in some cases some of this oil has been seen being
released (separated from the bacterial mass) after some time, or after an increase in injection rate. We will call this third dark
6 SPE 110134
phase bacterial mass, and it is most likely composed of bacteria cells, and the viscous material (polymeric stabilizer) reported
by Bredholt, et al.
In the experiments where bacteria mass appear, it becomes almost impossible to differentiate between the different
phases, so calculating the saturations and the CDC is no longer possible.
In some oil drops it can be observed how the growth starts in one place in the border, and starts spreading around the
whole drop, changing dramatically its shape, and eventually covering the whole drop (Figure 10-e-2 to 10-g-2). This is also
consistent with the bacteria staying in contact with the dodecane drop as the bacterial population increases. The bacteria are
not able to survive in the oily phase, and therefore needs to remain in the water, while one extreme of the bacterium is in
contact with the dodecane.
Additional proof of the biofilm theory is that after the experiment was finished, remnants of the growth were still in the
micromodel, and special care was necessary to extract it. When it came out from the micromodel, it was confirmed that it was
solid. Also, in experiments reported by Crescente et al
1,2
, bacterial cake was observed, which is also probably composed of
bacteria and biofilm.
The change of shape of the oil drops before any bacterial mass is visible (Figure 9-d-4 to 9-e-4 for example) is likely an
effect of bacteria reducing the interfacial tension.
Changes in wettability from the strongly water wet condition observed during waterflooding to a more oil wet condition
are also observable, for example in Figures 9-a-1 and 9-e-1. This is a case of dodecane becoming in contact with a grain
surface that was previously water wet. Cases where bacterial mass becomes in contact with the grain surface, due to the
sessile characteristic of the bacteria, as seen in Figure 10-g-3 are more complicated, as the bacterial mass is neither water nor
oil, and it is not clear whether its attachment to the grain surface should be accounted as a change in wettability, as is
apparently seen in Figure 10-a-4 to 10-g-4.
A problem with the bacterial mass is that it makes it impossible to measure oil saturation, since the drops are no longer
visible and using the bacterial mass as basis for the calculation overestimates the amount of oil.
It must be stated that an important difference was that a magnetic stirrer was used to agitate the brine with suspended
bacteria inside the bacteria bottles. This was not done on the previous experiments, where no visible changes were observed.
This highlights the importance of oxygen as the limiting factor for bacterial growth, since the stirring allows for oxygen to be
evenly dissolved in the brine with suspended bacteria, and therefore, allows for a continuous supply of oxygen into the
micromodel, and also hinders sedimentation in the bacteria bottles.
Experiment 8 was very similar to Experiment 7 but it was performed on Micromodel 1. Additional care was taken into
keeping the system as sterile as possible. This experiment was carried out mainly to confirm whether the results from
Experiment 7 were reproducible. Despite not having as good contrast as Micromodel 2, and being less realistic, some of the
same effects should be observable. Figures 11-a to 11-e show despite the lower contrast, bacterial mass surrounding residual
dodecane, and changes in wettability. Reduction of the flow area is also clearly visible.

Bacterial flooding with NSPB over prolonged period
Some differences were observed when using NSPB instead of SPB. From the obtained images it is visible that the process
seems to be more evenly spread out, as bacterial mass is seen over a broader area (as can be seen in the area between the
grains marked as 1 4 in figures 12-c to 12-f). The bacterial mass is clearer in color, which probably means it is less dense.
Another important difference is that when NSPB is used, the oil appears to divide in smaller drops at a much higher
frequency than when SPB is used. During NSPB floodings, abundant small drops were frequently seen throughout the
surveillance points of the micromodel.
Experiment 9 was performed on Micromodel 2 with NSPB. During the waterflooding, a rate of 3.5 ml/min was used
during 30 hours to achieve residual oil saturation. The procession from Figure 12-a shows how the oil thins out first (12-b),
then atomizes into smaller drops (12-c), just as bacterial mass starts to grow in some points, and from there the bacterial mass
continues growing and covering the surface of many grains (12-d to 12-f). During the growth of this bacterial mass, though,
some of the remaining oil apparently is released, so in this case the bacterial mass appears to be less effective than that of
SPB in capturing oil drops.
Experiment 10 was also carried out on Micromodel 2. The model was first waterflooded for 20 hours at 2.5 ml/min to
establish residual oil saturation. Then it was flooded with NSPB. Figure 13 shows surveillance point 8 of this experiment.
Once again, division of the remaining oil into small droplets was observed (Figures 13-a to 13-d)

Proposed Mechanism
From these results, considered together with the results from Crescente et al.
1,2
, it is possible to propose a theory of how
Rhodococcus sp. 094 improves the oil recovery. This theory assumes that either SPB or NSPB is used, and that the brine with
suspended bacteria has enough nutrients and oxygen, it is fresh and is agitated so that the bacteria remain suspended and with
enough oxygen throughout the injection:
From the start there is a great difference whether SPB or NSPB is being injected. SPB is hydrophobic and will
tend to form clumps so that each cell has the smallest possible part of its surface in contact with water. This
means that the bacteria travel through the porous space in larger pieces than the NSPB. This has four main
implications: (1) the bacteria will be less able to penetrate in smaller pores (2) it will block the pores quicker (3)
SPE 110134 7
its effects will be more localized, and (4) The effects will be much faster on the places where it occurs, since
more bacteria is present at initial conditions. NSPB, on the other hand travels as loose particles, since its cells are
hydrophilic, this means that: (1) the bacteria are able to penetrate into very small pores (2) pores will be
tightened at a slower pace than with SPB and it will occur through a larger area from the injection point (3) the
effects will be more spread out, and (4) the effects will occur more gradually, and it is likely that an activation
time is required for the NSPB to locally change into SPB upon contact with dodecane in the pore space.
Some activation time is apparently needed for the bacteria to adjust to the new conditions. Bredholt et al.
3

reported that 16 hours had to pass before most of the cells in a culture attached to the oil phase, and that
exponential growth took place over 22 h, and produced 30% of the biomass, and that the most intensive
emulsification took place between 16 and 22 h. Then from 22 to 36 h linear growth occurred, producing 70% of
the biomass. However these results were for a static culture, whereas the micromodels were continuously
flooded with brine containing cells in suspension, the necessary nutrients and dissolved oxygen. Despite these
differences, some time dependency is also observed.
Interfacial tension reduction is visible in many instances from the changes of the interfaces of oil drops/brine, as
oil droplets are seen to start changing in shape and losing the smooth rounded shape they normally have, and
getting an irregularly shaped interface which changes over time, until bacterial mass is seen growing on it.
Unusual changes, such as cut-ins where the oil drops appear to start to get divided are also observed and points
towards a sharp reduction of IFT. Another factor pointing in this direction is the greater incidence of small
droplets (from the division of larger drops) appearing at late stages in the experiments.
The hydrophobicity of the SPB also means that when a group of SPB comes in contact with a drop of dodecane,
their covering of the drop will occur much faster than with NSPB, as each cell will try to have as much of its
hydrophobic surface in contact with dodecane as possible. Another factor contributing to this is that the bacteria
will likely start reproducing much faster as it has a larger initial population, and it is already adapted to
metabolize and reproduce on dodecane. As this process is unfolding, the drop might be cut, and squeezed by the
bacteria both factors that contribute to release additional oil. NSPB on the other hand, will have a smaller
population when it comes in contact with dodecane, and it still needs some time to be able to produce surfactant
that will allow it to attach more efficiently to the dodecane. After this adaptation occurs it will have become
SPB, but more time will have passed and there will be fewer initial cells. Another factor is that since less pore
blocking is likely to occur, the flow is probably more evenly spread out throughout different pore network paths,
which means that fewer nutrients and less oxygen are available at a single growth spot. This slower growth is
probably also related to the fact that the occurrence of small droplets was more common with NSPB than with
SPB, since the bacterial mass is probably covering the oil drops slower than SPB, so the interfacial tension
reduction would have more time to act on, and divide the drops into smaller droplets of dodecane.
From the previous point it can be deducted that SPB will engulf the drops of oil it comes in contact with much
faster than NSPB, increasing the chance of the oil drop being trapped. The bacteria are sessile and it has been
observed in many instances (Figures 10-g-3 to 10-h-3 and 12-d-4 to 12-f-4) that once bacterial mass comes in
contact with grain surfaces, it does not move throughout the rest of the experiment. The drops covered in
bacterial mass are also irregular in shape and can be of considerable size, so in those cases when it is still mobile,
it will easily block a pore throat. NSPB will spread much slower on the surface of the dodecane, giving more
chance of interfacial tension reduction to change the shape and placement of the oil drop, allowing more of the
oil to be released.
It was also observed that biofilm seemed to appear and grow slightly with time around grains, so pore blocking
occurs mostly by reduction of the effective diameter of the pore throat, and less frequently by blocking of a pore
throat by large particles. However, with smaller pore throats the importance of blocking by moving pieces of
bacterial mass should increase.
After increasing the flooding rate it is observed that some dodecane is released from some of the oil drops
covered in bacterial mass (Figure 10-h). It is also seen that some bacterial masses that have attached to the glass
surface in the micromodel will remain attached after further increases. These attached bacterial masses, are later
difficult to remove during the cleaning of the micromodels.
Some wettability change occurs, mostly by bacterial mass attaching to the grain surfaces, but also some instances
of dodecane coming in contact with the grain surfaces. It is not clear, though, whether the surfaces covered by
bacterial mass should be considered a change of wettability.

Conclusions
Glass micromodels have been used to observe the effects of bacteria on remaining oil saturation, allowing to gain
additional insight on the mechanisms through which Rhodococcus sp. 094 is able to improve oil recovery
Bacterial mass, composed of bacteria and bioproducts is visible to the naked eye, and is seen growing in the
interface between dodecane and brine, and with time tends to cover complete drops of oil.
8 SPE 110134
Previously suspected mechanisms: Interfacial tension reduction, wettability changes and pore blocking are
confirmed, but the three are interlinked and seem to be an effect of the bacterial mass.
No gas was observed throughout any of the experiments.
The process appears to have some dependency on time
A method for calculating saturations from micromodel images is developed, and tested.
Saturations are not measurable when bacterial mass appears and makes it difficult to differentiate between the
different phases.
A hypothesis of how the bacteria achieve increased oil recovery is presented, and has numerous coincidences with
the process of utilization of crude oil as a growth substrate by Rhodococcus sp. 094 presented by Bredholt et al.
3


Acknowledgment
We would like to thank SINTEF for providing us with the bacterial strain studied here, Roger Overaa at NTNU for his
valuable help in running the lab and solving the problems that appeared throughout the laboratory work, Gunnar Bjerkan at
NTNU for his help in setting up the video system and solving its failures, and Siri Stavrum at NTNU for preparing the media.

References
1. Crescente, C., Torster, O., Hultmann, L., Strm, A., Rasmussen, K. and Kowalewski, E. An Experimental Study of
Driving Mechanisms in MIOR Processes by Using Rhodococcus sp. 094. SPE 100033. Presented at the 2006
DOE/SPE Symposium on Improved Oil Recovery held in Tulsa, Oklahoma, U.S.A. 22-26 April 2006.
2. Crescente, C., Rasmussen, K., Torster, O., Strm, A. and Kowalewski, E. An Experimental Study of Microbial
Improved Oil Recovery by Using Rhodococcus sp. 094. SCA2005-45. Presented in the SCA Annual Conference in
Toronto, Canada 21-25 September 2005.
3. Bredholt H., Josefsen K., Vatland A., Bruheim P. and Eimhjellen K. Emulsification of crude oil by an alkane-
oxidizing Rhodococcus species isolated from seawater. Can J Microbiol (1998) 44, 330-340.
4. Bryant, R.S. Review of Microbial Technology for Improving Oil Recovery. SPE paper 16646, SPE Reservoir
Engineering Journal, Vol 4, Number 2, May, 1989.
5. Kowalewski, E., Ruesltten, I., Gilje, E., Sunde, E., Bdtker, G., Lilleb, B.L.P., Torsvik, T., Stensen, J..,
Bjrkvik, B. and Strand, K.A.,

2005, Interpretation of Microbial Oil Recovery from Laboratory Experiments,
Paper presented at the 13
th
European Symposium on Improved Oil Recovery, Budapest, Hungary, Apr 25-27.
6. Deng, D., Li, C., Ju, Q., Wu, P., Dietrich, F.L. and Zhou, Z.H., Systematic Extensive Laboratory Studies of
Microbial EOR Mechanisms and Microbial EOR Application Results in Changquing Oilfield, SPE paper 54380,
presented at the SPE Asia Pacific Oil and Gas Conf. and Exhibition, Jakarta, Indonesia, Apr 20-22, 1999.
7. Sunde, E., Beeder, J., Nilsen, R.K., Torsvik, T. Aerobic Microbial Enhanced Oil Recovery for Offshore Use, SPE
paper 24204, presented at the SPE/DOE English Symposium on Enhanced Oil Recovery, Tulsa, Oklahoma, USA,
April 22-24, 1992.
8. Herd, M.D., Lassahn, G.D., Thomas, C.P., Bala, G.A. and Eastman, S.L., Interfacial Tensions of Microbial
Surfactants Determined by Real-Time Video Imaging of Pendant Drops, SPE paper 24206, presented at the Eight
Symposium on Enhanced Oil Recovery, Tulsa, USA, April 22-24, 1992.
9. Sugihardjo, E.H. and Pratomo, S.W. Microbial Core Flooding Experiments Using Indigenous Microbes, SPE paper
57306, presented at the SPE Asia Pacific Improved Oil Recovery Conference, Kuala Lumpur, Malaysia, October 25-
26, 1999.
10. Mu, B., Wu, Z. Chen, Z., Wang, X., Ni, F. and Zhou, J. Wetting Behaviour on Quartz Surfaces by the Microbial
Metabolism and Metabolic Products, paper presented at the 7th International Symposium on Wettability and its
Effect on Oil Recovery, Tasmania, Australia, March 12-14, 2002.
11. Polson, E.J., Buckman, J.O., Bowen, D., Todd, A.C., Gow, M.M. and Cuthbert, S.J., An ESEM investigation into
the effect of microbial biofilms on the wettability of quartz, presented at the 7th International Symposium on
Wettability and its Effect on Oil Recovery, Tasmania, Australia, March 12-14, 2002.
12. Bryant, R.S. Potential Uses of Microorganisms in Petroleum Recovery Technology, Proceedings of the Oklahoma
Academy of Science, Oklahoma, Vol. 67, 1987.
13. Gandler, G.L., Gbosi, A., Bryant, S.L. and Britton, L.N. Mechanistic Understanding of Microbial Plugging for
Improved Sweep Efficiency. SPE 100048. Symposium on IOR. Tulsa, Oklahoma. April 2006.
14. Gullapalli, I.L., Bae, J.H., Hejl, K. and Edwards, A., Laboratory Design and Field Implementation of Microbial
Profile Modification Process SPE paper 60910, SPE Reservoir Eva. & Eng. Vol. 3, No. 1, February, 2000.
15. Yusuf, A., Kadarwati, S., Nurkamelia and Sumaryana, Field Test of the Indigenous Microbes for Oil Recovery,
Ledok Field, Central Java, SPE No 57309, SPE Asia Pacific Improved Oil Recovery Conf., Kuala Lumpur,
Malaysia, Oct 25-26, 1999.
16. Stepp, A.K., Bryant, R.S., Llave, F.M., Evans, D.B. and Bailey, S.A., Microbial Methods for Improved
Conformance Control in Porous Media, SPE paper 35357, SPE/DOE Improved Oil Recovery Symposium, Tulsa
OK, USA, Apr 21-24, 1996.
SPE 110134 9
17. Stepp, A.K., Bailey, S.A., Bryant, R.S. and Evans, D.B., Alternative Methods for Permeability Modification Using
Biotechnology, SPE paper 36747, presented at the 71st Annual Technical Conference and Exhibition, Denver CO,
USA, Oct 6-9, 1996.
18. Udegbunam, E.O., Adkins, J.P., Knapp, R.M., McInerney, M.J. and Tanner, R.S., Assessing the Effects of
Microbial Metabolism and Metabolites on Reservoir Pore Structure, SPE paper 22846, presented at the 66th
Annual Technical Conference and Exhibition, Dallas TX, USA, Oct 6-9, 1991.
19. Nagase, K. Zhang, S.T., Asami, H., Yazawa, N., Fujiwara, K., Enomoto, H., Hong, C.X. and Liang, C.X., A
Successful Field Test of Microbial EOR Process in Fuyu Oilfield China, SPE paper 75238, presented at the
SPE/DOE Improved Oil Recovery Symposium, Tulsa, OK, USA, Apr 13-17, 2002.
20. Lake, L. Enhanced Oil Recovery. Prentice Hall, New Jersey, 1996.


















































10 SPE 110134
























Figure 1. A drop of oil is captured in the porous channel, decreasing IFT can help move the drop

Figure 2. While the wetting phase is continuous, the non-wetting phase is distributed in (1) disconnected drops, which
will tend not to move, and (2) continuous fluid which is easily moveable

Grain
Wetting phase
Non-wetting
phase
1
1
2 2 2
r
2
r
1

P
w1

P
w2
Rock
Rock
Rock
Brine
Oil

= =
2 1
/ 2 1
1 1
2
r r
P P P
w o w w w

Where:
P
w
is the pressure difference across the drop
P
w1
is the pressure behind the drop
P
w2
is the pressure ahead of the drop

o/w
is the IFT between oil and water
r
1
is the curvature radius of the back end of the drop
r
2
is the curvature radius of the front end of the drop

SPE 110134 11

Figure 3. (a) Selective plugging caused by bacterial growth on pore walls. (b) By sudden blocking by circulating bacterial
mass















a) Micromodel 1 b) Micromodel 2

Figure 4. Typical images obtained from both micromodels.

Table 1 Micromodel Characteristics

Parameter Micromodel 1 Micromodel 2
Length (cm) 1.95 7
Width (cm) 1.95 4.3
Depth (cm) 1.50 x 10
-2
1.50 x 10
-2

Porosity () 0.71 0.38
PV (cm
3
) 4.72 x 10
-2
1.72 x 10
-1

Cross sectional area (cm
2
) 3.41 x 10
-2
6.45 x 10
-2

A (cm
2
) 2.42 x 10
-2
2.45 x 10
-2







a
b
c
a
b
c
a
b
c
1
2
3
Legend
Brine
Rock Surface
Bacterial mass
Individual pore flow
Total flow
Circulating bacterial mass
a
a
b
c
a
b
c
1
2
b
12 SPE 110134
























Figure 5. Experimental Setup

Table 2 Growth media
Medium concentration
Component Formula Dodecane Acetate Pre-culture
Ammonium Chloride NH
4
Cl 0.6 g/l 4.6 g/l 4.6 g/l
Bicine [N.N-bis(2-
hydroxyethyl)glycine]
C
6
H
13
NO
4
10.0 g/l 10.0 g/l 10.0 g/l
Magnesium sulfate-7-hydrate MgSO
4
x 7H
2
O 0.05 g/l 0.05 g/l 0.05 g/l
Calcium sulfatedihydrate CaSO
4
x 2H
2
O 0.21 g/l 0.21 g/l 0.21 g/l
Potassium Chloride KCl 0.20 g/l 0.20 g/l 0.20 g/l
Sodium Chloride NaCl 30.0 g/l 30.0 g/l
Phosphate stock solution
1
5 ml/l 5 ml/l 5 ml/l
Trace mineral stock solution
2
5 ml/l 5 ml/l 5 ml/l
Sodium Acetate CH
3
COONa 6.83 g/l 6.83 g/l
Dodecane CH
3
(CH
2
)
10
CH
3
5.0 g/l
All media adjusted to pH 8.3 at 30
o
C before autoclaving.















1
The phosphate stock solution contained a mixture of 1M K
2
HPO
4
3H
2
O and 1M KH
2
PO
4
in a ratio of 8:1.
2
The trace mineral stock solution contained (gl
-1
distilled water): ZnSO
4
7H
2
O 0.5; FeSO
4
7H
2
O 0.5; MnSO
4
5H
2
O 0.5; and
concentrated H
2
SO
4
1.0 mll
-1
.
SPE 110134 13





































Figure 6. Process for calculating the oil saturation

c) Image is changed to black and white to allow
easier treatment by the software
d) The oil area is calculated and by repeating the
process with the grains, the saturation can be calculated
a) Original untreated image b) Magnetic lasso filter allows to easily fill in the oil
drops with white color.
14 SPE 110134

Figure 7. Process for calculating the oil saturation and Capillary Number
SPE 110134 15


















Figure 8-a CDC for experiments 2. 3 and 4 (Micromodel 1)




















Figure 8-b CDC for experiment 5 and 6 (Micromodel 2)




















16 SPE 110134












a) Time = 0 hours. Note the strongly water wet character b) Time = 65 hours. Very little change.













c) Time = 71 hours. Slight changes d) Time = 89 hours. Isolated drops














e) Time = 117 hours. Note cut-ins and irregular shape f) Time = 134 hours. Bacterial mass already visible
of oil drops. probably due to reduced IFT












g) Time = 158 hours. Further desaturation h) t = 185 h. Higher rate. smaller drops
and bacterial mass more visible Darker bacterial mass


Figure 9. Selected images from Experiment 7. point 7
1
2
4
2
1
7
5
6
3
4
2
1
7
5
6
3
4
2
1
7
5
6
3
4
2
1
7
5
6
3
4
2
1
7
5
6
3
SPE 110134 17














a) t = 0 h. Note the strongly water wet character b) t = 71 h. Barely any changes












c) t = 89 h. Slight changes. apparent d) t = 92 h. Thinning of dodecane.
bacterial mass visible further bacterial mass. snap-off












e) t = 112 h. Easily observed bacterial mass f) t = 119 h. Gradual evolution of the interface
Cut-ins and abrupt interface ends. Wettability in very irregular shape. Ever darker bacterial mass
change. Pore blocking.












g) t = 158 h. Further widespread bacterial h) t = 185 h. higher rate. Some of the dodecane
growth is displaced. bacterial mass remains.

Figure 10. Selected images from Experiment 7. point 8
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
1 2
3 4
5
18 SPE 110134













a) t = 0 h. Note the strongly water wet character b) t = 42 h some bacterial mass













c) t = 58 h. Additional bacterial mass d) t = 65 h. apparently wettability changes in some
of the surfaces. Additional bacterial mass












e) t = 84 h. Additional growth. clearer wettability f) t = 105 h. Additional growth. Foreseeable pore
changes blockage if growth continues.

Figure 11. Selected images from Experiment 8. point 4















SPE 110134 19













a) t= 0 h b) t = 24 h















c) t = 96 h d) t = 168 h















e) t = 264 h f) t = 360 h

Figure 12. Selected images from Experiment 10. point 3












1
2 3
4
1
2 3
4
1
2 3
4
1
2 3
4
1
2 3
4
1
2 3
4
20 SPE 110134


















a) t = 0 h. b) t = 96 h.
















c) t = 168 h. d) t = 264 h. Oil drops are divided in smaller drops.

Figure 13. Selected images from point Experiment 9. point 8