IPTC 13861

Estimation of the Potential of an Oil-Viscosity-Reducing Bacteria,

Petrotoga sp., Isolated from an Oilfield for MEOR
Isty A. PURWASENA, Yuichi SUGAI and Kyuro SASAKI, Earth Resources Engineering Department, Kyushu
University Japan

Copyright 2009, International Petroleum Technology Conference

This paper was prepared for presentation at the International Petroleum Technology Conference held in Doha, Qatar, 7–9 December 2009.

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Abstract
One thermophilic anaerobic oil-degrading bacterium was successfully isolated from reservoir brine of Yabase oilfield,
INPEX Corp., Akita, Japan. The potential of the isolated bacterium as a candidate for Microbial EOR (MEOR) was estimated
in this study.
This bacterium was identified as Petrotoga sp. by DNA sequencing analysis. The isolated bacterium can degrade long
chain hydrocarbons in crude oil into shorter chain hydrocarbons in reservoir brine containing 2 (v/v) % of crude oil as a
carbon source and 0.1 g/l of yeast extract as a nitrogen source. As a result, the viscosity of crude oil decreased. The isolated
bacterium can form their bacterial colonies on a solid medium exclusively under the presence of CO2 in gas phase. In
addition, the results of GC-MS analyses of crude oil showed that the isolated bacterium can degrade longer chain of n-
alkanes more selectively under 10 % CO2 atmospheric condition. These results show that CO2 stimulate the growth of the
isolated bacterium and selective degradation of longer chain of n-alkanes by the bacterium.
The suitable nitrogen source for the isolated bacterium was evaluated by reduction of viscosity of crude oil. Ammonium
nitrate, urea, and yeast extract were evaluated for the nitrogen source and added into the medium containing crude oil with 2
(v/v) %. The highest growth rate of the bacterium was observed in the medium containing yeast extract. Viscosity of crude
oil reduced by 40 % of its original viscosity in this case while ammonium nitrate, urea and non-nitrogen source addition gave
38 %, 35 % and 12 % oil viscosity reduction after 3 weeks incubation. 0.05 g/l of yeast extract is enough for the growth of
the isolated bacterium.
The isolated bacterium can grow under high salinity condition such as 90 g/l of NaCl and it can grow under the
temperature between 50 to 80 oC. These results show that the isolated bacterium can be applied to wide range of reservoirs.

Introduction
Microbial enhanced oil recovery (MEOR) is a tertiary oil recovery technique based on the application of biotechnology or
biological activities of microorganisms. In general, effective microorganisms is injected into reservoir with its nutrient
sources and it is incubated in reservoir. Viscosity reduction of crude oil by bio-degradation of crude oil and/or effective bio-
products such as surfactants and polymers lead to the improvement of oil recovery1). Molasses which is produced in sugar
refinery process as a byproduct has been used for a nutrient of microorganisms in many MEOR field trials2). Molasses is
carbon source which digested by the bacteria to generate gases such as hydrogen, carbon dioxide and other chemicals, which
in turn increase the mobility of oil in the reservoir and as a result the flow of oil increases. However, the cost of molasses has
been progressively increased over the past decades due to extensively usage as raw material for bio-ethanol production. For
example, Chinese molasses rose in its price from 40 US dollars at 2000 to 200 US dollars at 20073). In the near future, it is
predicted that the use of molasses in MEOR will be dissatisfied economically.
Within the oil industry, MEOR is desirable as this process could lead to a substantial economical gain. The use of
microorganisms which specifically consume crude oil as its sole carbon source in MEOR process could be one alternative for
molasses substitution. Those microorganisms can attack long chain hydrocarbon of crude oil into shorter chains resulting in
oil viscosity decreased and improved oil mobility4),5). Lazar et al. (1999)4) has been suggested to employ microorganism
which able to reduce the fraction of long chain n-alkanes in biotechnological process for waxy crude oil recovery. The
current identified thermophilic strain NG80-2 of Geobacter thermodenitrificans6) with its capabilities to degrade long chain
n-alkanes from C15 to C36 crude oil into shorter chain may provide an excellent basis for further application leading to
2 IPTC 13861

facilitate oil recovery7). However, to date, no MEOR field application using such microorganism acted as crude oil viscosity
reducer has been described or implemented.
Although a large number of microorganisms with a wide range of physiological properties have been isolated from oil
reservoirs, only very limited number of thermophilic anaerobe oil-degrading bacteria have been reported. Until now, only one
thermophilic anaerobic hydrocarbon degrader potentially capable of living in the deepest degraded reservoirs has been
identified8) and none have been isolated which has been shown to degrade hydrocarbons under conditions found in deep
petroleum reservoirs9).
This study describes the isolation and initial characterization of indigenous thermophilic anaerobe oil-degrading
bacterium which was isolated in Yabase oilfield in Japan. This study also investigated the effect of nitrogen sources and
influences of reservoir conditions such as salinity and temperature on the growth of isolated bacterium dealing with viscosity
reduction of crude oil.

Materials and methods

Brine samples
Reservoir brine was extracted from producing well heads in Yabase oilfield, INPEX Corp., Akita, Japan (Fig. 1).
Reservoir temperature of Yabase oilfield is 70°C. Salinity of the brine in Yabase oilfield is 1 % approximately. Brine was
collected in sterile plastic bottle containing deoxidizer. The bottle was completely filled with the mixture of brine and oil.
They were sealed tightly and transported to our laboratory as soon as possible.

Enrichment cultivation experiments and bacterial isolation

Brine extracted in Yabase oilfield was applied to enrichment culture experiments. Enrichment culture medium consists of
sterile brine supplemented with 0.1 g/l of yeast extract as nitrogen source and 2 % of crude oil as carbon source. Brine
supplemented with yeast extract was sterilized by filtration by using 0.2 µm pore size membrane filter while crude oil was
sterilized by autoclaving. 50 ml of enrichment culture medium was put into 100 ml serum bottles and sealed with butyl
rubber caps and aluminum crimps. The head spaces in the serum bottle were replaced by pure CO2, pure N2, and/or 10 % CO2
(N2 balanced). 0.1 ml of non-sterilized brine was inoculated into the medium and incubated at 50°C. After 1 week later, 0.1
ml of enrichment culture solution was inoculated into new serum bottles containing the flesh medium solidified by 2 %
gellan gum under the same atmospheric condition as above. Some colonies that were formed on the plates were picked and
inoculated into flesh liquid medium.

Analyses of microbial community structure in enrichment culture solution

The liquid culture solution was filtered through a 0.2 µm pore size membrane filter in order to concentrate all the bacterial
cells which inhabit each solution on a filter. Total Bacterial DNA was extracted and purified by using Ultra Clean Soil DNA
Isolation Kit (MO BIO Laboratories, Inc.) according to manufacturer’s instructions. Extracted and purified DNA was
amplified by polymerase chain reaction (PCR) targeting the 16S rDNA using Premix Taq (TAKARA BIO Inc., Japan) and
some primers. The first PCR using universal primers (forward EU27f [5’-AGA GTT TGA TCC TGG CTC AG-3’], reverse
EU1525r [5’-AAA GGA GGT GAT CCA GCC-3’]) amplifies almost all regions of 16S rDNA of Eubacteria. Amplified
DNA in the first PCR was used for the second PCR. The second PCR was performed using primers (forward EU341f-GC
[5’- GC clamp (CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G) -CC TAC GGG AGG CAG
CAG-3’], reverse EU534r [5’-ATT ACC GCG GCT GCT GG-3’]) amplifies a variable region of Eubacteria in 16S rDNA.
The thermal cycle profile for first PCR was as follows: initial denaturation at 94°C for 10 min; 30 cycles of denaturation at
94°C for 1 min, annealing at 52°C for 2 min, extension at 72°C for 2 min; final extension at 72°C for 7 min, and cooling at
4°C. The second PCR is consisted of initial denaturation at 94°C for 10 min; 25 cycles of denaturation at 94°C for 20 sec,
annealing at 55°C for 45 sec, extension at 72°C for 1 min; final extension at 72°C for 7 min, and cooling at 4°C.
PCR products were given the analyses of their microbial community structure by DGGE method. DGGE method was
performed using a D-code universal mutation system (Bio-Rad Laboratories, USA) according to the instruction manual. The
conditions for separation were as follows: running at 75 V for 16 hours in a 10 % polyacrylamide gel with the denaturing
gradient from 30 % to 70 % at 60°C. After electrophoresis, gels were stained with ethidium bromide and the DNA bands
were visualized with an UV transilluminator. Each different nucleotide sequence DNA can be separated on the gels and the
intensity of each DNA band means the dominance of each microbe in a sample. Microbial community structure in a sample
can be estimated by DGGE method.
After DGGE analyses, major DNA bands on the gels were excised by fresh sterile scalpels. Each DNA which was
extracted from each excised gel was sequenced by using MicroSeq 500 16Sr DNA Sequencing Kit (Applied Biosystems Inc.,
USA). Sequence reactions were electrophoresed using the ABI Prism 3130 Genetic Analyzer. Homology searches of the
GenBank (www.ncbi.nlm.nih.gov) DNA database were performed with BLAST web-based software.

Identification of the isolated bacterium

The isolated bacterium was also identified by using molecular biology technique. Bacterial DNA was extracted from
single bacterial colony by using PerpMan Ultra Reagent (Applied Biosystems Inc., USA) according to manufacturer’s
IPTC 13861 3

instructions. Extracted DNA was amplified by PCR targeting the top 500 region in 16S rDNA by using MicroSeq 500 16S
rDNA PCR kit (Applied Biosystems Inc., USA) according to the manufacturer’s instructions. The thermal cycle profile for
the PCR was as follows: initial denaturation at 95 degrees for 10 min; 35 cycles of denaturation at 95 degrees for 30 sec,
annealing at 60 degrees for 30 sec, extension at 72 degrees for 45 sec; final extension at 72 degrees for 10 min, and cooling at
4 degrees. PCR product was purified and directly sequenced by using MicroSeq 500 16Sr DNA Sequencing Kit (Applied
Biosystems Inc., USA). Sequence reactions were electrophoresed using the ABI Prism 3130 Genetic Analyzer. Homology
searches of the GenBank (www.ncbi.nlm.nih.gov) DNA database were performed with BLAST web-based software.

Pure cultivation experiments of isolated bacteria

Pre-incubation was carried out in order to prepare the inoculums. 10 % of bacterial cell culture solution was transferred to
100 ml serum bottle containing 50 ml of sterile brine which was supplemented with 19 g/l of RCM (reinforced clostridial
medium). This bacterial culture solution was incubated at 50 oC for 2 days. 100 ml of culture medium was put into sterile 200
ml serum bottles. The serum bottles were sealed with rubber caps and the headspaces were replaced by pure CO2, 50 % CO2
(N2 balanced), 10 % CO2 (N2 balanced) and/or pure N2. 10 ml of cell suspension was inoculated into the medium and
incubated at 50 oC for 3 weeks. The initial cell concentration in pure cultivation experiments was adjusted to 1.0x106 CFU/ml
approximately. Uninoculated blank was set and incubated in parallel. Bacterial cell numbers were counted directly by using a
phase difference microscope occasionally during the experiment. The pH of culture solution was measured by using compact
pH meter (HORIBA Inc., Japan) and viscosity of crude oil was also measured by using a viscometer (Programmable
Rheometer DV-III, Brookfield, USA) at 50 oC.

Gas chromatography-mass spectroscopy (GC-MS) analysis

Biodegradation of crude oil was estimated by GC-MS (QP5050A, Shimazu, Japan) analysis of crude oil after the
incubation. DB-5 silica capillary column (Agilent Technologies, Inc., USA) was used for the analyses. Helium gas was used
as a carrier gas with the flow rate of 1.5 ml/min. Temperature of coloum oven was increased from 60 oC to 300 oC with the
rate of 5oC/minute and maintained at final temperature for 30 minutes. Quantification of each alkane was based on the peak
area compared to pristane which was regarded to be a hard-to-degrade alkane by microorganisms compare to other alkanes.

Optimization of nitrogen source and influence of salinity and temperature for isolated bacteria
The suitable nitrogen source for the isolated bacterium was evaluated by reduction of viscosity of crude oil. Ammonium
nitrate, urea, and yeast extract were evaluated for the nitrogen source and added into the medium with 0.03 g/l, 0.025 g/l, and
0.1 g/l respectively in order to adjust the same concentration of nitrogen (∼10 %) in each medium. The serum bottles were
sealed with rubber caps and the headspaces in the bottles were replaced by 10 % CO2 (N2 balanced). 10 % inoculum was
inoculated into 100 ml serum bottle containing 50 ml of brine including 1 ml of crude oil and each nitrogen source. 10 %
inoculum was also inoculated into the medium without nitrogen source as a control. Uninoculated blank was also set and
incubated in parallel. The isolated bacterium was incubated at 50 oC for 14 days. Cell concentration and pH of culture
solution were measured periodically every 48 hours while oil viscosity was measured once in a week during the incubation.
The optimum concentration of the suitable nitrogen source was evaluated by changing the amount of the nitrogen source.
Influence of salinity on the growth of the isolated bacterium was estimated by incubation experiments by using the
medium containing a nitrogen source with the optimum concentration and 10 g/l, 30g/l, 60 g/l, and 90 g/l of NaCl. Influence
of temperature on the growth of the isolated bacterium was estimated by incubation under 50 oC, 60 oC, 70 oC, and 80 oC by
using the medium containing a nitrogen source and NaCl with the optimum concentration.

Results and Discussion

Isolation and identification of bacteria

One strain of thermophilic anaerobic bacteria which can utilize hydrocarbon as a carbon source was obtained from an
enrichment culture solution under the atmospheric condition of pure CO2. The isolated bacterium formed tiny whitish colony
on the solid medium. As determined by microscopic observation, it is a rod-shaped bacterium of 0.5-0.7µm width and 2.0-7.0
µm length in size with an outer sheath-like structure (toga), occurring singly or in sheaths (Fig. 2). This the isolated
bacterium was identified as Petrotoga halophilla, P. mexicana, and P. mobilis indicating the isolate belonged to the genera of
Petrotoga with 92 % homologous. Olliver and Cayol, 2005, revealed that Petrotoga species found exclusively oil reservoirs,
and it has been suggested that they are native to these environments10). Oil biodegradation study of Petrotoga sp. only
focused on n-alkanes. Those compounds were selected as representative hydrocarbon for its characteristic as either the major
constituents of most crude oil6) or the most biodegradable compounds under anoxic condition11).
DGGE profiles of enrichment culture solutions which were inoculated to the solid mediums are shown in Fig. 3.
Petrotoga sp. was detected exclusively from culture solution which was incubated under pure CO2 condition. It is assumed
that CO2 stimulate the growth of Petrotoga sp. while there are no previous reports which discuss about the influence of CO2
to the growth of Petrotoga sp.
4 IPTC 13861

Degradation of crude oil by the isolated bacterium

Reduction of each n-alkane after 14 days incubation is shown in Fig. 4. Heavier hydrocarbon fractions of C27-32 under
10 % CO2 was reduced selectively to 50 % or less in comparison with control, while this fractions in other treatments reduced
slightly. It is assumed that the selective degradations of heavier hydrocarbons are stimulated by the presence of CO2.
Reduction of hydrocarbon fractions of C14-20 was observed under pure N2, 50 % CO2, and pure CO2. In contrast, increment
of hydrocarbon fractions of C21-26 was observed in all treatments. Significant alteration of lighter hydrocarbon fractions of
C14 or less was not observed in comparison with control except pure N2 treatment.

Optimum nitrogen source for the growth of the isolated bacterium

Ammonium nitrate, urea, and yeast extract were evaluated as nitrogen source to stimulate the growth of the isolated
bacterium. Growth curve of the isolated bacterium in each medium was shown in Fig. 5. The isolated bacterium can grow
well without lag phase in all mediums. The highest bacterial growth rate was observed in the medium including yeast extract
as a nitrogen source. Bacterial growth rate of brine medium and each medium including yeast extract, ammonium nitrate, and
urea were 0.056 h-1, 0.154 h-1, 0.149 h-1, and 0.055 h-1, respectively. The maximum cell concentration in the medium
including yeast extract is 10 times higher than that in other mediums. These results show that yeast extract is the most
suitable nitrogen source to stimulate the growth of the isolated bacterium. In addition, the isolated bacterium can grow in
brine medium which is its original habitat. According to previous experiments, the isolated bacterium neither can grow in
RCM medium nor synthetic brine medium which are made from ion exchange water (data not shown). According to these
results, the isolated bacterium can utilize some nitrogen sources which are contained in the brine and/or crude oil. Table 1
shows the elemental composition of brine which was used in this study. Brine contains little nitrogen source, therefore, it is
assumed that the isolated bacterium utilizes some nitrogen sources in crude oil. Many studies reported that there are some
nitrogen sources in high molecular weight fraction of crude oil as aromatic nitrogen-containing compounds such as non basic
pyrrolic and carbazole12).
The effect of each nitrogen source on viscosity reduction of crude oil is shown in Fig. 6. Viscosity reductions of crude oil
were observed in all the treatments in comparison with blank. In particular, significant reduction of viscosity of crude oil was
observed in the medium which includes each nitrogen source during the incubation. The highest reduction of viscosity of
crude oil was observed in the medium including yeast extract as a nitrogen source and the viscosity of crude oil in such
medium decreased approximately 40 % compared to that in control after 3 weeks incubation. According to the results of GC-
MS analysis, in case of CO2-10 % condition, the isolated bacterium can degrade heavier hydrocarbon in crude oil. Growth of
the isolated bacterium was stimulated by yeast extract and bio-degradation of long chain hydrocarbons were also stimulated.
As a result, the viscosity reduction of crude oil was stimulated. In addition, the viscosity of crude oil decreases significantly
during the logarithmic growth phase. It is considered that the isolated bacterium attacks long chain hydrocarbons into shorter
chain hydrocarbons actively during their logarithmic growth phase. According to these results, it is expected that yeast extract
is quite effective as a nitrogen source for the stimulation of bacterial growth so that the viscosity reduction of crude oil is also
stimulated.
The optimum concentration of yeast extract for the growth of the isolated bacterium was estimated by pure cultivation
experiments by using the culture mediums which contain 0.05 g/l, 0.1 g/l, 0.2 g/l and 0.5 g/l of yeast extract. Growth curves
of the isolated bacterium in each medium were shown in Fig. 7. The isolated bacterium grow well in all treatments. It was
observed that the bacterial growth rate became faster by increase of yeast extract. In case of the highest concentration of yeast
extract, the bacterial growth rate was 0.92 h-1 while the growth rates were 0.81 h-1, 0.76 h-1, and 0.71 h-1 in 0.2 g/l, 0.1 g/l, and
0.05 g/l of yeast extract respectively. Although the bacterial growth rate was quite different, the maximum bacterial cell
number was almost similar among each treatment. Therefore, 0.05 g/l of yeast extract could be the most appropriate
concentration for stimulating the growth of the isolated bacterium. 0.05 g/l of yeast extract was put into the medium in
following experiments.

Influence of salinity and temperature for the growth of the isolated bacterium
Growth curves of the isolated bacterium under the different salinity were shown in Fig. 8. The growth rate of the isolated
bacterium became lower by increase of salinity. In case of the lowest salinity, which is original salinity of the brine, bacterial
growth rate was 0.66 h-1 while the growth rates were 0.49 h-1, 0.31 h-1, and 0.22 h-1 in 30 g/l, 60 g/l, and 90 g/l of salinity
respectively. Growth of the isolated bacterium becomes better with the decrease of salinity, however, the isolated bacterium
can grow even under higher salinity such as 60 g/l and 90 g/l. Therefore, it is expected that the isolated bacterium can be
applied to not only low-salinity reservoirs but also high-salinity reservoirs for MEOR.
Growth curves of the isolated bacterium under the different temperature were shown in Fig. 9. The growth rate of the
isolated bacterium became lower by increase of temperature. In case of the lowest temperature, bacterial growth rate was 0.66
h-1 while the growth rates were 0.48 h-1, 0.25 h-1, and 0.15 h-1 in 60 oC, 70 oC, and 80 oC respectively. Growth of the isolated
bacterium becomes worse with the increase of temperature significantly, however, it can be improved by adding more yeast
extract. Growth curves of the isolated bacterium under higher temperature and different concentration of yeast extract were
shown in Fig. 10. In case of both 70 oC and 80 oC, growth rate of the isolated bacterium in the medium containing higher
IPTC 13861 5

yeast extract became higher than that in the medium containing lower yeast extract. In particular, in case of 70 oC, the
maximum cell concentration in the medium including 0.1 g/l of yeast extract reached 10 times higher than that in another
medium. These results show that the isolated bacterium can be applied to high-temperature reservoirs for MEOR by adding
more amount of nitrogen source.

Conclusions
1) One thermophilic anaerobic oil-degrading bacterium was isolated from brine which was extracted in Yabase oilfield,
Akita, Japan. According to the sequencing analysis, the isolated bacterium was identified as a Petrotoga sp.
2) Petrotoga sp. was detected exclusively from enrichment culture solution which was incubated under pure CO2 condition.
It is assumed that CO2 stimulate the growth of Petrotoga sp..
3) According to the GC-MS analyses, the isolated bacterium can degrade long chain hydrocarbons in crude oil selectively
under 10 % CO2 condition. It is assumed that the selective degradations of heavier hydrocarbons are stimulated by the
presence of CO2. Viscosity reduction of crude oil is led by above degradation and it reduced by 40 % of its original
viscosity.
4) Yeast extract was the most suitable nitrogen source to stimulate the growth of the isolated bacterium and 0.05 g/l of yeast
extract is enough for the growth of the isolated bacterium. It is assumed that the isolated bacterium can utilize some
nitrogen sources which are contained in brine and/or crude oil.
5) Growth of the isolated bacterium becomes better with the decrease of salinity, however, the isolated bacterium can grow
even under higher salinity such as 60 g/l and 90 g/l. Therefore, it is expected that the isolated bacterium can be applied to
not only low-salinity reservoirs but also high-salinity reservoirs for MEOR.
6) Growth of the isolated bacterium becomes worse with the increase of temperature significantly, however, it can be
improved by adding more yeast extract. Therefore, the isolated bacterium can be applied to reservoirs whose temperature
is 50-80 oC.

Acknowledgements
We gratefully acknowledge Global COE Kyushu University for financial support. The authors are also grateful to the INPEX
Corporation in Japan for their support and cooperation in providing the samples.

References
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occurring selectively isolated bacteria for inhibiting paraffin deposition”. J. Petrol. Sci. Eng.,vol. 22,pp. 161-169, 1999.
5. Smith, T.L.,and Trebbau, G.: “MEOR treatments boost heavy oil recovery in Venezuela”, Petrol. Eng. Int., July, 45-50,1998.
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thermophilic Bacillus strain degrading long-chain n-alkanes”, Extremophiles, vol. 10, 347-356, 2006.
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Sci USA 104: 5602–5607.2007.
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oil by new types of sulfate-reducing bacteria”. Nature 372:455–458. 1994.
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71–88. Edited by B. Ollivier & M. Magot. Washington, DC: American Society for Microbiology, 2005.
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6 IPTC 13861

Yabase oilfield, INPEX Corp.

Tokyo

10 µm

Fig. 1 Location map of Yabase oilfield in Japan Fig. 2 Micrograph of isolated bacterium under 600x magnification
CO2 100 %
CO2 10 %
N2 100%

12
Pure N2 10% CO2
50% CO2
Percentage of remaining n-alkanes (%)

10 Blank
Pure CO2

Thermotoga sp. 8

Petrotoga sp. 6
Moorella sp.
Geobacter sp.
4

0
< 14 14 - 20 20 - 26 27 - 32
Carbon number distribution of n-alkanes in crude oil

Fig. 3 DGGE profile of enrichment Fig. 4 Percentage of n-alkanes remaining in crude oil after 2 weeks
culture of Yabase brine incubation of isolated bacterium
IPTC 13861 7

109
30

Bacterial Cell Concentration (cells/ml)

Yeast extract Control
25

Viscosity of crude oil (cP)

108 20
Control Urea

15

107 Ammonium nitrate

10
Ammonium nitrate
Yeast extract
Urea
5

106
0 5 10 15 20 25 0
0 5 10 15 20 25
Incubation time (days) Incubation time (days)
Fig. 5 Bacterial growth curve under different nitrogen source Fig. 6 Viscosity of crude oil under different nitrogen sources

109 109
0.2g/l 0.5g/l

Bacterial Cell Concentration (cells/ml)

Bacterial Cell Concentration (cells/ml)

3% 1%

108 108
0.05g/l
6%
0.1g/l

9%
107
107

106
106 0 5 10 15 20 25
0 1 2 3 4 5 6 7
Incubation time (days) Incubation time (days)
Fig. 7 Bacterial growth curve under different concentration of yeast extract Fig. 8 Bacterial growth curve under different salinity

109
109
Bacterial Cell Concentration (cells/ml)
Bacterial Cell Concentration (cells/ml)

50oC 70oC, 0.1g/l

108
108 80oC, 0.1g/l

60oC
80oC, 0.05g/l
107
107
70oC, 0.05g/l
o
70 C
80oC
106
106 0 2 4 6 8 10 12 14 16
0 5 10 15 20 25
Incubation time (days)
Incubation time (days)
Fig. 10 Bacterial growth curve under higher temperature with
Fig. 9 Bacterial growth curve under different temperature different concentration of yeast extract
8 IPTC 13861

Table 1. Elemental composition of brine used in the culture medium

Element Concentrations(mg/L)
+
Na 3075
K+ 30
2+
Ca 5
Mg2+ 3
-
Cl 2500
I- 1.5
+
NH4 15
HBO22- 250
T-FE 2
-
HCO3 4000