SPE 138135

Research for Microbial Conversion of Residual Oil into Methane in Depleted

Oil Fields to Develop New EOR Process
Haruo Maeda, Yoshihiro Miyagawa , Masayuki Ikarashi : INPEX Corporation, Daisuke Mayumi, Hanako Mochimaru,
Hideyoshi Yoshioka, Susumu Sakata: AIST, Hajime Kobayahsi, Hideo Kawaguchi, Kozo Sato: University of Tokyo

Copyright 2010, Society of Petroleum Engineers

This paper was prepared for presentation at the Abu Dhabi International Petroleum Exhibition & Conference held in Abu Dhabi, UAE, 1–4 November 2010.

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
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Abstract
We are trying to develop a methane-producing system using indigenous microbes in depleted oil fields as a new microbial
enhanced oil recovery process. In particular, we aim to combine a microbial conversion of the residual oil into methane with the
geological sequestration of carbon dioxide. The mechanism is as follows: Hydrocarbon-degrading bacteria are harnessed to
produce hydrogen and/or acetate from residual oil in the depleted oil reservoir. Then, methane-producing microbes (methanogens)
utilize the produced acetate or hydrogen and carbon dioxide, which is injected for geological sequestration, to generate methane.
We successfully isolated hydrogen- and methane-producing microbes (hydrogen-producing bacteria and methanogens) from oil
fields (Yabase and other oil fields) in Japan. Our analysis of microbial cultures incubated under high temperature and high
pressure, the condition similar to in situ petroleum reservoir conditions, revealed that indigenous microbes in the reservoir brine
are capable of generating methane by utilizing crude oil and carbon dioxide. Consumption/production rate of gases (methane and
carbon dioxide) and acetic acid indicated that the methane production under reservoir conditions is likely mediated through two
major pathways; the acetoclastic (acetic-acid utilizing) and the hydrogenotrophic (hydrogen and carbon-dioxide utilizing)
pathways. Furthermore, by analyzing methane-producing ability of isolated microbes, we found that the syntrophic
cooperation between hydrogen-producing bacteria and methanogens was critical for the methane producing under the reservoir
condition.
0%.tures with carbon dioxideent Strikingly, addition of carbon dioxide accelerated methane production of the cultures. The
methane production rate of the cultures, in which high concentration (10%) of carbon dioxide was supplied into the head spaces,
was 0.30 mmol/L/Day. On the other hand, the cultures without the addition of carbon dioxide showed the methane production rate
of 0.12 mmol/L/Day, significantly slower (ca. 40%) than the production rate of the cultures with carbon dioxide. These results
suggested that addition (injection) of carbon dioxide into reservoirs might accelerate the microbial methane production.
We further investigated the methanogenic communities and pathways in petroleum reservoirs by incubating the reservoir brine
from the Yabase oil field, combined with radiotracer experiments and molecular biological analyses. The brine samples were
incubated without exogenous-nutrient supplementation under the high-temperature and high-pressure condition (the in-reservoir
condition). The radiotracer analysis (using 14C-biocarbonate and 14C-acetate) indicated that the methane production rate of
hydrogenotrophic methanogenesis was 50-fold higher than that of acetoclastic methanogenesis, suggesting dominance of methane
production by syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis in reservoir.
In this study, we assessed the rate of oil biodegradation coupled with methanogenesis by using 14C-labeled toluene and
hexadecane as tracers. The analysis revealed that the rate was very low, being only about one thousandth of that of the
hydrogenotrophic methanogenesis. We are currently trying to enhance the crude-oil biodegradation for effective conversion of
crude oil to methane. Our goal is to establish effective microbial conversion system from residual oil into methane in depleted oil
fields as a new EOR technology.
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Introduction
The increased concentration of atmospheric carbon dioxide is considered as a major cause of the global warming. Therefore, the
subsurface Carbon dioxide Capture and Storage (CCS) technology, which is currently being developed around the world, could
become a practical countermeasure to reduce emission of the greenhouse gas into the atmosphere. It has been proposed that
depleted gas and oil reservoirs are employed as storage sites for carbon dioxide. Oil and gas fields have reservoir formations with
impermeable cap rocks, which has been stably storing gas and liquids over several million years and is therefore expected to
prevent leakage of stored carbon dioxide.
On the other hand, natural gas (methane, as the main component for fuel) is an environmentally friendly and highly demanded
fuel. Hence, natural gas consumption has risen significantly on a worldwide basis, and the development of new sources of natural
gas will become increasingly valuable in future. 20-65% of the earth’s methane deposit is estimated to be biogenic2. The duration
of gas generation and contribution of real-time biogenic methane formation (methanogenesis) are not well understood, however. In
this study, we aim to develop a technology, which can solve both of those issues (reduction of carbon dioxide emissions and
development of new natural gas sources) simultaneously, as a new EOR method.
The reactions of microbial conversion of crude oil (assuming as alkanes) to methane is proposed as follows:
Acetic acid producing reaction from alkanes
CnH2n+2 + nH2O → n/2CH3COOH + (n+1) H2
Hydrogen producing reaction from acetic acid
CH3COOH + 2 H2O → 4 H2 + 2CO2
Methane producing reaction from acetic acid
CH3COOH → CH4 + CO2
Methane producing reaction from hydrogen and carbon dioxide
CO2 +4 H2→ CH4 + 2 H2O
In petroleum reservoirs, where electron acceptors (such as oxygen, iron, nitrate and sulfate) are generally limited, methanogenic
conditions predominate. Under such condition, heterotrophic metabolism of bacteria is mostly accomplished by symbiotic
cooperation between bacteria and methanogenic microorganisms. Methanogenic microorganisms (methanogens) are obligate
anaerobes that produce methane as a metabolite. Methanogens are able to utilize a limited number of compounds with relatively
simple chemical structures (such as hydrogen plus carbon dioxide, acetate, formate, methanol and methylamines) as substrates.
The bacterial partners are capable of catalyzing degradation of complex organic matter(s) (such as alkanes) into, ultimately, CO2
and H2. Due to thermodynamic constraints, however, the reaction can occur and provide energy for the bacteria only if the
hydrogen and/or other metabolites are eliminated as soon as they are produced, by the methanogens.
Although the occurrence of both hydrogenotrophic and acetoclastic methanogens has been reported in the brines from many oil
fields, it is yet to be clarified which methanogenic pathway is dominant in a high-temperature petroleum reservoir. To address this
issue, we collected the reservoir brine and crude oil from a production well in Yabase oil field (Japan) (Fig. 1), and constructed
three microcosms only comprising the brine and oil (YAB1, 2, and 3) as follows.
The brine containing indigenous microbes was injected into the sterile stainless-cylinder bottles (1000 ml vessel volume) with
valves on both up and down sides of the bottle. After adding 8 ml of the crude oil, the headspace was flushed and pressurized with
nitrogen gas to 5 MPa.
The bottles were incubated at 55°C in the air bath, simulating the actual oil reservoir conditions. Aliquots of the incubated brine
samples were measured for methanogenic rates by radiotracer experiments and also analysed for the microbial communities by
molecular methods. The results (details to be published elsewhere 4) are briefly described as below.

Methane production and acetate degradation under high-temperature and high-pressure conditions
In all three microcosms, the methane concentration in the head spaces began to increase from day 20, and increased steadily with
time until day 96 (Fig 2). The acetate concentrations in the brines decreased from 8.83 mM (the initial concentration of acetic acid
in the brine) simultaneously with the methane production. In the microcosm YAB1, concentrations of methane and acetate reached
to 7.90 mM and 0.58 mM, respectively, on day 140, when methane production appeared to be ceased. Although we continued to
incubate the samples for further 90 days, methane production was no longer observed (data not shown). The consumed acetate
(8.25 mM) was nearly equivalent to the methane produced, suggesting that most of the produced methane was derived from
acetate. Although we monitored H2 and CO2 concentrations in the samples, no H2 could be detected (detection limit: 0.1 mM), and
CO2 was consistently at low concentration (average 0.32 mM; data not shown). Similar results were observed in the other
microcosms.

Evaluation of methane production rates from radiotracer experiments

In acetoclastic methanogenesis, the methyl group of acetate is directly converted into methane. Thus, we directly evaluated the
rate of acetoclastic methanogenesis in the samples by radiotracer experiments using [2-14C]-acetate.
SPE 138135 3

In the incubated brine from YAB2 (day 96), the rate of acetoclastic methanogenesis was relatively low (1.63 nmol/l/day). On the
other hand, by adding [14C]-bicarbonate to the same brine, we found the rate of hydrogenotrophic methanogenesis to be distinctly
higher (112.3 nmol/l/day) (Table 1). This result suggested that, in the oil reservoir, methane was produced from acetate via CO2
reduction (syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis). It is well known that, in syntrophic
acetate oxidation, the hydrogen concentration remains low, which can explain our observation that hydrogen was consistently
undetectable in the samples during incubation experiment.

Microbial diversity analysis

In order to evaluate microbial basis of methanogenesis in the cultures, the brine sample from YAB3 at day 140 was analyzed for
phylogenetic diversity of microorganisms (Table 2). For methanogens, Methanothermobacter, a representative of
hydrogenotrophic methanogenesis, was a dominant species. For bacteria, Thermacetogenium was detected as an dominant species
in the sample. Thermacetogenium is a syntrophic acetate-oxidizing bacterium, which is able to grow on acetate in association with
hydrogenotrophic methanogens.

All the above results consistently showed that syntrophic acetate oxidation coupled to carbonate reduction was the main pathway
of methanogenesis in the microcosms. It is unclear, however, how the acetate was produced. One possible process for acetate
production in the brine would be the biodegradation of petroleum hydrocarbons. To examine such possibility, we conducted
radiotracer experiments using 14C-labeled hydrocarbons in this study. We further examined the effect of exogenous nutrients
supplementation on the methanogenic and oil-biodegradation rate by microorganisms indigenous to the reservoir.

Materials and Methods

Collecting of microbes inhabiting oil fields
The reservoir brine and crude oil were collected from the well AR-80 of the Yabase oil field (Fig. 1). The Yabase oil field is one
of the oldest and largest onshore oil fields in Japan, located in Akita Prefecture (39°42′N, 140°5′E). Currently, the oil field is
almost depleted and producing by high overall water cut (about 90% BS&W). The main oil-bearing formation is tuffaceous
sandstone of Miocene-Pliocene age. At the well AR-80, pumping unit has been used to produce crude oil along with formation
water. The depth of the oil horizon ranges from 1293 to 1436 m, with in situ temperature 40-82°C and pressure 5 MPa. Water
injection for enhance oil recovery has never been applied at or around this well.
Produced water and crude oil were collected at the well head by discharging the fluid mixture through a metal tube into gas-tight
glass bottles, which was flushed in advance with nitrogen gas. Immediately before each bottle was sealed, the headspace was
flushed again with nitrogen gas to minimize exposure of the samples to oxygen. The samples were kept at ca. 50°C for three days
until they were used for the incubation experiments. Concentrations of chemical components in the reservoir brine4 are shown in
Table 3. The sulfate and nitrate levels were low, indicating that a methanogenic condition predominate in the reservoir.
The concentrations of organic-acids in the brine4, determined by HPLC analysis, are shown in Table 4. Among organic acids,
only acetic acid was detected at high concentration in the brine.

Measurements of methanogenesis rates from petroleum hydrocarbons

The rates of methanogenesis coupled with oil biodegradation were determined by radiotracer experiments using 14C-labeled
substrates. The incubated brine was collected from microcosms YAB2 (day 96) and YAB3 (day 140) into sterile 1-L gas-tight
glass bottles, and then 10-ml aliquots of water from each microcosm and 0.1 ml of crude oil were transferred into sterile 30-ml
vials flushed in advance with nitrogen gas. [1-14C] hexadecane was injected into the vials containing the brines from YAB2 and
YAB3, while [ring-14C(U)] toluene was injected into the vials containing the brines from YAB3. Each set of vials with the same
water and radioisotope was divided into one time-zero control vial and triplicate vials for each of specific incubation periods (10,
20, 40, and 60 days for YAB2 amended with [1-14C] hexadecane; 28, 56, and 89 days for YAB3 amended with [1-14C] hexadecane
and with [ring-14C(U)] toluene). The vials were incubated at 55°C and atmospheric pressure. Time-zero controls and all
incubations were terminated by injection of 5 ml of 1M NaOH.
Production of 14CH4 was determined as described by Yoshioka et al. (2009)11. The effluent gas from the vials amended with
[ring-14C(U)] toluene was pretreated with a Molecular Sieve 5A column to remove the toluene, which otherwise might be
combusted to yield 14CO2.

Enhancement of oil biodegradation and methanogenesis by supplementation of exogenous nutrient

The brines from the well AR-80 were aliquoted into 250-ml serum bottles (75 ml of the water samples per each bottle), following
addition of 0.4% (w/v) NaHCO3 and 0.025% (w/v) L-cysteine HCl for a buffing and reducing agents, respectively. 1.0% (w/v)
yeast extract was added to the samples #3, 4, 7, 8, 11, and 12. To examine the effect of petroleum compounds on methane
production, 750 μl of crude oil (collected from the Yabase oilfield), a mixture of n-alkanes and toluene [comprising 25% (w/w)
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tetradecane, 25% (w/w) hexadecane, 25% (w/w) octadecane and 25% (w/w) toluene], or distilled water (for control) were also
added, respectively (samples #1-4 with crude oil; samples #5-8 with the alkane-toluene mixture; samples #9-12 with distilled
water). The bottles were sealed with thick rubber stoppers and incubated anaerobically [N2 (for samples #2, 4, 6, 8, 10, 12) or N2-
CO2 (80:20, v/v) (for samples #1, 3, 5, 7, 9, 11, 13), 0.2 MPa] at 55°C without agitation. (shown in Fig.3) Two types of anoxic
gases [N2 and N2-CO2 (80:20, v/v)] were used to fill the head spaces of the bottles to examine the effect of exogenous addition of
CO2 on the microbial activity.

Results and Discussion

Rates of methane production associated with oil biodegradation assessed by radiotracer experiments
The production rates of methane from hexadeacane and toluene assessed by radiotracer experiments are shown in Table 5. The
production rate of methane from hexadecane in the incubated brine from YAB 2 at day 96 (0.08 nmol l-1 d-1) was very low, being
about one thousandth of the rate of hydrogenotrophic methanogenesis (112 nmol l-1 d-1) in the same brine. The rates of methane
production from hexadecane and toluene in the incubated brine from YAB 3 at day 140 were similarly low. These results indicated
that the conversion of crude oil into methane (methanogenic biodegradation of crude oil) was a very slow process under the
reservoir condition in nature. This was consistent with our geochemical analysis, which showed the crude oil in the Yabase oil
field was not subjected to biodegradation.

Enhancement of oil biodegradation and methanogenesis by supplementation of exogenous nutrient

In an effort to enhance methanogenic biodegradation of crude oil for EOR, the effect of exogenous nutrient on the metabolic
activity of the indigenous microorganisms was assessed. In microbial EOR (MEOR) process, exogenous nutrient(s) were injected
into the reservoir to promote microbial propagation as well as production of metabolites (such as gases, acids, biopolymers and
biosurfactants) within the reservoir. We hypothesized that supplementation of nutrient(s) might activate microbial activity, which
was involved in crude-oil biodegradation.
As an experimental model of exogenous nutrients, yeast extract was added to the samples #3, 4, 7, 8, 11, and 12 (Fig. 3). To
examine the effect of petroleum compounds on methane production, crude oil and a mixture of n-alkanes and toluene were also
added, respectively (samples #1-4 with crude oil; samples #5-8 with the alkane-toluene mixture; samples #9-12 with distilled water
for negative control: Fig. 3).
Production of methane from the samples was monitored using a gas chromatography at 117, 224, 330 and 419 days. In all
samples, active production of CH4 (increased concentration of CH4 in the head spaces of the bottles) was observed. Generally, the
samples with yeast extract (#3, 4, 7, 8, 11 and 12 in Fig. 3) produced larger amount of methane than the samples without nutrient
supplementation (#1, 2, 5, 6, 9 and 10 in Fig. 3) did, indicating that addition of the nutrient to the formation water could induce
methane production by indigenous microorganisms. The difference of the head-space gases [N2 or N2-CO2 (80:20, v/v)] did not
significantly affect the gas production, suggesting that addition of exogenous CO2 did not inhibit methane-producing activity of
reservoir microorganisms.
Among the yeast-extract-supplemented samples, the samples supplied with the petroleum compounds (crude oil and the alkanes-
toluene mixture) (#3, 4, 7 and 8 in Fig. 3) produced higher amount of methane than the samples without the petroleum compounds
(#11 and 12 in Fig. 3). However, no obvious effect of petroleum compounds was observed in the samples without yeast-extract
supplementation. These observations suggested that exogenous nutrients (in this experiment, yeast extract) induced microbial
activity to utilize petroleum compounds as the substrate for methanogenesis (methanogenic degradation of petroleum compounds).

Conclusions
(1) Radiotracer experiments adding 14C-labeled hexadecane or toluene to the incubated reservoir brines indicated that the
methanogenic biodegradation of crude oil was a very slow process under the reservoir condition
(2) Supplementation of exogenous nutrients can potentially induce microbial activity to utilize petroleum compounds as the
substrate for methanogenesis (methanogenic degradation of petroleum compounds).

Acknowledgement
The authors wish to thank the management of INPEX/U of TOKYO/AIST for permission to present the results of this research.

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