Bioresource Technology xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Development of a microbial process for the recovery of petroleum oil

from depleted reservoirs at 9196 C
Preeti Arora, Dilip R. Ranade, Prashant K. Dhakephalkar
Microbial Science Division, Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India

h i g h l i g h t s
 Hyperthermophilic, alkalophilic, halophilic bacteria enriched from oil well.
 Bacterial consortium enhanced oil recovery at 96 C in core ood studies.
 96 C is the highest temperature reported for Microbial Enhanced Oil Recovery.
 Microbial metabolites aiding oil recovery at 96 C were identied.

a r t i c l e

i n f o

Article history:
Received 10 January 2014
Received in revised form 12 March 2014
Accepted 15 March 2014
Available online xxxx
Keywords:
Microbial Enhanced Oil Recovery (MEOR)
Sand pack
Core ood
Hyperthermophile

a b s t r a c t
A consortium of bacteria growing at 91 C and above (optimally at 96 C) was developed for the recovery
of crude oil from declining/depleted oil reservoirs having temperature of more than 91 C. PCR-DGGESequencing analysis of 16S rRNA gene fragments of NJS-4 consortium revealed the presence of four
strains identied as members of the genus Clostridium. The metabolites produced by NJS-4 consortium
included volatile fatty acids, organic acids, surfactants, exopolysaccarides and CO2, which reduced viscosity, emulsied crude oil and increased the pressure that facilitated displacement of emulsied oil towards
the surface. NJS-4 enhanced oil recovery by 26.7% and 10.1% in sand pack trials and core ood studies
respectively in optimized nutrient medium comprised of sucrose and sodium acetate as carbon/energy
source and urea as nitrogen source (pH 79, 96 C, and 4% salinity). Nutrient medium for MEOR was
constituted using commercial grade cheap nutrients to improve the economic viability of MEOR process.
2014 Published by Elsevier Ltd.

1. Introduction
A petroleum reservoir is a subsurface pool of hydrocarbons contained in porous or fractured rock formations. There are three main
stages by which oil is recovered from reservoir. The rst stage is
the primary recovery stage where oil is recovered due to natural
energy inherent in the reservoir. Expansion of the dissolved gas
following reduced pressure as a consequence of drilling operations
is usually responsible for the driving energy that carries oil to the
surface. When inherent pressure of reservoir tends to fall, secondary recovery methods are applied. In this method external uid or
gases are injected to maintain reservoirs pressure. Primary and
secondary oil recovery processes can account for 3040% oil
productions leaving behind about 55% of oil as residual oil in the
reservoirs (Sen, 2008). A third stage of oil recovery called tertiary

Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542.

E-mail addresses: pkdhakephalkar@aripune.org, pkdhakephalkar@gmail.com
(P.K. Dhakephalkar).

recovery or enhanced oil recovery involves chemical ooding, thermal recovery and miscible displacement involving carbon dioxide
(CO2), hydrocarbon or nitrogen injection. Enhanced oil recovery
methods are not cost effective (Al-Sulaimani et al., 2011). So there
is need to develop alternate strategy for oil recovery.
Microbial Enhanced Oil Recovery (MEOR) is one among the
most promising novel approaches that can be potentially
implemented with an exceptionally low operating cost (Lazar et
al., 2007). MEOR processes can be categorized into three main
types. (i) In the rst type of MEOR process, bacteria are grown ex
situ. The culture broth, with or without bacterial cells, is injected
in the oil wells in a process similar to chemical ooding. The
microbial metabolites such as organic acids, biosurfactants and
solvents, then remove adhered oil as well as parafn deposits from
well surfaces including tubing, rods, etc. Such type of MEOR
process helps overcome clogging related problems which reduce
oil production. This process is usually applicable to oil reservoirs
rich in parafn contents where parafn clogs the well bores
decreasing the efciency of oil recovery. (ii) In the second type of

http://dx.doi.org/10.1016/j.biortech.2014.03.109
0960-8524/ 2014 Published by Elsevier Ltd.

Please cite this article in press as: Arora, P., et al. Development of a microbial process for the recovery of petroleum oil from depleted reservoirs at 91
96 C. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.109

P. Arora et al. / Bioresource Technology xxx (2014) xxxxxx

MEOR process, a combination of anaerobic bacteria and nutrient

media supplemented with cheap substrates (such as molasses)
are injected in depleted oil wells. Bacterial fermentation of nutrient
media occurs within oil wells producing bacterial metabolites
which stimulate displacement of oil entrapped by capillaries and
brine. (iii) The third MEOR process is a water ooding process
boosted with microbial cultures but not supplemented with nutrient media. Microorganisms used in this type of MEOR process must
be capable of using reservoir hydrocarbons as source of carbon and
energy and produce desired metabolites. This type of MEOR process is practiced late in the course of a water ood and stimulates
microbial activity throughout the reservoir.
MEOR methods were usually limited to oil reservoir having
temperature up to 70 C (Sen, 2008) but there are many oil reservoirs world over having temperature higher than 90 C. So there
is need to develop a microbial process which can be applied to such
oil reservoirs characterized by extreme high temperature, pressure
and salinity. In this study a unique combination of novel indigenous micro-organisms optimally growing at 96 C and producing
biosurfactant, exopolysaccharide, CO2, organic acids and solvents
was developed from formation water sample. The efciency of consortium for MEOR was evaluated using simulated sand pack column and core ood assay.
2. Methods
2.1. Enrichment of the hyperthermophilic consortia at temperatures
above 90 C
Crude oil or formation water samples collected from oil wells in
Ahmadabad and Mehsana oil elds in Western India were used as
inoculum for the enrichment of hyperthermophilic bacteria.
Enrichment for hyperthermophilic bacteria was set up in peptoneyeast extract base medium [in g/L: peptone, 10; yeast extract,
10; NaCl 10; Salt Solution SS30 (K2HPO4 1 g/L, KH2PO4 1 g/L,
NaHCO3 10 g/L, NaCl 2 g/L, CaCl2 anhydrous 0.2 g/L, MgSO4
7H2O
0.2 g/L), 40 ml] pH 7.0. Enrichments were incubated at 91 or
96 C in hot air oven for 1421 days.
2.2. Optimization of nutritional and growth parameters
Carbon and nitrogen source were optimized by using basal
medium (K2HPO4 7 g/L, KH2PO4 3 g/L, yeast extract 10 g/L, NaCl
10 g/L) supplemented with carbon source (1% w/v) and nitrogen
source (0.5% w/v). Actively growing culture (ca. 106 cells/ml) was
inoculated at 1% v/v and incubated at 96 C for 14 days. Temperature, pH and salinity were optimized using basal medium
supplemented with 1% sucrose as carbon source and 0.5% urea as
nitrogen source. Growth was evaluated at 80, 91 and 96 C at pH
511 and at salinity 4, 7 and 11 g%.

2.4. Sand-pack column assays

Vertically oriented jacketed glass columns (500 ml) were
uniformly packed with sterilized dry sand. Temperature was maintained at 96 C by circulating heated oil through the jacketed walls
of the glass column using a circulating oil bath. Sand used to pack
the glass column was washed with dilute acid (1) then with
deionized water (3) and dried in hot air oven at 80 C for 18 h.
The column was then ooded with brine (2% NaCl) followed by
oil under pressure till brine was washed out. The sand pack was
subsequently ooded with nutrient medium to ensure maximum
possible displacement of oil till saturation of residual oil was
reached. The column was inoculated with 100 ml inoculums
(106 cells/ml) in test and sterile distilled water in control column.
2.5. Core ood assay
Characteristics of Barea core used in the Core ood study are
described in Table 3. The core fragment was placed inside a
stainless steel core holder and placed in the oven at 96 C. Fluids
(brine, oil, and microorganisms plus nutrients) were injected using
an injection pump. Core ood studies were performed at 96 C, 700
psi and 40 g/L NaCl. Core was initially saturated with brine (2 g%
NaCl) and then with oil. Brine was again injected to displace
removable oil and allow only residual oil in place. Subsequently,
nutrient medium with NJS4 consortium having a cell density of
8  106 cells/ml was injected. The sealed core was incubated at
96 C for 14 days. Subsequently oil was recovered by displacement
at the same temperature with brine and analyzed.
2.6. Metabolite analysis
Cell free supernatant was used for the metabolite analysis. Gas
analysis for carbon dioxide, hydrogen, methane and nitrogen was
performed using Gas chromatography (PerkinElmer) equipped
with thermal conductivity detector (TCD), Porapak Q column (SS,
1/800 _ 80 mesh) with argon as carrier gas (ow rate:
40 ml/min). Temperature settings for TCD analysis were as follows:
oven at 40 C, injector and detector at 70 C, injector at 100 C.
Biosurfactant production was analyzed in terms of emulsication
index (Satpute et al., 2010). Exopolysaccharide was determined
by precipitation by isopropanol (Pawar et al., 2013). Cell surface
hydrophobicity of the consortium was determined by MATH measurement as per protocol described by Xiao et al. (2013). Organic
acids and solvents were analyzed using high-performance liquid
chromatography system (Dionex, USA) using Aminex HPX-87H,
Biorad column, equipped with refractive index (RI) detector. The
working conditions were: 5 mM H2SO4 as a mobile phase with a
ow rate of 0.7 ml/min at 40 C.
3. Results and discussion

2.3. Phylogenetic afliation of microbial species present in NJS-4

consortium
Bacterial species present in NJS-4 consortium were identied by
PCR-DGGE-Sequencing of 16S rRNA gene fragments of NJS-4 consortium as described previously by Sachdev et al. (2010) with following modications. Genomic DNA extraction of NJS4 consortium
was done by CTAB method (Zhou et al., 1996). PCR amplication of
16S rRNA gene was carried out using GC341F and 907R primers
described by Muyzer et al. (1995). PCR amplicons were resolved
by DGGE performed using DCode system (Bio-Rad, USA) over a
denaturing gradient of 3070% at 65 C and 100 V for 14 h. Separated bands were excised, reamplied, puried and sequenced on
an ABI 3100 sequencer as described earlier (Sachdev et al., 2010).

Hyperthermophilic microbial consortia were enriched from the

formation water samples collected from oil wells (in situ temperature 90 C or more) in western India. Only those oil wells were
selected where water injection was not practiced. This assured
enrichment of only native ora and ruled out contamination of
heterologously introduced microorganisms. Growth of hyperthermophiles was monitored in terms of cell density as well as
production of CO2 and desired metabolites which included biosurfactant, organic acids, solvents, exopolysaccharide and volatile
fatty acids. One such consortium designated as NJS4 was selected
for further studies as it showed maximum growth and production
of desired metabolites (data not shown). PCR-DGGE analysis of 16S
rRNA gene fragments performed to determine the composition of

Please cite this article in press as: Arora, P., et al. Development of a microbial process for the recovery of petroleum oil from depleted reservoirs at 91
96 C. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.109

P. Arora et al. / Bioresource Technology xxx (2014) xxxxxx

NJS4 consortium revealed four bands. Three prominent bands were

identied as the phylogenetic afliates of Clostridium sp. (>98% 16S
rRNA V3V6 sequence homology with reference sequences in GenBank). All three sequences were deposited in the GenBank database under the accession numbers KJ561456, KJ522756 and
KJ522757. Member of Clostridium such as Clostridium acetobutylicum, Clostridium tyrobutiricum, and others have been extensively
used in Microbial Enhanced Oil Recovery in USA, Russia, Germany,
Hungary, and Poland as reviewed by Lazar et al. (2007). Hyperthermophilic strain Clostridium bifermentans was used in MEOR
performed at 7080 C by Babcock et al. (2009). However, this is
the rst report of Clostridium sp. growing at temperature above
90 C.
Growth and nutritional parameters including temperature, pH,
and salinity, redox potential, as well as source of carbon and
nitrogen were optimized for NJS4 consortium (Table 1). Maximum
growth of NJS4 was observed at 96 C (2.1  106 cells/ml) which
was the highest temperature tested in the present study.
Approximately 28% less growth was observed at 91 C. Growth
was substantially lower (<104 cells/ml) at 80 C. Growth was not
observed at lower temperature. NJS4 could grow optimally at pH
9 (2.2  107 cells/ml). However, signicant growth (50% of the
optimum growth) was observed over a wide range of pH i.e.
511. Growth of NJS4 remained unaffected over a wide range of
salinity (2.32.5  107 cells/ml over a salinity range of 0.8511%).
Most of the high temperature reservoirs in Indian subcontinents
are characterized by temperature above 85 C, 47% (w/v) salinity
and slight alkaline, oxygen limiting environment. NJS4, characterized as hyperthermophilic, alkalophilic, halophilic anaerobic consortium, was suitable for application in such high temperature
oil reservoirs. NJS4 was categorized as oxygen tolerant anaerobic
consortium as it showed comparable growth in nonushed as well
as nitrogen ushed media supplemented with increasing
concentration of cysteine HCl (1.12  107 cells/ml over 00.5 g
Cysteine-HCl/L). Dissolved oxygen concentration at 96 C was
2 ppm.
Inuence of various carbon and nitrogen sources was
investigated to determine the optimal conditions that yielded the
highest growth and desired metabolites by NJS4 (Table 2). NJS4
was able to grow in all carbon sources tested. Growth of

Table 1
Nutritional and growth parameters affecting growth of NJS4.
Parameters

Characteristics

Temperature range
Optimum
temperature
pH range
Optimum pH
Salt range
Optimum salt
concentration
Cystine HCL
requirement
Utilizable carbon
source
Most suitable
carbon source
Utilizable nitrogen
source

85107 C
96

Most suitable
nitrogen source
Optimized medium
for growth
Growth period
Pathogenicity

511
79
111% w/v
4% w/v
00.5 g/L
Glucose, sucrose, lactose, starch, sodium citrate and
sodium acetate
Sucrose
Ammonium sulphate, urea, potassium nitrate,
ammonium nitrate, Di-ammonium hydroxy
orthophosphate (DAHOP), ammonium chloride
Urea
Sucrose (1%), sodium acetate (%), urea (0.5%), K2HPO4
(0.7%), KH2PO4 (0.3%), trace element solution (1 ml/L),
pH = 9
14 days
Non toxic in oral and dermal pathogenicity testing

NJS4 and CO2 production was the highest when glucose was used
as carbon- and urea as nitrogen source. Growth was the lowest
when Di-hydrogen ammonium phosphate was used as a source
of nitrogen. Several species of Clostridium have been reported to
utilize a wide range of carbohydrate substrates (Rainey et al.,
2009). Interestingly, sodium acetate has not been included as an
utilizable substrate for Clostridium in the same description.
Evolution of large quantities of gases is a desired trait in MEOR
as it increases the reservoir pressure and the dissolution of carbon
dioxide which is known to cause oil swelling, viscosity reduction
and increase permeability due to solubilization of carbonate rocks
(Al Sulaimani et al., 2011). In the present investigation NJS4 consortium produced signicant quantities of gas when grown in presence of fermentable substrates (56 ml/g sucrose at 96 C). Carbon
dioxide was the major component (>48 mM) of gas produced by
NJS4. Hydrogen was produced in traces and Methane was not detected in the GC analysis. CO2 yield reported here was signicant
as compared to the previous report wherein Bacillus licheniformis
produced 21.4 mM CO2 at 50 C and reported oil recovery of
922% (Yakimov et al., 1997). In another study, genetically
engineered bacteria produced 11.44 mM CO2 at 45 C resulting in
1725% oil recovery (Xu and Lu, 2011). Dissolution of carbonaceous rocks is also caused by organic acids produced by
microorganisms resulting in increased reservoir permeability and
porosity. Enhanced organic acid production by NJS4 was observed
in this study when sucrose was the source of carbon and ammonium sulfate/potassium nitrate/ammonium nitrate as nitrogen
source (Table 2). NJS produced 508 mg/L of exopolysaccharide
(EPS) when sucrose and acetate were used as carbon- and urea
as nitrogen source (Table 2). EPS has been reported to improve
sweep efciency and enhance oil recovery by selective plugging
of high permeability zone redirecting the water-ood to oil-rich
zones in the reservoir and by altering the wettability of reservoir
to displace more oil from the formation rocks (Lazar et al., 2007).
Biosurfactant production was monitored in terms of emulsication index (EI). The highest EI (43%) obtained with sodium acetate
as carbon source provided evidence that the NJS4 had the ability to
produce surface active agents, which can reduce the interfacial
tension, thus enhancing oil mobility. Higher biosurfactant production by NJS4 with sodium acetate as compared to that with
carbohydrate substrates was contradictory to previous reports
where biosurfactant production is enhanced by mono-, di- or
polysaccharides (Prasad et al., 2013). EI has been repeatedly used
to evaluate the potential of microorganisms to produce biosurfactants. Prasad et al. (2013) reported high EI of 85% for Pseudomonas
aeruginosa MTCC2297. The lower EI observed in the present
investigation could be attributed to the lower growth of
hyperthermophilic consortium at 96 C as compared to that of
mesophilic MTCC2297 strain at 37 C. NJS4 also produced organic
solvents including methanol (12,650 mg/L) and ethanol (77 mg/L)
in a medium containing sucrose & sodium acetate as carbon source
and urea as nitrogen source (Table 2). These organic solvents also
aided in solubilization of oil.
Ability of NJS4 to enhance crude oil recovery was evaluated in
sand pack trials and core ood studies using medium that consisted of sucrose (supported high growth and CO2 production)
and sodium acetate (high biosurfactant production) as carbon
source, urea as nitrogen source and yeast extract in trace amount
to satisfy growth factor requirement. Precipitation resulting from
the partial denaturation of carbohydrate at high temperature was
negligible when urea was used as a nitrogen source. Oil recovery
by NJS4 was 26.7% over oil in place as against to only 1% in uninoculated control (Table 3). Shavandi et al. (2011) reported 70%
residual oil recovery in sand pack studies. Signicantly higher oil
recovery reported in that study may be attributed to MEOR
performed in mesophilic temperature (30 C) using light oil. It

Please cite this article in press as: Arora, P., et al. Development of a microbial process for the recovery of petroleum oil from depleted reservoirs at 91
96 C. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.109

P. Arora et al. / Bioresource Technology xxx (2014) xxxxxx

Table 2
Metabolite analysis of NJS4 consortium grown in media supplemented with various carbon or nitrogen sources. Metabolites were analyzed after incubation of 14 days at 96 C.
CO2 (mM)

a
b
c
d

Cell density

E.Ib (%)

EPSc

Maleic

Carbon source
Glucose
Lactose
Sucrose
Starch
Sodium acetate
Sodium citrate

48.6
39.8
36.5
33.2
29.58
22.6

16.0  107
8.0  107
4.0  107
1.6  107
4.0  106
2.4  107

20

43.3
40

+
+
+

Nitrogen source
Urea
Ammonium sulphate
Potassium nitrate
DAHOPd
Ammonium chloride
Ammonium nitrate
Sucrose + Na acetate + Urea

48.1
23.7
19.1
14.6
19.3
22.6
49.02

4.0  107
3.2  107
8.0  106
1.2  107
1.6  107
2.8  107
8.0  106

9.4

10.3
6.6
26

+
+
+
+
+
+
+(508 mg/L)

Solventsa (mg/L)

Organic acids (mg/L)

Malic

Formic

Acetic

Met

Eth

273.6
47.34
448

56.77

301.2

104

41.6

2193
5301
859
160
1974

737
753
602
50
828

927.7

404
454

720

308.3

193
354

5900

12,650

77

Met methanol; Eth ethanol.

E.I emulsication index.
EPS exopolysaccharide.
DAHOP di-ammonium hydrogen orthophosphate.

Table 3
Oil recovery in sand pack trials and core ood studies.
Sand pack study
Volume of sand
Void volume
Volume of residual oil in column after water ooding
Oil recovered after 14 days of incubation at 96 C
Percent oil recovery

500 ml
192 ml
90 ml
24 ml
26.7%

Characteristics of crude oil

Value

Type of oil
Pour point
Specic gravity
Viscosity gravity constant (VGC)

Heavy
31 C
0.842
0.7873

Core ood study

Type of rock
Length
Diameter
Pore volume
Permeability
Original oil in place (OIIP)
Residual oil saturation (ROS)
Oil recovery over ROS

Sandstone Berea
7.0 cm
3.8 cm2
23.8 ml
620 mD
18.3 ml
37.40%
10.10%

Sand pack study

Volume of sand
Void volume
Volume of residual oil in column after water ooding
Oil recovered after 14 days of incubation at 96 C
Percent oil recovery

500 ml
192 ml
90 ml
24 ml
26.7%

may be noted that the oil used in the present study was heavy oil
and also MEOR was performed at 96 C.
Efciency of NJS4 to enhance oil recovery was also investigated
by core ood trials. Characteristics of the crude oil and core used in
this experiment are described in Table 3. In the core ood study, oil
recovery was 10.1% over residual oil saturation (ROS). Previous report has described 5.6% increase in oil recovery at 70 C in an oil
displacement system employing the injection of bacteria (Jinfeng
et al., 2005). MEOR at temperatures exceeding 82 C was never
reported in 1970s. Wilhelms et al. (2001) suggested that MEOR
related activities such as partial degradation of hydrocarbons were
inhibited at temperatures higher than 8090 C. Failure to cultivate
reservoir organisms at temperatures above 82 C was reported by
Bernard et al. (1992). Stetter (2006) could cultivate reservoir

microorganisms up to 113 C. However, these microorganisms

could not be employed in MEOR process. In the view of these
studies reporting application of MEOR technology to 85 C or lower
temperature, MEOR at 96 C reported in the present study is of
special signicance with potential applications in high temperature oil reservoirs in gulf and Indian subcontinent.

4. Conclusion
The results show that the consortium, characterized as hyperthermophilic, halophilic, and anaerobic microorganisms, presented
the ability to improve the recovery of heavy oil in sand pack and
core ood assay. This culture produced metabolic products which
interacted with oil and increased its mobility. The mechanisms
involved were the reduction of interfacial tension by surface-active
agents, reduction of oil viscosity by acids and solvents, repressurization by gases and wettability change by biopolymer.

Acknowledgements
Gratefully acknowledged: (i) Financial assistance from Institute
of Reservoir Studies, ONGC, Ahmedabad and (ii) CSIR-JRF to Preeti
Arora by Government of India.

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96 C. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.03.109