Bioresource Technology 51 (1995) 1-12
/
0960-8524(94)00
ELSEVIER
BIOSURFACTANTS PRODUCTION A N D POSSIBLE USES IN
MICROBIAL E N H A N C E D OIL RECOVERY A N D OIL
POLLUTION REMEDIATION: A REVIEW
I. M. Banat
Department of Biology, United Arab Emirates University, PO Box 17551, Al-Ain, Abu-Dhabi, UAE
(Accepted 4 October 1994)
Abstract
Surfactants are widely used for various purposes in
industry, but for many years were mainly chemically syn-
thesized. It has only been in the past few decades that
biological surface-active compounds (biosurfactants)
have been described. Biosurfactants are gaining promin-
ence and have already taken over for a number of
important industrial uses, due to their advantages of
biodegradability, production on renewable resources
and functionality under extreme conditions; particularly
those pertaining during tertiary crude-oil recovery. Con-
flicting reports exist concerning their efficacy and the
economics of both their production and application. A t
present, their uses are mainly in the oil and petroleum
industries, where they are employed primarily for their
emulsification capacity in both tertiary recovery and pol-
luted-sites remediation. However, caution is frequently
exercised with respect to their use because of possible
subsequent microbial contamination of either under-
ground oil reservoirs or products. The limited successes
and applications for biosurfactants' production,
recovery, use in oil pollution control, oil storage tank
clean-up and enhanced oil-recovery are reviewed from
the technological point of view.
Key words: Biosurfactants, emulsification, biodegrada-
tion, bioremediation, microbially-enhanced oil
recovery, MEOR.
INTRODUCTION
Biosurfactants are a heterogeneous group of surface-
active molecules produced by microorganisms. These
molecules reduce surface tension, critical micelle con-
centration (CMC) and interracial tension in both aque-
ous solutions and hydrocarbon mixtures. These
properties create micro-emulsions in which micelle
formation occurs where hydrocarbons can solubilize in
water, or water in hydrocarbons. The properties of the
various biosurfactants have been extensively reviewed
(Cooper, 1986; Rosenberg, 1986; Haferburg et al.,
1990; Fiechter, 1992a; Georgiou et al., 1992; Kosaric,
1995 Elsevier Science Limited
Printed in Great Britain. All rights reserved
0960-8524/95/$9.50
I 0 1-4
1993). Generally the structure of biosurfactants
includes a hydrophilic moiety composed of amino acids
or peptides, anions or cations, or mono-, di-, or poly-
saccharides. The hydrophobic portion is often made
up of saturated, unsaturated or hydroxylated fatty acids
(Georgiou et al., 1992), or composed of amophophilic
or hydrophobic peptides. World-wide interest in bio-
surfactants has increased immensely due to their ability
to meet most synthetic surfactants' requirements
(Morkes, 1993).
Biosurfactant(s) spontaneous release and function
are often related to hydrocarbon uptake; therefore,
they are predominantly synthesized by hydrocarbon-
degrading microorganisms. Some biosurfactants, how-
ever, have been reported to be produced o n
water-soluble compounds, such as glucose, sucrose,
glycerol or ethanol (Guerra-Santos, 1986; Cooper &
Goldenberg, 1987; Palejwala & Desai, 1989; Passeri et
al., 1992; Hommel & Huse, 1993). In some instances,
these compounds have antibiotic properties which may
serve to disrupt membranes of microorganisms com-
peting for food. Examples of these include the lipo-
peptides of the iturin family produced by Bacillus
subtilis, which have powerful anti-fungal properties
(Sandrin et al., 1990; Thimon et al., 1992), Candida
antarctica, which have antimicrobial activity (Kitamoto
et al., 1993), and Bacillus licheniformis, which inhibit
bacteria, yeast and filamentous fungi (Fiechter, 1992a).
Chemically-synthesized surfactants have been used
in the oil industry to aid the clean up of oil spills, as
well as to enhance oil recovery from oil reservoirs.
These compounds are not biodegradable and can be
toxic to the environment. Biosurfactants, however,
have been shown in many cases to have equivalent
emulsification properties and are biodegradable. Thus,
there is an increasing interest in the possible use of bio-
surfactants in mobilizing heavy crude oil, transporting
petroleum in pipelines, managing oil spills, oil-pollu-
tion control, cleaning oil sludge from oil storage faci-
lities, soil/sand bioremediation and microbially-
enhanced oil recovery (MEOR). M E O R offers major
advantages over conventional E O R in that lower capi-
tal and chemical/energy costs are required (Sarkar et
al., 1989).
2 I.M. Banat

BIOSURFACTANT-PRODUCING
MICROORGANISMS
A large variety of biosurfactants is known; their type,
quantity and quality are influenced by the nature of the
carbon substrate (Georgiou et al., 1992), the con-
centration of N, P, Mg, Fe and Mn ions in the medium
(Atlas, 1981; Cooper et al., 1981 a, b; Guerra-Santos et
al., 1984 & 1986; Haferburg et al., 1986; Abu-
Ruwaida et aL, 199 lb) and culture conditions, includ-
ing pH, temperature, agitation and dilution rate
(Guerra-Santos e t al., 1984 & 1986; Abu-Ruwaida et
al., 1991a; Fiechter, 1992a; Drouin & Cooper, 1992;
Lin etal., 1994).
When considering which microorganisms to use for
MEOR, the varying conditions in which they will be
used, such as temperature, pressure, pH and salinity,
must be given priority (Khire & Khan, 1994b). Typic-
ally, microorganisms injected into an oil well should
endure high temperatures, pressures and salinity, and
be capable of growth under anaerobic or microaero-
philic conditions. It has been estimated that 50-70% of
oil wells in the USA could support microbial growth
with pH values from 4 to 8, temperatures < 75C and
salinity < 10% (Clark et al., 1981 ).
Sources of cultures
Several types of biosurfactant have been isolated and
characterized, including glycolipids, phospholipids,
neutral lipids, fatty acids, peptidolipids, lipopoly-
saccharides and others not fully characterized. Micro-
organisms which produce biosurfactants, and their
structures, are listed in Table 1. Certain micro-
organisms are likely to be found to be better adapted to
particular environments, such as oil reservoirs, soil or
the ocean. A strain of Pseudomonas aeruginosa iso-
lated from crude oil-associated injection water in
Venezuelan oil fields was found to be adapted to the
conditions prevalent in this oil reservoir (Rocha et al.,
1992). Further, the biosurfactants (rhamnolipids) pro-
duced by this strain were not inactivated by pH, tem-
perature, salinity, calcium or magnesium at
concentrations in excess of those found in many oil
reservoirs in Venezuela (Rocha et al., 1992). A halo-
tolerant Bacillus licheniformis strain JF-2, was also iso-
lated from oil field injection water, and was found to
produce biosurfactant under both aerobic and anaero-
bic conditions (Jenneman et al., 1983; Javaheri et al.,
1985). Bacillus strain SP018 was found to tolerate
anaerobic conditions at 50C and < 10% NaCl while
producing biosurfactants (Pfiffner et al., 1986). Two

Table 1. Various biosurfactants produced by microorganisms

Microorganism Biosuffactant Reference
A rthrobacter RAG- 1 hetropolysaccharides Rosenberg et al. (1979)
A rthrobacter MIS 38 lipopeptide Morikawa et al. (1993)
Arthrobacter sp. trehalose, sucrose and Suzuki et al. (1974)
fructose lipids Itoh et al. (1974)
Bacillus licheniformis JF-2 lipopeptides Mclnerney et al. (1990)
Bacillus licheniformis 86 lipopeptides Horowitz et al. (1990)
Bacillus subtilis surfactin Arima et al. (1968)
Bacillus pumilus A1 surfactin Morikawa et al. (1992)
Bacillus sp. AB-2 rhamnolipids Banat (1993)
Bacillus sp. C- 14 hydrocarbon-lipid-protein Eliseev etal. (1991)
Candida antarctica mannosylerthritol lipid Kitamoto et al. ( 1992)
Candida bombicola sophorose lipids Gobbert et al. (1984)
Candida tropicalis marman-fatty acid Kappell and Fiechter
(1976)
Candida lipolytica Y-917 sophoros lipid Lesik etal. (1989)
Clostridium pasteurianum neutral lipids Cooper et al. (1980)
Corynebacterium hydrocarbolastus protein-lipid-carbohy. Zajic etal. (1977)
Corynebacterium insidiosum phospholipids Akit etal. (1981)
Corynebacterium lepus fatty acids Cooper etal. (1979)
Strain MM1 glucose, lipid and
hydroxydecanoic acids Passeri (1992)
Nocardia erythropolis neutral lipids MacDonald et al. (1981)
Ochrobactrum anthropii protein Wasko and Bratt (1990)
Penicillium spiculisporum spiculosporic acid Ban and Sato (1993)
Pseudomonas aeruginosa rhamnolipid Robert etal. (1989)
Pseudomonas fluorescens lipopeptide Neu etal. (1990)
Phaffta rhodozyma carbohydrates-lipid Lesik etal. (1991)
Rhodococcus erythropolis trehalose dicorynomycolate Shulga etal. (1990)
Rhodococcus sp. ST-5 glycolipid Abu-Ruwaida et al. ( 1991 a)
Rhodococcus sp. H13-A glycolipid Singer and Finnerty (1990)
Rhodococcus sp. 33 polysaccharide Neu et al. (1992)
Torulopsis bombicola sophorose lipids Inoue and Ito (1982)
 Biosurfactants production and applications
biosurfactant-producing Bacillus strains, AB-2 and
Y 12-B, were also isolated from oil sludge-mixed sand
and had the ability to grow on hydrocarbon-containing
medium at -<50C (Banat, 1993).
Other microorganisms have been isolated from
hydrocarbon-contaminated soils, such as Rhodococcus
(Singer & Finnert, 1990; Abu-Ruwaida et al.,
1991a, b), Bacillus pumilus (Morikawa et al., 1992),
Arthrobacter sp. strain MIS38 (Morikawa et al., 1993);
others were isolated from contaminated fuels, such as
Ochrobactrum anthropii (Wasko & Bratt, 1990) or
from oceanic oil spillage, such as Pseudomonas aeru-
ginosa (Shafeeq et aL, 1989).
Techniques for identifying biosurfactant producers
Techniques have been developed to help identify bio-
surfactant-producing microbes or quantify their pro-
duction capabilities. One such technique is called the
axisymmetric drop shape analysis by profile (ADSA-
P), which simultaneously determines the contact angle
and liquid surface tension from the profile of a droplet
resting on a solid surface (Van der Vegt et aL, 1991).
Drops containing biosurfactant-producing microbes
are placed on a fluoroethylene-propylene surface and
the profile of the droplet determined with a contour
monitor. Surface tensions are then calculated from the
droplet profiles with ADSA-P. Surface tensions deter-
mined by this method were only found to be reduced
by biosurfactant-producing bacteria. Shnlga et al.
(1993) described a method for determining aniono-
genie bacterial peptidolipid biosurfactants based on the
ability of the anionic surfactants to form a coloured
complex with the cationic indicator methylene.
Other, simpler, methods have been described, such
as blood haemolysis (a known characteristic of some
biosurfactant compounds) (Banat, 1993) and an
emulsification index value (E-24) obtained on kero-
sine, as described by Cooper and Goldenberg (1987).
A rapid drop-collapsing test was also employed to
screen bacterial colonies for biosurfactant production
(Jain et al., 1991). Drops of cell suspensions were
placed on an oil-coated surface and if the drop con-
tained biosurfactants it was observed to collapse,
whereas non-surfactant-containing drops remained
stable.
BIOSURFACTANT PRODUCTION
The fermentation of biosurfactant-producing micro-
organisms varies greatly and has been reviewed previ-
ously (Georgiou et al., 1992; Desai & Desai, 1993).
Conditions which promote biosurfactant production
have been determined for several microorganisms.
Pseudomonas aeruginosa has been shown to produce
rhamnolipids on C12 n-alkanes (Robert et aL, 1989),
and increased production was noted on a phosphate-
limited medium (Mulligan et al., 1989) or upon the
exhaustion of nitrogen in the medium (Venkata
Ramana & Karanth, 1989). The structure of surfactin
has been shown to be influenced by amino acid con-
3
centration in the media to produce a Val-7 or Leu-7
surfactin (Peypoux & Michel, 1992). Rhodococcus sp.
had maximum growth and biosurfactant production on
medium containing 2% (v/v) n-paraffin and nitrate as
the N source and its product was found to be a primary
metabolite that could be produced in continuous cul-
ture (Abu-Ruwaida et aL, 1991 b).
Other substrates, such as olive-oil mill effluent
(Oome), whey and peat pressate, have also been used
for biosurfactant production (Mercade et al., 1993;
Mercade & Manresa, 1994). Mannosylerythritol lipid
biosurfactants were produced at the highest level and
accumulated by resting Candida antarctica cells in a
medium containing only the carbon source (Kitamoto
et aL, 1992a, b). Similar results were observed with
sophorose lipids produced by resting Candida apicola
(Hommel & Huse, 1993). A Pseudomonas strain Pet-
1006 required two carbon sources; a readily available
one (glucose) and a hydrocarbon (oleic acid) to be
utilized upon glucose exhaustion (Banat et aL, 1991).
The results of a typical batch fermentation experiment
on a biosurfactant-producing Rhodococcus ST-5 grow-
ing on paraffin oil and demonstrating biomass, surface
tension, emulsification and critical micelle dilution
(CMD) measurements against time, are shown in
Fig. 1.
Sophorose lipids were produced by Candida bombi-
cola CBS 6009 which was grown in fed-batch fer-
mentation (Davila et aL, 1992). Candida bombicola
had previously been shown to produce sophorose
lipids during a resting stage under conditions of
nitrogen limitation (Gobbert et al., 1984). Davila et aL
(1992) demonstrated that ethyl esters of rapeseed-oil
fatty acids and glucose could be utilized to produce a
high yield of sophorose lipids, with exclusion of the
lipids from the aqueous phase. This lipid layer is easily
separated by centrifugation and has the advantage of
avoiding limitations in product concentration due to
accumulation inhibition.
An integrated system for biosurfactant production
using P. fluorescens has also been developed (Fiechter,
1992b). In this system, HPLC is utilized to monitor
biosurfactant production instead of CMC and surface-
tension measurements, and a highly effective mem-
brane technology for cell retention is employed to
maximize the product formation rate (7"7 mg/1/h).
Biosurfactant recovery
In many cases the recovery of biosurfactants from
medium or microorganisms may be desirable. Several
procedures exist: biosurfactants from Nocardia
amarae, for example, have been isolated by methanol
precipitation (Sutton, 1992), while rhamnolipids from
P. aeruginosa have been isolated by acidification of
culture media followed by extraction with chloroform/
methanol solvent (Robert et al., 1989). Improved isola-
tion of glycolipids from Rhodococcus sp. H13A was
accomplished using XM 50 diafiltration and isopropa-
nol precipitation (Bryant, 1990). This technique has
the advantage of separating the glycolipid from co-iso-
4 L M. Banat
100 - 100
x Biomass
0 CMD -l
~ 80 - 80 --
CMD -2
CMD -3
E24
Surface tension
60 - 60
.~ 40 - ~ 4o
20 - 20
0 - 0 I " I-.~~
0 8 16
Fig. 1. The results of a typical batch fermentation of n-paraffin mineral medium by Rhodococcus sp. ST-5 culture at 37C, pH
6-8 and dissolved oxygen 30-50% saturation (Abu-Ruwaida et al., 1991 b). Symbols: x = biomass (g/l); o --critical mieelle dilu-
tion (CMD -1) (mN/m) at 1:10 culture broth to control medium; =CMD -2 (mN/m) at 1:100 culture broth to control
medium; = CMD -3 (mN/m) at 1:1000 culture broth to medium; =E24 (emulsification index %); = surface tension
(mN/m).
lated proteins. An aqueous two-phase fermentation
system has been developed which separates suffactants
on the basis of their charge (Drouin & Cooper, 1992).
This two-phase system, employing polyethylene glycol
and dextran, was found to partition cationic surfactants
to the bottom phase and anionic surfactants to the top
phase. Bacillus subtilis cells partitioned into the bottom
phase, while the biosurfactant (surfactin) partitioned
into the top phase.
Synthesis and modification
Laboratory synthesis and modifications of biosur-
factants have also been carried out. Chemical conver-
sion of the carboxylic moiety of rhamnolipid to a
non-ionic methyl ester resulted in enhanced interfacial
lowering and wetting action (Ishigami et al., 1993). It
was also noted that the modified rhamnolipid CMC
value had increased significantly. Phospholipids were
successfully modified by lipase-catalyzed transesterifi-
cation to incorporate n-3 polyunsaturated fatty acids
(Mutua & Akoh, 1993a). Transesterifications were
also used to prepare polyunsaturated phospholipids
(Totani & Hara, 1991). Candida cylindracea and
Rhizopus delemar lipases were utilized to prepare
polyunsaturated phospholipids from soy phospholipid
and sardine oil. Alkyl glycoside fatty acid esters were
also synthesized by lipase-catalyzed transesterification
of methyl glucoside, methyl galactoside and octyl
glucoside with methyl oleate (Mutua & Akoh, 1993b).
A fixed-bed reactor has been utilized for continuous
enzymatic transesterification of rapeseed oil and lauric
acid (Forsell et al., 1993). These techniques may prove
useful in designing biosurfactants or in producing
larger quantities of relatively pure products.
100 1 5
x~,~..- x -------.~. x
/x
X
[
-- 60 3 ~
- 4o .,, --, ~_
-- 20 11
I I I I 0 0
24 32 40 48
Time (h)
BIOSURFACTANTS IN POLLUTION CONTROL
Oil pollution accidents have become numerous and
have caused ecological and social catastrophes (Burger,
1993; Shaw, 1992; Burns et al., 1993). The ability of
biosurfactants to emulsify hydrocarbon-water mix-
tures has been widely reported (Broderick & Cooney,
1982; Harvey et al., 1990; Oberbremer et al., 1990;
Wasko & Bratt, 1990; Francy et al., 1991; Zhang &
Miller, 1992). These emulsification properties have
also been demonstrated to enhance hydrocarbons
degradation in the environment, hence making them
potentially useful tools for oil spill pollution-control
(Atlas & Bartha, 1992; Atlas, 1993; Bertrand et al.,
1994).
Bioremediation (laboratory experiments)
Experiments with P. aeruginosa isolate $8, isolated
from oil-polluted seawater, showed its ability to
degrade hexadecane, heptadecane, octadecane and
nonadecane in seawater by up to 47, 58, 73 and 60%,
respectively, after a 28 day incubation period (Shafeeq
et al., 1989). Tensiometric studies indicated the pre-
sence of biosurfactants in the culture medium. Oil-pol-
luted seawater, when supplemented with nitrogen and
phosphate, was able to reduce the quantities of n-para-
ffins present in the crude oils (Vrdoljak et al., 1992).
The addition of Pseudomonas aeruginosa UG2 bio-
surfactant to soil contaminated with a hydrocarbon
mixture of tetradecane, hexadecane, pristane and
2-methylnaphthalene, followed by a 2 month incuba-
tion period, also showed enhanced degradation of all
hydrocarbons except 2-methylnaphthalene (Jain et al.,
1992).
 Biosurfactants production and applications
In another experiment, contaminated soil, packed
into 8 ml solid-phase extraction columns, was ino-
culated with 108 cells/ml of Pseudomonas ML2 or
Acinetobacter haemolyticus. Other treatments with
ML2 biosurfactant product (at 41 or 82/~g/ml) were
used in addition to the buffer controls. After 2 months,
39-71% reduction in hydrocarbons was achieved by
A. haemolyticus, while the Pseudomonas ML2 showed
11-72% reduction (a very wide range). The treatment
with the ML2-biosurfactant product gave the best
results, yielding 44-46% reductions (when used at 41
/~g/ml) and 32-34% reductions (when used at 8 2 / t g /
ml). Hydrocarbon reduction was believed to be due to
degradation. These results suggested that using cell-
free biosurfactant stimulated degradation by indige-
nous microorganisms in the soil, giving closely
consistent results as compared to using actual micro-
bial cells. This might have been due to poor adaptation
and limited survival abilities of non-indigenous bac-
teria in the contaminated soil.
Biosurfactants have also been demonstrated to suc-
cessfully solubilize and remove hydrocarbon pollutants
from contaminated substrate. Studies employing bio-
surfactant-containing culture broths from Rhodo-
coccus ST-5 (Abu-Ruwaida et al., 1991a, b) and the
thermophilic Bacillus AB-2 (Banat, 1993) were carried
out to ascertain their effectiveness in removing residual
oil from sand-packed columns. Sandpacks composed
of 45-mesh acid-cleaned sand were saturated with oil
and then flooded with three pore volumes of water to
release unbound oil. Biosurfactant-containing culture
media (ST-5, AB-2 and Pet 1006) were then compared
to 0.1% SDS, 1% spolene and 1% petroleum sulfonate
for their ability to remove the residual oil. Both Pet
1006 and AB-2 cultures broths were found to release
95% of the residual oil (Table 2), compared to 63 and
58% for the surfactants spolene and petroleum sul-
fonate, respectively (Banat, 1993). Eliseev et al. ( 1991 )
also reported the ability of a biosurfactant produced
from Bacillus sp. C-14 to release oil from oily sand at a
concentration of 0"04 mg/ml. These experiments
demonstrated the effectiveness of biosurfactants in
removing residual oil from sand and are suggestive of
possible applications in EOR and in oil removal from
oil-polluted sand.
Numerous biosurfactant-producing microorganisms
were screened for their ability to solubilize C 14 labelled
Table 2. Effectiveness of various surfactant solutions in releasing residual light crude oil from sand (Abu-Ruwaida et aL, 1991;
Banat etaL, 1991; Banat, 1993)
Surfactant ST (mN/m)
0"1% SDS
1% Spolene
1% Petroleum sulfonate
ST-5 (from Abu-Ruwaida et aL, 1991a)
Pet 1006 (from Banat etal., 1991)
AB-2 (from Banat, 1993)
ST = surface tension; RO = residual oil; RRO = recovered residual oil.
5
3,3', 4,4', 5,5'-hexachlorobiphenyl in hexane (Van
Dyke et al., 1993 b). Their filtered biosurfactant-con-
taining culture broths were used to solubilize the
hydrocarbon mixed with soil in centrifuge tubes which
were incubated with shaking for 2 h at 22C, centri-
fuged and tested for solubilized hexachlorobiphenyl.
Of the organisms tested, Acinetobacter calcoaceticus
RAG-1 and Pseudomonas aeruginosa UG2 demon-
strated the best solubilization, at 41-9 and 48"0%,
respectively, compared to controls.
Rhanmolipid biosurfactants from Pseudomonas
aeruginosa were characterized for their ability to
remove hydrocarbons from sandy-loam soil and silt-
loam soil (Van Dyke et al., 1993a). Soil containing
hydrocarbon mixtures (naphthalene, anthracene, phen-
anthrene, fluorene, 2,2',5,5'-tetrachlorobiphenyl,
3,3',4,4',5,5'-hexachiorobiphenyl) plus partially-puri-
fied biosurfactant solutions were incubated before test-
ing hydrocarbon removal. Distilled water was found, in
most cases, to remove < 10% of the hydrocarbons
from the soil. The rhamnolipids at a concentration of
5 g/1 were found to increase recovery of the hydro-
carbons to 25-70% in silt-loam soil and 40-80% in
sandy-loam soil. However, substantial adsorption of
the rhamnolipid to the soil was observed and such a
high concentration of biosurfactant (5 g/l) was used as
to be highly impractical.
Similar experiments with a mixture of aliphatic and
aromatic hydrocarbons, mixed with sandy-loam soil
yielded similar results (Scheibenbogen et al., 1994). In
this study, a 0"08% mixture of rhamnolipids removed
36 and 40% of the aliphatic and aromatic hydro-
carbons, respectively, compared to 8.9 and 7.2% for
water. The addition of 0"1% pyrophosphate to the
rhamnolipid mixture was found to enhance aliphatic
and aromatic hydrocarbon removal to 56 and 73%,
respectively. These results compared favourably with
2% Triton X-100 (43 and 71%) and 2% Tween 60 (7.9
and 32%), where the Tween 60 had the disadvantage of
clogging the column after several total pore-volume
passages.
Bioremediation (field experiment)
Oil-contaminated soil is a common problem and its
treatment techniques, including excavation, incinera-
tion, landfarming and landfilling, can be difficult or
economically prohibitive. The other most economical
RO (%) RRO (%)
27"1 35 0"0
28"0 33 63"0
27"5 33 58"0
27-6 35 80"0
29"0 35 95"0
29"0 35 95"0
6 I.M. Banat
methods include in situ bioremediation. Machine-oil-
contaminated soil has been shown to be remediated by
microbial inoculation and by biosurfactant treatment
(Fry et al., 1993b). Furthermore, Fry et al. (1993a)
demonstrated the successful bioremediation of oil-con-
taminated soil and groundwater from a US Army engi-
neering plant using natural surfactants produced by
indigenous microorganisms. The addition of bio-
surfactant can increase the bioavailability of hydro-
phobic compounds to receptive bacteria.
Biosurfactants from Pseudomonas aeruginosa SB30
were tested for their abilities to remove oil from the
Exxon Valdez Alaskan contaminated gravel in the
laboratory (Harvey et al., 1990). A 1% biosurfactant
solution was found to consistently yield three-times
higher oil removal at temperatures > 40C and 1 min
contact time compared to water controls. These results
demonstrate the capacity of biosurfactants to remove
environmental pollutants, such as oil, from naturally-
occurring substrates. In a recent large-scale investiga-
tion, Bragg et at (1994) reported the effectiveness of
bioremediation activities on the Exxon Valdez oil spill
in situ. This was carried out through treatment of the
contaminated shore lines with an oleophilic liquid ferti-
lizer containing N and P to accelerate the growth of the
natural ~ hydrocarbon-degrading microorganisms;
chemical surfactant agents, in comparison, were found
to be ineffective in removing oil from sediments. Other
extensive bioremediation studies were successfidly
carried out on oil-contaminated desert sand in Kuwait,
both in situ and on-site (AI-Awadhi et al., 1994). All
these techniques directly involved utilizing indigenous
microbial populations, through the introduction of
specific nutrients and oxygen to encourage bio-
surfactant production and hydrocarbon utilization
(Miiller-Hurtig et al., 1993).
BIOSURFACTANTS IN OIL STORAGE TANK
CLEAN-UP
Field tests, utilizing biosurfactants produced from a
proprietary bacterial strain (Pet 1006), were performed
to test their ability to clean oil storage tanks and to
recover hydrocarbons from the emulsified sludge
(Banat et al., 1991). Pilot-plant-scale production of the
biosurfactant using a 1500 1up-lift fermenter produced
2 tonnes of culture broth. The biosurfactant-containing
broth was used as a substitute for chemical surfactants
in a test carried out on an oil storage tank belonging to
Kuwait Oil Company, Kuwait. Basal salt medium con-
taining 2% w/v glucose as a readily available carbon
source was used and oleic acid, a hydrocarbon source
(2% v/v), was added after glucose consumption. Bio-
surfactant production reached a maximum after
18-19 h, as measured by reductions in the surface and
interracial tension in the broth (Banat et al., 1991). At
the end of the production run, the culture broth was
sterilized in the fermenter and stored in 200 1 sterile
drums.
Surveys conducted of the 44 m diameter floating-
roof tank showed the presence of approximately
750 m 3 of sludge. After installation of circulation
pumps, hoses, circulation boxes and connections to
manholes, 1"5 tonnes biosurfactant were added to the
tank, in addition to a 1 : 1 crude oil to sludge ratio and a
1.25:1 brackish water to total hydrocarbon
(sludge + fresh crude) ratio. Circulation of the mixture
was carried out for 5 days, at ambient temperatures of
between 40 and 50C, after which most sludge had
been resuspended. An emulsion breaker was added to
separate the water from the crude oil and the two
layers were easily extracted from the tank. After clean-
ing, the total sludge was determined to be 850 m 3, and
nearly 91% (774 m 3) was recovered as crude oil,
whereas 76 m 3 remained as impurities at the tank
bottom and consisted mainly of non-hydrocarbon
materials. The characteristics of the recovered crude-
oil samples are shown in Table 3. The crude oil
extracted after cleaning was found to have API values
ranging between 27.6 and 29.8, similar to the API
range for the standard Kuwaiti crude, and a total
hydrocarbon content of 100%. Approximately 5550
barrels of saleable crude oil were produced. This oil
may be sold to cover costs of the cleaning at approxi-
mately S100 000-150 000 (US) per storage tank. Such
a clean-up process is highly desirable as it is economi-
cally rewarding, environmentally sound and is less
hazardous for the persons involved than the conven-
tional process (Lillienberg et al., 1992). In addition it is
a limited application for biosurfactants that can be
easily controlled and, therefore, would be a suitable
way for progressing towards involving the oil com-
panies who are usually reluctant to adopt this tech-
nology.
MICROBIALLY-ENHANCED OIL RECOVERY
(MEOR)
M E O R is an important tertiary recovery technology
utilizing microorganisms and/or their metabolic end-
products for recovery of residual oil. It is generally
accepted that approximately 30% of the oil present in a
reservoir can be recovered using current EOR tech-
nology (Singer & Finnerty, 1984). Poor oil recovery in
existing producing wells may be due to several factors.
The main factor is the low permeability of some reser-
voirs or the high viscosity of the oil which results in
poor mobility. High interfadal tensions between the
water and oil may also result in high capillary forces
retaining the oil in the reservoir rock (Bubela, 1987).
Since most of the oil remains in the reservoir following
primary and secondary recovery techniques, interest
has evolved in tertiary recovery techniques (Morkes,
1993). Techniques involving the use of chemical or
physical processes such as pressurization, waterflood-
ing or steaming, however, are generally unapplicable to
most oil reservoirs. The use of chemical surfactants for
cleaning-up oil reservoirs is an unfavourable practice
 Biosurfactants production and applications 7

Table 3. Characteristics of biosurfaetant-recovered crude oil samples before and after blending with fresh crude and after long
storage (Banat etat, 1991)

Property Test method Unit Sample

1 2 3
Specific gravity IP 160/D1298 g/ml 0-883 0"877 0"888
API gravity IP 200/D1250 API 28.6 29.8 27"6
Viscosity kinematic at 20C IP 71/D445 cSt 166.6 54.1 196.6
Water content (A) IP 74/D95 % vol ND ND ND
Sediment (B) IP 53 % vol ND ND ND
Water & sediment
(BS&W) (A+ B) -- % vol ND ND ND
Asphaltic material IP 143 % wt 2.09 2-14 1"05
Oil content
100 - (A+ B) -- % vol 100 100 100
ND = None detected.
Sample 1; biosurfactant-extracted crude oil before blending.
Sample 2; biosurfactant-extracted crude oil after blending.
Sample 3; biosurfactant-extracted crude oil after blending and 2 months storage.

that is hazardous, costly and will leave undesirable 2. The second involves the injection of selected
residues which are difficult to dispose of without nutrients into a reservoir, thus stimulating the
adversely affecting the environment. growth of indigenous biosurfactant-producing
microorganisms.
Strategies and factors affecting M E O R 3. The third mechanism involves the production of
The appropriate remedy for any given oil reservoir will biosurfactants in bioreactors ex situ and subsequent
vary and be based on the conditions present. Tempera- injection into the reservoir.
ture, pressure, pH, porosity, salinity, geologic make-up
of the reservoir, available nutrients and the presence of
Laboratory studies on M E O R
indigenous flora must all be taken into consideration. It
Laboratory studies on M E O R have typically utilized
is estimated, based on criteria developed by the
core samples and columns containing the desired sub-
National Institute for Petroleum & Energy Research,
strate. These substrates have been utilized to demon-
that 27% of the oil reservoirs in the major oil-produc-
strate the usefulness of biosurfactants in oil recovery
ing states in the USA may be suitable for M E O R
from sand and limestone. Similarly, core samples have
(Bryant, 1991 ). It has also been estimated that 40% of
been used to model the movement of microorganisms
the oil-producing carbonate reservoirs in the USA may
and nutrients through substrates to ascertain their use-
also be suitable for M E O R (Tanner et al., 1991 ).
fulness after injection into oil reservoirs.
The mechanisms of MEOR's action in situ are most
In an investigation in which B. subtilis was injected
probably due to multiple effects of the microorganisms
through sand-packed columns (2-5 cm diameter x 28
on the environment and oil. These mechanisms
cm length) with a permeability of 4000 md, a release of
include: gas formation and pressure increases; acid
35% residual oil, compared to 21% using the nutrient
production and degradation of limestone matrices;
solution control, was observed. Similar experiments
reduction in oil viscosity and interfacial tension by bio-
with C. acetobutylicum using molasses (4%) and 0"5%
surfactant; solvent production; plugging by biomass
ammonium diphosphate nutrient medium yielded 66
accumulation or polymer formation; and degradation
and 50% oil recovery in the presence and absence of
of large organic molecules in oil, resulting in decreases
pyrophosphate, respectively. It was suggested that C.
in viscosity (Jack, 1988; Khire & Khan, 1994a).
acetobutylicum has an advantage in M E O R because of
The presence of different types of microorganisms
its anaerobic growth and gas-producing capabilities
with varying growth properties and metabolite produc-
(Chang, 1987). The added oil was not sterile, however,
tion will have different effects on the reservoir environ-
and the role of indigenous bacteria in the oil recovery
ment. Thus, it is important to consider all aspects of
was not monitored.
M E O R when trying to influence oil production by one
mechanism, such as the use of biosurfactants. There
Vibrio aspartigenicus strain GSP-1 and Bacillus
licheniformis JF-2 were also tested for their ability to
are several strategies involving the use of biosurfactants
recover residual oil from crushed unconsolidated Viola
in M E O R (Shennan& Levi, 1987):
limestone (20-50 mesh) cores (Adkins et al., 1992).
1. The first involves injection of biosurfactant-pro- Vitrio aspartigenicus (an acid-producing, halophilic,
ducing microorganisms into a reservoir through the anaerobic, motile bacterium) was observed to recover
well, with subsequent propagation in situ through 32-36% more oil from saturated cores than the control
the reservoir rock (Bubela, 1985). columns after three treatments (injection of nutrients,
8 I.M. Banat
incubation, flooding with brine). In this experiment a
27-38% increase in dissolved calcium concentration
was observed, due to acid production which can
degrade the limestone matrix and cause release of the
residual oil. In comparison, B. licheniformis increased
oil recovery by 27%, with a 13% increase in dissolved
calcium which was also attributed to biosurfactant
production (Adkins et al., 1992).
Plugging and clogging investigations
One of the methods of MEOR involves using micro-
organisms in in situ selective plugging through the
introduction of viable bacteria in the aqueous displac-
ing-fluid injected into the oil well water-swept zones.
Investigations involving high-pressure consolidated
systems were employed using 1:1 mixtures of a
cement-sand mixture with powdered aluminum and
shaped into cores. These oil-saturated cores were
mounted in a Hessler cell and subjected to high pres-
sure sweeps (250-350 kPa). Water resulted in a 15%
recovery of OIP, whereas injections of microorganisms
resulted in plugging of the cores. Experiments using
brine-saturated Berea sandstone cores showed that
injecting nutrients and viable bacterial cells resulted in
permeability reductions (clogging) of 60-80% (Jenne-
man et al., 1984).
To facilitate microbial movements and formation of
plugs deep within the reservoir, strata studies examin-
ing the injection of nutrient-starved ultramicrobacteria
(UMB) into three-dimensional sandpack-reservoir
simulations have been carried out (Cusack et al., 1992).
Starved Pseudomonas sp. FC3 cells were found to
penetrate a sandpack 45 cm in diameter and 38 cm in
length. The UMB were then resuscitated with nutrients
to grow in situ and form a confluent bacterial plug.
Scanning electron microscopy and carbohydrate assays
revealed glycocalyx formations in all sections
examined. UMB are effective for deeper penetration
into oil reservoirs as they are small in size, have high
shear-stress tolerance and are useful for microbial
plugging in reservoirs with a permeability range of 200
mD-5.9D (Cusack et al., 1992) which can facilitate
secondary oil-recovery techniques involving water
sweeps.
Studies to determine the factors influencing micro-
bial movement through anaerobic, nutrient-saturated,
unconsolidated Ottawa sand-packed cores were car-
ried out under static conditions (Reynolds et al., 1989).
Gas-producing motile Escherichia coli were found to
penetrate cores saturated with galactose-peptone up to
six times faster than non-gas-producing motile or non-
motile mutants. Similarly, motile strains of E. coli were
found to penetrate the core four times faster than
mutant strains defective in flagellar formation. Further-
more, motile strains with faster growth-rates pene-
trated the cores at a more rapid rate. Chemotaxis was
not found to influence the penetration rate of these
bacteria. Such factors are important to consider in the
choice of bacterium to employ in in situ MEOR.
Field studies of MEOR
One of the early field studies on MEOR involved an
unconsolidated waterflooded sandstone reservoir in
Union County, Arkansas, USA (Yarbrough & Coty,
1983; Hitzman, 1988). Molasses was injected along
with an inoculum of Clostridium acetobutylicum into
the reservoir. After treatment, carbon dioxide produc-
tion increased and the production of oil increased by
250% (Tanner et al., 1991). Metabolite production,
however, was found to match that of an indigenous
microorganism, C. kluyveri, detected in the reservoir
water. The mechanisms by which these micro-
organisms acted were believed to result from acid
production.
Reports of successful MEOR use in carbonaceous
reservoirs have been reviewed (Tanner et al., 1991).
Injection of molasses and anaerobic acid- and gas-
producing microorganisms resulted in increased oil
recovery. A decrease in water pH and an increase in
carbon dioxide production indicated active growth of
microorganisms in the reservoir. Acid and gas produc-
tion were believed to be important elements in the oil
recovery (Lazar et al., 1988; Tanner et al., 1991).
Other M E O R investigations in carbonate reservoirs
showed an increase of 60-126% in oil production in
Hungary (Hitzman, 1983) and a 200% increase in
Germany (Wagner, 1991). Injection of nutrients and
Desulfovibrio desulfuricans (Hitzman, 1988) obtained
from sewage sludge, followed by a shut-down of oil
production for approximately 6 months, resulted in a
60-200% increase in oil recovery. After production
resumed, a reduction in pH, an increase in carbon
dioxide and a decrease in oil viscosity were observed.
Increased numbers of D. desulfuricans in the water
produced from the reservoir were also detected. It is
the author's belief, however, that the use of sulphate-
reducing bacteria such as D. desulfuricans would not be
favoured in, or recommended to, the oil industries, due
to the adverse effects an increase in sulphur content
could have on the oil quality.
Three wells on the Burnett J lease (USA) were used
in a pilot investigation of M E O R which involved the
injection of 80 1of kerosene, followed by 15 I of a com-
mercial biosurfactant product (RAM Biochemicals'
WelPrep 5). The wells were then flushed with saltwater
and shut down for 48-96 h. Oil production increased
five-fold from 0.3 barrel/day before treatment, to 1"6
barrel/day after treatment (Nelson & Launt, 1991). In
another set of experiments involving the injection of
microorganisms and nutrients into the wells, followed
by a 3 day shut-down period; an 11-25% increase in
weekly oil production was detected (Nelson & Launt,
1991; Bryant etaL, 1993).
Similarly, injections of P. aeruginosa, Xanthomonas
campestris and B. licheniformis through the well casing,
followed by longer shut-down periods of 40 and 64
days for two wells at the Daqing oil field in China,
resulted in an increase in the numbers of injected bac-
teria, organic acids and carbon dioxide and a slight
 Biosurfactants production and appfications
decrease in the interfacial tension in the oil-water
mixtures. The two wells tested showed an increase
from 3"5 to 5"5 t/day in the first and from 7"6 to 10-11
t/day in the second (Zhang & Zhang, 1993).
In all the previously-mentioned studies, however, it
is not known what effect the shut-down of the oil pro-
duction well itself may have had on the increase in oil
production, which is a serious drawback in the
methodology. Furthermore, a field experiment carried
out at the Romashkino oil field in Russia, where wells
were injected with phosphate and nitrogen to stimulate
the growth of native microflora (Ivanov et al., 1993)
resulted in an increase of 32"9% in oil production, the
mechanisms of which are not yet clear.
Nevertheless, carefully controlled field studies of
M E O R at the Alton Field in Queensland, Australia
were carried out (Sheehy, 1990). The M E O R strategy
used was to introduce microorganisms that were pre-
screened in sandpacks designed to simulate the
physical parameters found in the reservoir. Laboratory
analyses of nutrient requirements, metabolite produc-
tion and interactions with the indigenous microbiota
were carried out. A mixture of microorganisms and
nutrients was filtered through 10 and 28 ~m filters and
injected into the well followed by a shut-down of 20
days. Control experiments were initiated in which the
natural baseline production was determined before and
after the trial shut-down period, as well as after a con-
trol injection of water. An approximately 40% ino ease
in oil production was observed, compared to the con-
trol baseline (Sheehy, 1990). Additionally, a decrease
in the level of the base sediment and water, with an
increased percentage of oil content was observed.
Increased gas produced in the reservoir was found to
be the result of increased carbon dioxide and methane.
Microbial numbers were found to rise from = 103/ml
in pre-injection water to > 105/ml after microbial
injection. Sulphate-reducing bacteria were not
observed to be stimulated and H2S production was not
detected.
In all the previous reports involving micro-
organisms, no changes in any physical characteristics of
the oil were reported or observed, however, occasion-
ally the interfacial tension of the water-oil interface
decreased compared to the control values. These
results are indicative of the effectiveness of M E O R in
obtaining oil with the same desired characteristics as
before the treatment.
CONCLUSION AND FUTURE O U T L O O K
The usefulness of biosurfactants in the emulsification
of aqueous hydrocarbon mixtures has been clearly
demonstrated. Biosurfactants in many cases have
proved to be more effective than chemical surfactants
and have the added benefit of being biodegradable.
Studies of oil- or hydrocarbon-contaminated sand or
soil have also indicated that microorganisms which
produce biosurfactants, when stimulated properly, can
aid bioremediation. Laboratory studies have shown
9
that the addition of biosurfactant mixtures alone may
be useful for stimulating biodegradation of these con-
taminants in the environment. Biosurfactants have also
demonstrated their usefulness in the solubilization and
removal of oil from sand and sludge in oil storage
tanks. Therefore, in ecological terms, the use of bio-
surfactants is obvious for closed systems but remains
speculative in the open environment.
The utility of M E O R has not been conclusively
documented in the field at this stage. Preliminary find-
ings from the few investigations carried out to date,
however, seem promising. The precise mechanism of
enhanced oil recovery in situ is unclear due to the lack
of controls in some cases, with the unforseen dif-
ficulties usually encountered in situ and insufficient
analyses in other cases. It would appear that in certain
circumstances M E O R could be a viable alternative,
which, if carefully applied, could prove to be an econo-
mically-feasible method of enhancing oil recovery.
However, the technologies involved require an inter-
disciplinary effort among microbiologists, biochemists,
geologists and petroleum engineers. An evaluation of
the different criteria for the application of the various
microbial methods needs to be extensively investigated.
Future efforts in strain improvement and development
with the aid of genetic engineering are expected to help
progress in this emerging technology.
REFERENCES
AI-Awadhi, N., Williamson, K. J. & Isok, J. D. (1994).
Remediation of Kuwait's oil-contaminated soils. In Hydro-
carbon Contaminated Soils and Groundwater, ed. P. T.
Kostecki & E. J. Calabrese, Vol. 3. Lewis Publisher,
Michigan, USA (in press).
Abu-Ruwaida, A. S., Banat, I. M., Haditirto, S., Salem, A. &
Kadri, M. (1991a). Isolation of biosurfactant-producing
bacteria -- product characterization and evaluation. Acta
Biotechnologica, 11, 315-24.
Abu-Ruwaida, A. S., Banat, I. M., Haditirto, S. & Khamis, A.
(1991 b). Nutritional requirements and growth charac-
teristics of a biosuffactant-producing Rhodococcus bacte-
rium. World J. Microbiol. & Biotech., 7, 53-61.
Adkins, J. P., Cornell, L. A. & Tanner, R. S. (1992). Micro-
bial composition of carbonate petroleum reservoir fluids.
GeomicrobioL J., 10, 87-97.
Adkins, J. P., Tanner, R. S., Udegbunam, E. O., Mclnerney,
M. J. & Knapp, R. M. (1992). Microbially enhanced oil
recovery from unconsolidated limestone cores. Geomicro-
biol. J., 10, 77-86.
Akit, J., Cooper, D.I., Manninen, K. I. & Zajic, J. E. (1981).
Investigation of potential biosurfactant production among
phytopathogenic Corynebacteria and related soil
microbes. Curr. Microbiol., 6, 145-50.
Arima, K., Kakinuma, A. & Tamura, G. (1968). Surfactin, a
crystalline peptidolipid surfactant produced by Bacillus
subtilis: isolation, characterization and its inhibition of
fibrin clot formation. Biochem. Biophys. Res. Comm., 31,
488-94.
Atlas, R. M. (1981). Microbial degradation of petroleum
hydrocarbons: an environmental perspective. Microbiol.
Rev., 45, 180-209.
Atlas, R. M. (1993). Bacteria & bioremediation of marine
oil-spills. Oceanus, 36, 71-81.
10 L M. Banat
Atlas, R. M. & Bartha, R. (1992). Hydrocarbon biodegrada-
tion and oil-spill bioremediation. Adv. Microb. Eco., 12,
287-338.
Ban, T. & Sato, T. (1993). Aqueous microbial biosurfactant
solutions exhibiting ultra-low tension at oil-water inter-
faces. Dev. Petr. Sci., 39, 115-25.
Banat, I. M. (1993). The isolation of a thermophilic bio-
surfactant producing Bacillus sp. Biotech. Lett., 15,
591-4.
Banat, I. M., Samarah, N., Murad, M., Horne, R. & Banerjee,
S. (1991). Biosurfactant production and use in oil tank
clean-up. World J. MicrobioL & Biotech., 7, 80-8.
Bertrand, J. C., Bonin, P., Goutx, M., Gauthier, M. & Mille,
G. (1994). The potential application of biosurfactants in
combatting hydrocarbon pollution in marine environment.
Res. MicrobioL, 145, 53-6.
Bragg, J. R., Prince, R. C., Harner, E. J. & Atlas, R. M.
(1994). Effectiveness of bioremediation for the Exxon
Valdez oil spill. Nature, 368, 413-18.
Broderick, L. S. & Cooney, J. J. (1982). Emulsification of
hydrocarbons by bacteria from freshwater ecosystems.
Dev. Ind. MicrobioL, 23,425-34.
Bryant, E O. (1990). Improved method for the isolation of
biosurfactant glycolipids from Rhodococcus sp. strain
H13A. AppL Environ. MicrobioL, 56, 1494-6.
Bryant, R. S. (1991). MEOR screening criteria fit 27% of
U.S. oil reservoirs. Oil & GasJ., 89, 56-9.
Bryant, R. S., Stepp, A. K., Bertus, K. M., Burchfield, T. E. &
Dennis, M. (1993). Microbial-enhanced waterflooding
field pilots. Dev. Petr. Sci., 39, 289-306.
Bubela, B. (1985). Effect of biological activity on the move-
ment of fluids through porous rocks and sediments and its
application to enhanced oil recovery. Geomicrobiol. J., 4,
313-27.
Bubela, B. (1987). A comparison of strategies for enhanced
oil recovery using in situ and ex situ produced biosur-
factants. Surfactant Science Series, 25, 143-61.
Burger, A. E. (1993). Estimating the mortality of seabirds
following oil-spills -- effects of spill volume. Mar. Poll.
Bull., 26, 140-3.
Bums, K. A., Garrity, S. D. & Levings, S. C. (1993). How
many years until mangrove ecosystems recover from
catastrophic oil-spills. Mar. Poll. Bull., 26, 239-48.
Chang, Y. (1987). Preliminary studies assessing sodium pyro-
phosphate effects on microbially mediated oil recovery.
Ann. New York Acad. Sci., 506, 296-307.
Clark, J. B., Munnecke, D. M. & Jenneman, J. I. (1981). In
situ microbial enhancement of oil production. Dev. Ind.
MicrobioL, 22,695-701.
Cooper, D. G. (1986). Biosurfactants. Microbiol. Sci., 3,
145-9.
Cooper, D. G. & Goldenberg, B. G. (1987). Surface-active
agents from two Bacillus species. AppL Environ. Micro-
bioL, 53,224-9.
Cooper, D. G., MacDonald, C. R., Duff, J. B. & Kosaric, N.
(1981 a). Enhanced production of surfactant from Bacillus
subtilis by continuous product removal and metal cation
addition. Appl. Environ. MicrobioL, 42,408-12.
Cooper, D. G., Zajic, J. E. & Denis, C. (1981b). Surface-
active properties of a biosurfactant from Corynebacterium
lepus. J. Amer. Oil Chem. Soc., 58, 77-80.
Cooper, D. G., Zajic, J. E. & Gerson, D. E (1979). Produc-
tion of surface-active lipids by Corynebacterium lepus.
AppL Environ. Microbiol., 37, 4-10.
Cooper, D. G., Zajic, J. E., Gerson, D. E & Manninen, K. I.
(1980). Isolation and identification of biosurfactants
produced during anaerobic growth of Clostridium pasteu-
rianum. J. Ferment. Tech., 58, 83-6.
Cusack, E, Singh, S., McCarthy, C., Grieco, J., De Rocco, M.,
Nguyen, D., Lappin-Scott, H. & Costerton, J. W. (1992).
Enhanced oil recovery three-dimensional sandpack simu-
lation of ultramicrobacteria resuscitation in reservoir for-
mation. J. Gen. Microbiol., 138, 647-55.
Desai, J. D. & Desai, A. J. (1993). Production of bio-
surfactants. In Biosurfactants Production -- Properties --
Applications, ed. N. Kosaric. Marcel Dekker, New York,
pp. 65-97.
Davila, A., Marchal, E & Vandecasteele, J. (1992). Kinetics
and balance of a fermentation free from product inhibi-
tion: soporose lipid production by Candida bombicola.
Appl. Microbiol. Biotech., 38, 6-11.
Drouin, C. M. & Cooper, D. G. (1992). Biosurfactants and
aqueous two-phase fermentation. Biotech. Bioeng., 40,
86-90.
Eliseev, S. A., Vildanova-Martsishin, R., Shulga, A., Shabo,
A. & Turovsky, A. (1991). Oil-washing bioemulsifier pro-
duced by Bacillus sp. Microbiol. J., 53, 61-6.
Falatko, D. M. & Novak, J. T. (1992). Effects of biologically
produced surfactants on the mobility and biodegradation
of petroleum hydrocarbons. Water Environ. Res., 64,
163-9.
Fiechter, A. (1992a). Biosurfactants: moving towards in-
dustrial application. Trends in Biotech., 10, 208-17.
Fiechter, A. (1992b). Integrated systems for biosurfactant
synthesis. Pure Appl. Chem., 64, 1739-43.
Forsell, P., Parovuori, P., Linko, P. & Poutanen, K. (1993).
Enzymatic transesterification of rapeseed oil and lauric
acid in a continuous reactor. J. Amer. Oil Chem. Soc., 70,
1105-9.
Francy, D. S., Thomas, J. M., Raymond, R. L. & Ward, C. H.
(1991). Emulsification of hydrocarbons by subsurface
bacteria. J. Ind. Microbiol., 8, 237-46.
Fry, I. J., Chakrabarty, A. M. & DeFrank, J. J. (1993a). In situ
bioremediation of oil-contaminated soil and ground water
of the Straford army engine plant using natural surfact-
ants. Proc. 1992 CRDEC Science Conf. on Chemical
Defense, Edgewood, Maryland, USA, p. 362.
Fry, I. J., Checkai, R. T., Lynch, M., Rahrbaugh, D. &
DeFrank, J. J. (1993b). Bioremediation of oil-con-
taminated soil by microbial inoculation or surfactant treat-
ment. Proc. 1993 CRDEC Science Conf. on Chemical
Defense, Aberdeen Proving Ground, Maryland, USA (in
press).
Georgiou, G., Lin, S. & Sharma, M. M. (1992). Surface active
compounds from microorganisms. Bio/Tech., 10, 60-5.
Gobbert, U., Lang, S. & Wagner, E (1984). Sophorose lipid
formation by resting cells of Torulopsis bombicola. Bio-
tech. Lett., 6, 225-30.
Guerra-Santos, L., Kappeli, O. & Fiechter, A. (1984).
Pseudomonas aeruginosa biosurfactant production in con-
tinuous culture with glucose as carbon source. Appl.
Environ. Microbiol., 48, 301-5.
Guerra-Santos, L., Kappeli, O. & Fiechter, A. (1986).
Dependence of Pseudomonas aeruginosa continuous
culture biosurfaetant production on nutritional and
environmental factors. Appl. Microbiol. Biotech., 24,
443-8.
Haferburg, D., Hommel, R., Claus, R. & Kleber, H. (1986).
Extracellular microbial lipids as biosurfactants. Adv. Bio-
chem. Engng/Biotech., 33, 53-93.
Harvey, S., Elashvili, I., Valdes, J. J., Kamely, D. & Chakra-
barty, A. M. (1990). Enhanced removal of Exxon Valdez
spilled oil from Alaskan gravel by a microbial surfactant.
Bio/Tech., 8, 228-30.
Hitzman, D. O. (1983). Petroleum microbiology and the
history of its role in enhanced oil recovery. In Proc. 1982
International Conf.: Microbial Enhancement of Oil
Recovery, ed. E. C. Donaldson & J. B. Clark. NTIS,
Springfield, USA, pp. 163-218.
Hitzman, D. O. (1988). Review of microbial enhanced oil
recovery field tests. In Proc. Syrup. on Applying Micro-
organisms to Petroleum Technology, ed. T. S. Burchfield &
R. S. Bryant. NTIS, Springfield, USA, pp. VI-1-VI-41.
 Biosurfactants production and applications
Hommel, R. K. & Huse, K. (1993). Regulation of sophorose
lipid production by Candida (Torulopsis) apicola. Biotech.
Lett., 15,853-8.
Horowitz, S., Gilbert, J. N. & Griffin, W. M. (1990). Isolation
and characterization of a surfactant produced by Bacillus
licheniformis 86. J. Ind. Microbiol., 6, 243-8.
Inoue, S. & Ito, S. (1982). Sophrolipids from Torulopsis bom-
bicola as microbial surfactants in alkane fermentations.
Biotech. Lett., 4, 3-8.
Ishigami, Y., Gama, Y., Ishii, E & Kook Choi, Y. (1993).
Colloid chemical effect of polar head moieties of a
rhamnolipid-type biosurfactant. Langmuir, 9, 1634-6.
Itoh, S. & Suzuki, T. (1974). Fructose-lipids of Arthrobacter,
Corynebacteria, Nocardia and Mycobacteria grown on
fructose. Agr. Biol. Chem., 38, 1443-9.
Ivanov, M. V., Belyaev, S. S., Borzenkov, I. A., Glumov, I. E
& Ibatullin, R. R. (1993). Additional oil production during
field trial in Russia. Dev. Petr. Sci., 39, 373-81.
Jack, T. R. (1988). Microbially enhanced oil recovery. Bio-
recovery, 1, 59-73.
Jain, D. K., Collins-Thompson, D. L., Lee, H. & Trevors, J. T.
(1991). A drop-collapsing test for screening surfactant-
producing microorganisms. J. Microbiol. Meth., 13,
271-9.
Jain, D. K., Lee, H. & Trevors, J. T. (1992). Effect of addition
of Pseudomonas aeruginosa UG2 inocula or biosur-
factants on biodegradation of selected hydrocarbons in
soft. J. Ind. Microbiol., 10, 87-93.
Javaheri, M., Jermeman, G. E., McInerney, M. J. & Knapp,
R. M. (1985). Anaerobic production of a biosurfactant by
Bacillus licheniformis JF-2. Appl. Environ. Microbiol., 50,
698-700.
Jenneman, G. E., Knapp, R. M., Mclnerney, M. J., Menzie,
D. E. & Revus, D. E. (1984). Experimental studies of in
situ microbial enhanced oil recovery. Soc. Petr. Engin. J.,
Feb., 33-7.
Jelmeman, G. E., McInerney, M. J., Knapp, R. M., Clark,
J. B., Ferro, J. M., Revus, D. E. & Menzie, D. E. (1983). A
halotolerant, biosurfactant-producing Bacillus species
potentially useful for enhanced oil recovery. Dev. Ind.
Microbiol., 24, 485-92.
Kappell, O. & Fiechter, A. (1976). The mode of interaction
between the substrate and cell surface of the hydrocarbon-
utilizing yeast, Candida tropicalis. Biotech. Bioeng., 18,
967-74.
Khire, J. M. & Khan, M. I. (1994a). Microbially enhanced oil
recovery (MEOR). Part 1. Importance and mechanism of
MEOR. Enzyme Microb. Tech., 16, 170-2.
Khire, J. M. & Khan, M. I. (1994b). Microbially enhanced oil
recovery (MEOR). Part 2. Microbes and the subsurface
environment for MEOR. Enzyme Microb. Tech., 16,
258-9.
Kitamoto, D., Fuzishiro, T., Yanagishita, H., Nakane, T. &
Nakahara, T. (1992a). Production of mannosylerythritol
lipids as biosurfactants by resting cells of Candida
antarctica. Biotech. Lea., 14, 305-10.
Kitamoto, D., Nakane, T., Nakao, N., Nakahara, T. &
Tabuchi, T. (1992 b). Intracellular accumulation of manno-
sylerythritol lipids as storage materials by Candida antarc-
tica. Appl. Microbiol. Biotech., 36, 768-72.
Kitamoto, D., Yanaglshita, H., Shinbo, T., Nakane, T.,
Kamisava, C. & Nakahara, T. (1993). Surface-active
properties and antimicrobial activities of mannosylerythri-
tol lipids as biosurfactants produced by Candida antarc-
tica. J. Biotech., 29, 91-6.
Kosaric, N. (1993). Biosurfactants Production -- Properties --
Applications. Marcel Dekker, New York.
Lazar, I., Dobrota, S. & Stefanescu, M. (1988). Some con-
siderations concerning nutrient support injected into
reservoirs subjected to microbiological treatment. In Proc.
Symp. on Applying Microorganisms to Petroleum Techno-
11
logy, ed. T. E. Burchfield & R. S. Bryant. NTIS, Spr-
ingfield, USA, pp. XIV- 1-XIV-6.
Lesik, O. Y., Karpenko, E. V., Elysseev, S. A. & Turovsky,
A. A. (1989). The surface-active and emulsifying propert-
ies of Candida lipolytica Y-917 grown on n-hexadecane.
Microbiol. J., 51, 56-9.
Lesik, O. Y., Elyseev, S. A., Polulyakh, O. V. & Karpenko,
E. V. (1991). Production of a surface-active complex by
the culture of carotene-synthesizing yeast Phaffia rhodo-
zyma and its emulsifying properties. Microbiol. J., 53,
36-40.
Lillienberg, L., Hogstedt, B., Jarvhohn, B. & Nilson, L.
(1992). Health-effects of tank cleaners. Amer. Ind.
Hygiene Assoc. J., 53, 375-80.
Lin, S.-C., Carswell, K. S., Sharma, M. M. & Georgiou, G.
(1994). Continuous production of the lipopeptide bio-
surfactant of Bacillus licheniformis JF-2. Appl. Microbiol.
Biotech., 41,281-5.
MacDonald, C. R., Cooper, D. G. & Zajic, J. E. (1981).
Surface-active lipids from Nocardia grown on hydro-
carbons. Appl. Environ. Microbiol., 41, 117-23.
Mclnerney, M. J., Javaheri, M. & Nagle, D. P. (1990).
Properties of the biosurfactant produced by Bacillus
licheniformis strain JF-2. J. Ind. Microbiol., 5, 95-102.
Mercade, M. E. & Manresa, M. A. (1994). The use of agro-
industrial by-products for biosurfactant production. J.
Amer. Oil Chem. Soc., 71, 61-4.
Mercade, M. E., Manresa, M. A., Robert, M., Espuny, M. J.,
Deandres, C. & Guinea, J. (1993). Olive oft mill effluent
(Oome) -- new substrate for biosurfactant production.
Biores. Tech., 43, 1-6.
Morikawa, M., Daido, H., Takao, T., Murata, S., Shimonishi,
Y. & Imanaka, T. (1993). A new lipopeptide biosurfactant
produced by Arthrobacter sp. strain MIS38. J. Bacteriol.,
175, 6459-66.
Morikawa, M., Ito, M. & Imanaka, T. (1992). Isolation of a
new surfactin producer Bacillus pumilus A-l, and cloning
and nucleotide sequence of the regulator gene, psf-1. J.
Ferment. Bioengng, 74, 255-61.
Morkes, J. (1993). Oil-spills -- whose technology will clean
up. R &D Magazine, 35, 54-6.
Miiller-Hurtig, R., Wagner, F., Blaszczyk, R. & Kosaric, N.
(1993). Biosurfactants for environmental control. In Bio-
surfactants Production -- Properties -- Applications, ed. N.
Kosaric. Marcel Dekker, New York, pp. 447-69.
Mulligan, C.N., Mahmourides, G. & Gibbs, B. E (1989). The
influence of phosphate metabolism on biosurfactant pro-
duction by Pseudomonas aeruginosa. J. Biotech., 12,
199-210.
Mutua, L. N. & Akoh, C. C. (1993a). Lipase-catalyzed
modification of phospholipids: incorporation of n-3 fatty
acids into biosurfactants. J. Amer. Oil Chem. Soc., 70,
125-8.
Mutua, L. N. & Akoh, C. C. (1993b). Synthesis of alkyl
glycoside fatty acid esters in non-aqueous media by
Candida sp. lipase. J. Amer. Oil Chem. Soc., 70, 43-6.
Nelson, S. J. & Launt, P. D. (1991 ). Stripper well production
increased with MEOR treatment. Oil & Gas J., 89,
114-18.
Neu, T. R., Dengler, T., Jann, B. & Poralla, K. (1992). Struc-
tural studies of an emulsion-stabilizing exopolysaccharide
produced by an adhesive, hydrophobic Rhodococcus
strain. J. Gen. Microbiol., 138, 2531-7.
Neu, T. R., Harmer, T. & Poralla, K. (1990). Surface active
properties of viscosin: a peptidolipid antibiotic. Appl.
Microbiol. Biotech., 32, 518-20.
Oberbremer, A., Muller-Hurtiq, R. & Wagner, E (1990).
Effect of the addition of microbial surfactants on hydro-
carbon degradation in a soil population in a stirred
reactor. Appl. Microbiol. Biotech., 32, 485-9.
Palejwala, S. & Desai, J. D. (1989). Production of an extra-
12 L M. Banat
cellular emulsifier by a Gram negative bacterium. Biotech.
Lett., 11, 115-18.
Passeri, A. (1992). Marine biosurfactants: IV. Production,
characterization and biosynthesis of an anionic glucose
lipid from the marine bacterial strain MM1. Appl. Micro-
biol. Biotech., 37, 281-6.
Peypoux, E & Michel, G. (1992). Controlled biosynthesis of
Val7- and Leu7-surfactins. Appl. Microbiol. Biotech., 36,
515-17.
Pfiffner, S. M., Mclnerney, M. J., Jenneman, G. E. & Knapp,
R. M. (1986). Isolation of halotolerant, thermotolerant,
facultative polymer-producing bacteria and charac-
terization of the exopolymer. Appl. Environ. Microbiol.,
51, 1224-9.
Reynolds, P. J., Sharma, P., Jenneman, G. E. & Mclnerney,
M. J. (1989). Mechanisms of microbial movement in sub-
surface materials. Appl. Environ. Microbiol., 55, 2280-6.
Robert, M., Mercade, M. E., Bosch, M. P., Parra, J. L.,
Espuny, M. J., Manresa, M. A. & Guinea, J. (1989). Effect
of the carbon source on biosurfactant production by
Pseudomonas aeruginosa 44T 1. Biotech. Lett., 11, 871-4.
Rocha, C., San-Bias, E, San-Bias, G. & Vierma, L. (1992).
Biosurfactant production by two isolates of Pseudomonas
aeruginosa. World J. Microbiol. Biotech., 8, 125-8.
Rosenberg, E. (1986). Microbial surfactants. CRC Crit. Rev.
Biotech., 3, 109-32.
Rosenberg, E., Zuckerberg, A., Rubinovits, C. & Gutnick,
D. L. (1979). Emulsifier of Arthrobacter RAG-1: Isolation
and emulsifying properties. Appl. Environ. Microbiol., 37,
409-13.
Sandrin, C., Peypoux, E & Michel, E (1990). Coproduction
of surfactin and iturin A, lipopeptides with surfactant and
antifungal properties, by Bacillus subtilis. Biotech. Appl.
Biochem., 12,370-5.
Sarker A, K., Goursaud, J. C., Sharma, M. M. & Georgiou,
G. (1989). A critical evaluation of MEOR processes. In
Situ, 13, 207-38.
Scheibenbogen, K., Zytner, R. G., Lee, H. & Trevors, J. T.
(1994). Enhanced removal of selected hydrocarbons from
soil by Pseudomonas aeruginosa UG2 biosurfactants and
some chemical surfactants. J. Chem. Tech. Biotech., 59,
53-9.
Shafeeq, M., Kokub, D., Khalid, Z. M., Khan, A. M. & Malik,
K. A. (1989). Degradation of different hydrocarbons and
production of biosurfactant by Pseudomonas aeruginosa
isolated from coastal waters. MIRCEN J. Appl. Microbiol.
Biotech., 5, 505-10.
Shaw, D. G. (1992). The Exxon-valdez oil-spill -- ecological
and social consequences. Environ. Conserv., 19, 253-8.
Sheehy, A. J. (1990). Field studies of microbial EOR. 7th
Symp. on Enhanced Oil Recovery. Tulsa, OK, USA, pp.
785-90.
Shennan, J. L. & Levi, J. D. (1987). In situ microbial
enhanced oil recovery. In Biosurfactants and Biotech-
nology, ed. N. Kosaric, W. L. Cairns & N. C. C. Gray.
Marcel Dekker, New York, pp. 163-81.
Shulga, A. N., Karpenko, E. V., Eliseev, S. A. & Turovsky,
A. A. (1993). The method for determination of anioiao-
genic bacterial surface-active peptidolipids. Microbiol. J.,
55, 85-8.
Shulga, A. N., Karpenko, E. V., Eliseev, S. A., Turovsky, A.
A. & Koronelli, T. V. (1990). Extracellular lipids and
surface-active properties of the bacterium Rhodococcus
erythropolis depending on the source of carbon nutrition.
Mikrobiologya, 59, 443-7.
Singer, M. E. Vogt & Finnerty, W. R. (1984). Microbial meta-
bolism of straight and branched alkanes. In Petroleum
Microbiology, ecl. R. Atlas. Collier MacMillan, New York,
pp. 1-59.
Singer, M. E. Vogt & Finnerty, W. R. (1990). Physiology of
biosurfactant synthesis by Rhodococcus species H13-A.
Can. J. MicrobioL, 36, 741-5.
Sutton, R. (1992). Use of biosurfactants produced by
Nocardia amarae for removal and recovery of non-ionic
organics from aqueous solutions. Water Sci. Tech., 26,
9-11.
Suzuki, T., Tanaka, H. & Itoh, I. (1974). Sucrose lipids of
Arthrobacteria, Corynebacteria and Nocardia grown on
sucrose. Agr. Biol. Chem., 33, 190-5.
Tanner, R. S., Udegbunam, E. O., Mclnerney, M. J. & Knapp,
R. M. (1991). Microbially enhanced oil recovery from
carbonate reservoirs. Geomicrobiol. J., 9, 169-95.
Thimon, L., Peypoux, E, Maget-Dana, R., Roux, B. &
Michel, G. (1992). Interactions of bioactive lipopeptides,
iturin A and surfactin from Bacillus subtilis. Biotech. Appl.
Biochem., 16, 144-51.
Totani, Y. & Hara, S. (1991). Preparation of polyunsaturated
phospholipids by lipase-catalyzed transesterification. J.
Amer. Oil Chem. Soc., 68, 848-51.
Van der Vegt, W., Vander Mei, H. C., Noordmans, J. &
Busscher, H. J. (1991). Assessment of bacterial biosur-
factant production through axisymmetric drop shape
analysis by profile. Appl. Microbiol. Biotech., 35, 766-70.
Van Dyke, M. I., Couture, P., Brauer, M., Lee, H. & Trevors,
J. T. (1993a). Pseudomonas aeruginosa UG2 rhamnolipid
biosurfactants: structural characterization and their use in
removing hydrophobic compounds from soil. Can. J.
Microbiol., 39, 1071-8.
Van Dyke, M. I., Gulley, S. L., Lee, H. & Trevors, J. T.
(1993b). Evaluation of microbial surfactants for recovery
of hydrophobic pollutants from soil. J. Ind. Microbiol., 11,
163-70.
Venkata Ramana, K. & Karanth, N. G. (1989). Factors affect-
ing biosurfactant production using Pseudomonas aeru-
ginosa CFTR-6 under submerged conditions. J. Chem.
Tech. Biotech., 45,249-57.
Vrdoljak, M. M., Marinkovic, G., Pavusek, I. & Johanides, V.
(1992). Capability for degradation of crude oil hydro-
carbons by sea water microbial association of yeasts and
bacteria from the Kvarner bay. Periodicum Biologorum,
94, 169-78.
Wagner, M. (1991). Microbial enhancement of oil recovery
from carbonate reservoirs with complex formation
characteristics. Dev. Petr. Sci., 31,387-98.
Wasko, M. P. & Bratt, R. P. (1990). Properties of a bio-
surfactant produced by the fuel contaminant Ochro-
bactrum anthropii. Inter. Biodeter., 27, 265-73.
Yarbrough, H. F. & Coty, V. E (1983). Microbially enhanced
oil recovery from the Upper Cretaceous Nacatoch forma-
tion, Union County Arkansas. In Proc. 1982 International
Conf. on Microbial Enhancement of Oil Recovery, ed. E. C.
Donaldson & J. B. Clark. NTIS, Springfield, VA, USA, pp.
149-53.
Zajic, J. E., Guignard, H. & Gerson, D. E (1977). Emulsify-
ing and surface active agents from Corynebacterium
hydrocarboclastus. Biotech. Bioeng., 19, 1285-301.
Zhang, C. Y. & Zhang, J. C. (1993). A pilot test of EOR by in
situ microorganism fermentation in the Daqing oil field.
Dev. Petr. Sci., 39, 231-44.
Zhang, Y. & Miller, R. M. (1992). Enhanced octadecane dis-
persion and biodegradation by a Pseudomonas rhamno-
lipid surfactant (biosurfactant). Appl. Environ. Microbiol.,
58, 3276-82.