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Available online at www.sciencedirect.com
Colloids and Surfaces B: Biointerfaces 63 (2008) 73–82
Microbial surfactant-enhanced mineral oil recoveryunder laboratory conditions
N.K. Bordoloi, B.K. Konwar ∗Department of Molecular Biology and Biotechnology, Tezpur University,
Napaam, Tezpur 784028, Assam, India
Received 17 July 2007; received in revised form 6 November 2007; accepted 11 November 2007Available online 19 November 2007
bstract
Microbial enhanced oil recovery (MEOR) is potentially useful to recover incremental oil from a reservoir being beyond primary and secondaryecovery operations. Effort has been made to isolate and characterize natural biosurfactant produced by bacterial isolates collected from various oilelds of ONGC in Assam. Production of biosurfactant has been considered to be an effective major index for the purpose of enhanced oil recovery.n the basis of the index, four promising bacterial isolates: Pseudomonas aeruginosa (MTCC7815), P. aeruginosa (MTCC7814), P. aeruginosa
MTCC7812) and P. aeruginosa (MTCC8165) were selected for subsequent testing. Biosurfactant produced by the promising bacterial isolatesave been found to be effective in the recovery of crude oil from saturated column under laboratory conditions. Two bacterial strains: P. aeruginosaMTCC7815) and P. aeruginosa (MTCC7812) have been found to be the highest producer of biosurfactant. Tensiometer studies revealed thatiosurfactants produced by these bacterial strains could reduce the surface tension (σ) of the growth medium from 68 to 30 mN m−1 after 96 h
f growth. The bacterial biosurfactants were found to be functionally stable at varying pH (2.5–11) conditions and temperature of 100 ◦C. Thereatment of biosurfactant containing, cell free culture broth in crude oil saturated sand pack column could release about 15% more crude oil at0 ◦C than at room temperature and 10% more than at 70 ◦C under laboratory condition. 2007 Elsevier B.V. All rights reserved.aseTfioaocte
eywords: Microbial surfactant; Mineral oil; Microbial oil recovery
. Introduction
Biosurfactants are amphipathic molecules with bothydrophilic and hydrophobic domains [1,2]. They are mainlyroduced by hydrocarbon utilizing microorganisms, exhibitingurface activity [3]. These molecules reduce surface tension (σ),ritical micelle concentration (CMC) and interfacial tension inoth aqueous solutions and hydrocarbon mixtures. These prop-rties create micro-emulsions leading to micelle formation inhich hydrocarbons can solubilize in water or water in hydro-
arbons. The properties of the various biosurfactants have beenxtensively reviewed by Fiechter [4], Georgiou et al. [5] and
osaric [6]. Chemically synthesized surfactants have been usedn the oil industry to aid the clean up of oil spills, as well aso enhance oil recovery from oil reservoirs. These compounds
∗ Corresponding author. Tel.: +91 3712 267172; fax: +91 3712 267005/6.E-mail addresses: [email protected], [email protected]
B.K. Konwar).
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927-7765/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2007.11.006
re not biodegradable and can be toxic to the environment. Bio-urfactants, however, have been shown in many cases to havequivalent emulsification properties and are biodegradable [41].hus, there is an increasing interest in the possible use of biosur-
actants in mobilizing heavy crude oil, transporting petroleumn pipelines, managing oil spills, oil-pollution control, cleaningf oil sludge from storage facilities [39], soil/sand bioremedi-tion and microbial enhanced oil recovery (MEOR). MEORffers major advantages over conventional EOR in that lowerapital and chemical/energy costs are required [7]. There arehree main strategies adopted for the use of biosurfactants innhanced oil recovery or mobilization of heavy oils [2]: (i)njection of biosurfactant producing microorganisms into theeservoir through the well, with subsequent multiplication oficroorganisms in situ through the reservoir rocks; (ii) injec-
ion of selected nutrients into a reservoir, thus stimulating therowth of indigenous biosurfactant producing microorganisms;iii) production of biosurfactants ex situ and their subsequentnjection into the reservoir.
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4 N.K. Bordoloi, B.K. Konwar / Colloids an
MEOR is an important tertiary recovery technology utilizingicroorganisms and/or their metabolite to recover incremental
il from a reservoir being beyond primary and secondary recov-ry techniques [2,8,9], therefore, in the recent years, worldwidenterest has been growing in tertiary recovery techniques forore residual oil recovery. The concept of microorganisms to
nhanced oil recovery, MEOR, was first proposed in 1926 byeckman; but this technology has advanced from laboratoryased studies in the early 1980s to field application in the 1990s.t is generally accepted that approximately 30% of the oil presentn a reservoir can be recovered using current EOR technology10]. There are several factors responsible for poor oil recoveryrom the existing oil producing wells. The main factor is the lowermeability of some reservoirs or the high viscosity of the oil,hich results in poor mobility. High interfacial tensions between
he water and oil may also result in high capillary forces retaininghe oil in the reservoir rock [11]. Techniques involving the usef chemical or physical processes such as pressurizations, waterooding or steaming are generally inapplicable to most oil reser-oirs. Laboratory studies on MEOR have typically utilized coreamples and columns containing the desired substrate. Theseubstrates have been utilized to demonstrate the usefulness ofiosurfactants in oil recovery from sand and limestone [2]. Forhe production of biosurfactants, usually the low cost substratesuch as molasses and inorganic nutrients are used. Therefore,ffort has been made to isolate and characterize natural biosur-actants produced by bacterial isolates collected from various oilelds of Assam Asset Basin, Oil and Natural Gas Corporation,ssam, India.
. Materials and methods
.1. Soil samples for microbial isolation
Crude oil-contaminated soil samples were collected fromhree different oil fields of Assam: Lakuwa, Gelekey andudrasagar. Collection sites were contaminated with crude oil
ince the time of drilling and subsequent production of crude oil.amples were taken from just below the soil surface. Moisture
evel in soil samples was maintained with the addition of 0.85%aline water at regular interval. Samples were stored at normaloom temperature (20 ± 5 ◦C) for subsequent use.
.2. Isolation of bacterial isolates
One gram of crude oil-contaminated soil sample from each ofamples collected from three different sites was serially dilutedith sterile distilled water. Dilutions 10−3–10−6 were plated on
gar solidified Bushnell-Hass [12] medium containing 1% n-exadecane. The cultures were incubated at 37 ◦C and observedfter 24 h. Subsequently, pure cultures were maintained at 37 ◦Cn nutrient agar and LB plates.
Another technique was also used for the isolation of cul-
urable bacterial isolates. Microbial strains were isolated fromnrichment cultures established in mineral medium supple-ented with n-hexadecane (1%, v/v). Pure cultures were grownn agar solidified mineral medium containing hexadecane as
wowe
faces B: Biointerfaces 63 (2008) 73–82
he sole source of carbon. Bacterial colonies surrounded by alearing zone indicated degradation of hexadecane.
Colony morphology and growth of each isolate wereecorded.
.3. Growth media
Bushnell-Hass medium (Difco) supplemented with 1% (v/v)-hexadecane was used for screening of potential hydrocar-on degrading and biosurfactant producing bacterial isolates.utrient agar and Luria–Bertani (LB) media (Merck, Germany
nd Himedia, India) were used for the maintenance of bacte-ial isolates. The mineral media used for enrichment cultureontained: urea – 2 g, (NH4)2SO4 – 2 g, Na2HPO4 – 3.61 g,H2PO4 – 1.75 g, MgSO4·7H2O – 0.2 g, CaCl2·2H2O – 50 mg,eSO47H2O – 1 mg, CuSO4·7H2O – 50 �g, H3BO3 – 10 (g,nSO4·5H2O – 10 �g, ZnSO4·7H2O – 70 �g, and MoO3 –
0 �g in 1 l of distilled water. The bacterial culture induced auto-atic maintenance of near neutral pH throughout the culture
eriod.
.4. Screening of potential biosurfactant producing isolates
Potential biosurfactant producing microbial isolates werecreened by a rapid drop-collapsing test [42]. In the technique, arop of culture broth was placed on an oil-coated paper surface.he absorption and spread of the droplet of the bacterial culture-roth confirmed the presence of biosurfactant, which causedenetration of the medium into the paper through the oil coat.
Along with the production of biosurfactant, each bacterialsolate also exhibited one more character in the formation ofoam during enrichment culture in mineral medium supple-ented with 2% (v/v) n-hexadecane, octadecane, dodecane or
lucose (w/v).
.5. Biomass determination
Aliquots measuring 2 ml at different time intervals of cultureere taken in Eppendorf tubes and centrifuged at 10,000 × g
or 10 min. Biomass obtained were dried overnight at 45 ◦C andeighed.
.6. Determination of biosurfactant yield
Biosurfactant produced by each bacterial isolate was sepa-ated and purified by the method described by Yakinov et al.13]. As per the method, bacterial cells were removed fromiosurfactant-containing medium by centrifugation (10,000 × g,5 min). The supernatant was subjected to acid precipitation bydding 6N HCl to obtain a final pH of 2.0 and allowing the pre-ipitate to form at 4 ◦C overnight. The precipitate thus obtainedas pelleted at 10,000 × g for 15 min. The pellet was washed
everal times with acidic water (pH 2.0, with HCl), again washed
ith alkaline water (pH 8.0, with NaOH) to achieve a final pHf 7.0 and then freeze dried overnight. The dried biosurfactantas extracted with solvent (chloroform–methanol, 65:15). Thextract was dried with the aid of rotary evaporator under vacuum.
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N.K. Bordoloi, B.K. Konwar / Colloids a
iosurfactant production was estimated following the method ofhang and Miller (1992) [40].
.7. Estimation of biosurfactant activity
The activity of the biosurfactant was determined by mea-uring changes in σ at air/water interface by using a Kruss K9T (Germany) tensiometer. Critical micelle dilution (CMD) isefined as the solubility of a surfactant in an aqueous phase ands commonly used to measure the efficiency of a surfactant [14].he σ of non-inoculated (control) culture medium as well as allacterial cultures was determined at the critical micelle dilutions0−1 and 10−2 i.e., CMD−1 and CMD−2, respectively, wereetermined using Du-Nouy Tensiometer (Kruss K9 ET Ten-iometer, Kruss, Germany) at room temperature (25 ◦C) usinghe ring correction mode of the instrument.
.8. Stability studies
Stability studies were carried out using the cell free brothbtained by centrifuging the cultures at 10,000 rpm for 10 min.ulture broth measuring 20 ml was heated to 100 ◦C in a boil-
ng water bath for different time intervals and cooled to roomemperature. σ values of each sample were measured at normaliosurfactant concentration, CMD−1 and CMD−2. To study theH stability of cell free culture broth, the pH of the cell freeulture broth was adjusted from 2 to 11 and then σ values wereetermined.
.9. Sand pack test
The potential application of the biosurfactant in MEOR wasvaluated using the ‘sand pack column’ technique described bybu-Ruwaida et al. [15]. A glass column (61 cm × 3.5 cm i.d.)as packed with 150 g of acid washed dry sand (cuttings col-
ected from a drilling site in Gelekey oil field, Assam Asset,ndia). The column was then saturated with crude oil (50 ml).he potential of the isolated surfactant for oil recovery wasstimated by pouring 50 ml of aqueous solution of biosurfac-ant (extraction from 100 ml culture broth) in the column. Themount of oil released was measured. The experiment was car-ied out at room temperature, 70 and 90 ◦C to assess the influencef temperature on biosurfactant-induced oil recovery.
.10. Statistical analysis
Statistical analysis was done by Student’s “t-test” [16] andactorial two-way ANOVA.
. Results
.1. Potential biosurfactant producing bacteria
Screening of bacterial cultures isolated from oil well-siteoil samples for the bioremediation of crude oil-contaminatedoils of Assam Asset and Assam Arakan basin resulted into2 strains capable of producing biosurfactant. The strains
stgp
rfaces B: Biointerfaces 63 (2008) 73–82 75
ere identified with the help of IMTECH, Chandigargh.hese 12 strains are Pseudomonas aeruginosa (MTCC7815),. aeruginosa (MTCC7812), Microbacterium (G35-I), P.eruginosa (MTCC8163), P. aeruginosa (MTCC8165), P. fluo-escens (L490-II), P. aeruginosa (L43-I), Bacillus licheniformisMTCC8166), B. circulans (MTCC8167), B. subtilis (R38-I),. aeruginosa (MTCC7814) and P. aeruginosa (MTCC7816).he bacterial isolates were not only found to degrade differentomponents of crude oil like hexadecane (aliphatic), ben-ene, toluene (aromatic), phenanthere and pyrene (polycyclicromatic), but also to produce biosurfactant in hydrocarbon-upplemented media. The ability of bacterial isolates toegrade crude oil components and glucose is presented inable 1. In the glucose-supplemented medium, the bacterial
solates P. aeruginosa (MTCC7815) followed by B. circu-ans (MTCC8167) and P. aeruginosa (MTCC8165) producedomparatively higher biomass of 3.5–3.1 g l−1, whereas, theemaining isolates possessed less biomass yield of 2.5–1.5 g l−1
or the entire growth period. In hexadecane-supplementededium, the isolate P. aeruginosa (MTCC8165), followed by
. aeruginosa (MTCC7815), P. aeruginosa (MTCC8163), B.irculans (MTCC8167) and P. aeruginosa (L43-I) exhibited bet-er growth with the biomass yield of 3.5–3.1 g l−1 in crude oilupplemented medium, the isolates B. subtilis (R38-I) followedy P. aeruginosa (MTCC7815), P. aeruginosa (MTCC8165)nd P. aeruginosa (MTCC8163) showed better growth with dryiomass yield of 1.4–1.0 g l−1; in benzene and toluene (aro-atic) hydrocarbon-supplemented media, the bacterial isolates
roduced comparatively low biomass of 0.8–0.2 g l−1. Similarly,n phenanthrene and pyrene-supplemented media (polycyclicromatic hydrocarbons), the isolates possessed comparativelyess growth with biomass yield of 1.0–0.1 g l−1.
Rapid drop-collapsing test and formation of foam duringnrichment culture in mineral media supplemented with dif-erent hydrocarbon components like n-hexadecane, octadecane,odecane as well as the simple carbon source glucose establishedhem to be potential for use in MEOR. Biomass and biosurfac-ant yield of these bacterial isolates in hexadecane-supplemented
edium are presented in Table 2. B. circulans (MTCC8167),. aeruginosa (MTCC7812) and B. subtilis (R38-I) exhibitedigher production of dry biomass. In the case of biosurfactantroduction, the strains of P. aeruginosa (MTCC7812), followedy MTCC7815, MTCC7814 and MTCC8165 were found toe superior. On the other hand, the biosurfactant produced byhe strains MTCC7816, MTCC8163, L43-I and MTCC8165aused the lowest surface tension of the culture medium. How-ver, on the basis of biosurfactant production potentiality thetrains MTCC8165, MTCC7815, MTCC7812 and MTCC7814ere selected for subsequent works. Each bacterial isolate was
lso cultured in MS medium without the addition of hexade-ane (control). But, none of the isolates could grow in theedium and produce biosurfactant. In Table 3, the growth of
he bacterial isolates and production of biossurfactant were pre-
ented in single control for all bacterial strains used. None ofhe bacterial isolates cultured in the medium devoid of glucose,lycerol and hexadecane could exhibit growth and biosurfactantroduction. The yield of biosurfactant was higher in hexadecane-
76 N.K. Bordoloi, B.K. Konwar / Colloids and Surfaces B: Biointerfaces 63 (2008) 73–82
Tabl
e1
Gro
wth
ofba
cter
iali
sola
tes
ondi
ffer
entc
arbo
nso
urce
sov
era
peri
odof
96h
cultu
re(m
ean
ofth
ree
expe
rim
ents
)
Car
bon
sour
ces
Dry
biom
ass
(gl−
1)
ofba
cter
iali
sola
tes
MT
CC
7815
G35
-IM
TC
C81
63M
TC
C81
66M
TC
C81
65M
TC
C81
67L
490-
IIL
43-I
R38
-IM
TC
C78
12M
TC
C78
14M
TC
C78
16
Glu
cose
3.5
±0.
42.
1±
0.1
2.5
±0.
22.
1±
0.2
3.1
±0.
23.
2±
0.2
2.2
±0.
12.
5±
0.1
1.5
±0.
12.
4±
0.2
2.3
±0.
11.
9±
0.1
Hex
adec
ane
3.1
±0.
32.
4±
0.1
3.1
±0.
12.
3±
0.1
3.5
±0.
13.
1±
0.1
2.5
±0.
23.
1±
0.2
1.8
±0.
12.
2±
0.3
2.1
±0.
12.
3±
0.1
Cru
deoi
l1.
2±
0.2
0.9
±0.
21.
0±
0.1
0.6
±0.
21.
1±
0.1
0.7
±0.
10.
9±
0.1
0.7
±0.
11.
4±
0.1
0.5
±0.
10.
6±
0.2
0.9
±0.
1B
enze
ne0.
6±
0.2
0.2
±0.
20.
7±
0.1
0.4
±0.
20.
5±
0.1
0.5
±0.
20.
4±
0.1
0.5
±0.
10.
5±
0.1
0.3
±0.
10.
4±
0.1
0.4
±0.
1To
luen
e0.
8±
0.2
0.2
±0.
10.
6±
0.1
0.5
±0.
10.
6±
0.2
0.6
±0.
40.
3±
0.2
0.3
±0.
20.
7±
0.2
0.5
±0.
10.
6±
0.2
0.5
±0.
1Ph
enan
thre
ne1.
0±
0.1
0.3
±0.
20.
9±
0.4
0.4
±0.
10.
8±
0.1
0.7
±0.
20.
5±
0.1
0.5
±0.
10.
6±
0.1
0.2
±0.
10.
4±
0.1
0.6
±0.
2Py
rene
0.7
±0.
10.
1±
0.1
0.8
±0.
20.
3±
0.2
0.6
±0.
20.
7±
0.3
0.6
±0.
10.
6±
0.1
0.8
±0.
10.
7±
0.3
0.6
±0.
10.
7±
0.1
Con
trol
0.1
±0.
010.
2±
0.01
0.1
±0.
010.
1±
0.01
0.2
±0.
010.
3±
0.01
0.2
±0.
010.
1±
0.01
0.1
±0.
00.
2±
0.01
0.1
±0.
010.
1±
0.01
Table 2Biomass and biosurfactant yield of bacterial isolates in (1%) hexadecane sup-plemented medium by bacterial isolates after 96 h of culture
Bacteria isolates MSM + 1% hexadecane in 96 h
DB (g l−1) BS (g l−1) ST (mN m−1)
P. aeruginosa (MTCC7815) 6.4 ± 1.2 4.6 ± 0.7 36.2 ± 0.3P. aeruginosa (MTCC7812) 8.6 ± 0.4 5.0 ± 0.8 38.4 ± 0.5Microbacterium (G35-I) 5.3 ± 0.4 0.8 ± 0.2 37.1 ± 0.4P. aeruginosa (MTCC8163) 6.2 ± 0.5 2.1 ± 0.3 33.5 ± 0.2P. aeruginosa (MTCC8165) 6.4 ± 0.6 2.9 ± 0.4 34.4 ± 0.3P. fluorescens (L490-II) 5.3 ± 0.4 1.8 ± 0.7 35.1 ± 0.5P. aeruginosa (L43-I) 6.1 ± 0.3 2.0 ± 0.8 34.2 ± 0.2B. licheniformis (MTCC8166) 4.2 ± 0.3 0.5 ± 0.6 37.4 ± 0.2B. circulans (MTCC8167) 8.7 ± 0.2 0.3 ± 0.1 37.2 ± 0.2B. subtilis (R38-I) 8.4 ± 0.1 0.3 ± 0.7 39.6 ± 0.3P. aeruginosa (MTCC7814) 6.4 ± 1.6 3.5 ± 0.9 39.6 ± 0.4P. aeruginosa (MTCC7816) 5.6 ± 1.3 2.0 ± 1.0 33.5 ± 0.3MSM with bacterial isolate
(control)0.1 ± 0.01 0.01 ± 0.02 68.01.0
Rd
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esults represented mean ± S.D. of three independent experiments. NB: DB,ry biomass; BS, yield of biosurfactant; ST, surface tension.
upplemented medium as compared to other two carbonources.
.2. Growth and biosurfactant production in differentarbon sources
The growth and biosurfactant production of the selectedacterial isolates in different carbon sources: glucose, glyc-rol and hexadecane were assessed. Biomass production andield of biosurfactant of each bacterial isolate in mediaupplemented separately with 2% glucose, glycerol and hex-decane are presented in Table 3. All four strains belongingo P. aeruginosa exhibited growth with biomass productionnd biosurfactant yield in glucose, glycerol and hexadecane-upplemented medium. But, the yield of biosurfactant wasigher in hexadecane-supplemented medium as compared tother two carbon sources.
.3. Efficacy of bacterial biosurfactant
Biosurfactant produced by four bacterial isolates weressessed for their ability to lower the surface tension (σ value)f culture media supplemented with carbon sources: glucose,lycerol and hexadecane. σ of culture media was determined byrawing samples up to 96 h with an interval of 24 h. Data thusbtained are presented in Table 4. Glucose supplemented cul-ure medium having the biosurfactant produced by P. aeruginosatrains MTCC7815 and MTCC7814 could lower down the sur-ace tension of the medium significantly to 32 and 34 mN m−1 in6 h as compared to the control medium having no biosurfactant71 mN m−1). In the glycerol-containing medium, the surface
ension reduction was the maximum in the case of the biosur-actant produced by the strains MTCC7815 and MTCC8165.ut, in the case of hexadecane supplemented medium the sur-ace tension reduction was significantly high all throughout 96 h
N.K. Bordoloi, B.K. Konwar / Colloids and Surfaces B: Biointerfaces 63 (2008) 73–82 77
Table 3Bacterial biomass and biosurfactant yield (g l−1) in media supplemented with different carbon sources after 96 h of incubation at 37 ◦C and 200 rpm
Bacterial isolates Glucose Glycerol Hexadecane
Biomass (g l−1) Yield of crudebiosurfactant (g l−1)
Biomass (g l−1) Yield of biosurfactant (g l−1)
Biomass (g l−1) Yield of biosurfactant (g l−1)
MSM without bacterial isolate (control) 0.2 ± 0.02 0.16 ± 0.03 0.2 ± 0.02 0.14 ± 0.03 0.2 ± 0.02 0.12 ± 0.03P. aeruginosa (MTCC8165) 5 ± 0.65*,** 2.45 ± 0.35*,** 5.3 ± 0.55*,** 3.5 ± 0.75*,** 6.4 ± 0.65*,** 2.95 ± 0.45*,**P. aeruginosa (MTCC7815) 10 ± 1.5*,** 3.65 ± 0.55*,** 6.9 ± 1.8*,** 4.25 ± 0.85*,** 6.4 ± 1.2*,** 4.66 ± 0.75*,**P , , 5 , , , ,
P 5
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. aeruginosa (MTCC7812) 5.4 ± 1.2* ** 4.0 ± 0.65* **
. aeruginosa (MTCC7814) 3.6 ± 1.1*,** 3.25 ± 0.58*,**
esults represented mean ± S.D. of three independent experiments; significanc
f incubation. The trend was similar in the case of glucose-upplemented medium.
.4. Properties of biosurfactant
.4.1. Critical micelle concentration (CMC) and dilutionCMD) of biosurfactant
CMC values of the biosurfactant were determined by mea-uring the σ values of water supplemented separately withifferent concentrations (log of mg l−1) of bacterial biosurfac-ant (data not presented). CMD values obtained are presented inables 5 and 6.
.4.2. Influence of pH on efficacy of biosurfactantThe pH of biosurfactant-containing culture media (96 h) in
ormal concentration, CMD−1 (10 times dilution) and CMD−2
100 times dilution) was altered from 2 to 11 and the σ val-es measured. Data thus obtained are presented in Table 5.he biosurfactants except that of MTCC7812 in the cell free
ulture medium supplemented with glycerol after 96 h of incu-ation and diluted to 10 (CMD−1) and pH adjusted over a rangerom 2 to 11 could not reduce the efficiency as depicted byhe surface tension values. But, 100 times (CMD−2) dilutiontPat
able 4eduction in surface tension of cell-free culture media supplemented with different c
ell free culture media of bacterial isolates in different carbon sources Surface t
0 h
arbon source: glucoseControl 71.2 ± 0P. aeruginosa (MTCC8165) 70.0 ± 1P. aeruginosa (MTCC7815) 70.6 ± 1P. aeruginosa (MTCC7812) 70.8 ± 1P. aeruginosa (MTCC7814) 70.7 ± 2
arbon source: glycerolP. aeruginosa (MTCC8165) 70.9 ± 0P. aeruginosa (MTCC7815) 70.2 ± 0P. aeruginosa (MTCC7812) 70.2 ± 0P. aeruginosa (MTCC7814) 68.4 ± 0
arbon source: hexadecaneP. aeruginosa (MTCC8165) 67.6 ± 0P. aeruginosa (MTCC7815) 67.0 ± 0P. aeruginosa (MTCC7812) 68.5 ± 0P. aeruginosa (MTCC7814) 68.0 ± 0
esults represented mean ± S.D. of three independent experiments; significance of d
.4 ± 1.5* ** 3.2 ± 0.95* ** 8.6 ± 1.8* ** 5.04 ± 0.85* **
.5 ± 1.3*,** 4.57 ± 0.75*,** 6.4 ± 1.6*,** 3.57 ± 0.95*,**
ifference with respect to control experiment: *p < 0.05 and **p < 0.01.
f all surfactant-containing medium over the pH range exhib-ted higher surface tension exhibiting less efficacy due to lowoncentration.
.4.3. Influence of high temperature on efficacy ofiosurfactant
Biosurfactant-containing bacterial culture media (96 h) withwo reduced concentrations CMD−1 and CMD−2 were exposedo 100 ◦C for different time periods from 5 to 60 min andheir σ measured. Data obtained are presented in Table 6. Theiosurfactant-containing cell free medium of each bacterial iso-ate at CMD−1 and CMD−2 on exposure to high temperature of00 ◦C over a period of 5–60 min exhibited almost the same sur-ace tension of 29–34 mN m−1 (CMD−1) and 46–67 mN m−1
CMD−2) depicting no loss of efficiency.
.5. Recovery of crude oil from the sand packed column
On treatment of crude oil-saturated sand packed column with
he bacterial biosurfactant produced by the bacterial isolates. aeruginosa (MTCC7815), P. aeruginosa (MTCC7814), P.eruginosa (MTCC7812) and P. aeruginosa (MTCC8165) andhen subsequently incubated at room temperature, 70 and 90 ◦Carbon sources over a period of 96 h
ension (mN rn−1) of the culture media
24 h 48 h 72 h 96 h
.7 71.1 ± 2.3 71.1 ± 1.8 70.9 ± 0.5 71.0 ± 0.3
.3 50.9 ± 1.5* 32.4 ± 0.6* 34.4 ± 0.7* 36.9 ± 0.8*
.6 31.7 ± 0.7* 31.5 ± 0.5* 31.1 ± 1.7* 31.9 ± 0.8*
.2 39.5 ± 0.8* 35.7 ± 0.6* 36.3 ± 0.5* 39.9 ± 0.4*
.8 37.3 ± 0.5* 33.6 ± 0.9* 34.7 ± 0.6* 33.9 ± 1.2*
.4 69.3 ± 1.3** 44.1 ± 0.7* 31.8 ± 0.9* 30.1 ± 1.1*
.5 68.5 ± 0.4* 32.5 ± 0.5* 31.8 ± 0.5* 29.7 ± 1.3*
.7 69.8 ± 0.2* 59.0 ± 1.0* 59.1 ± 0.4* 39.4 ± 0.6*
.9 64.2 ± 0.6* 31.4 ± 0.9* 34.1 ± 0.7* 31.1 ± 1.3*
.8 35.5 ± 0.7* 32.2 ± 0.2* 31.5 ± 0.3* 34.4 ± 0.6*
.8 34.4 ± 0.3* 33.3 ± 0.5* 32.6 ± 0.4* 36.2 ± 0.6*
.4 34.2 ± 0.4* 33.4 ± 0.6* 31.7 ± 0.6* 38.4 ± 1.2*
.9 48.6 ± 0.6* 44.1 ± 0.7* 34.6 ± 0.7* 39.6 ± 0.6*
ifference with respect to control experiment: *p < 0.05 and **p < 0.01.
78 N.K. Bordoloi, B.K. Konwar / Colloids and Surfaces B: Biointerfaces 63 (2008) 73–82
Table 5Effect of pH on biosurfactant produced by bacterial isolates in 2% glycerol supplemented medium and critical micelle dilutions (CMD−1 and CMD−2 g l−1) culturedfor 96 h
Bacterial isolates pH of media Surface tension (mN m−1)
Culture medium CMD−1 CMD−2
P. aeruginosa(MTCC8165)
2 29.1 ± 0.14 29.5 ± 0.78 29.6 ± 0.614 29.4 ± 0.08 29.6 ± 0.66 45.7 ± 0.616.5 30.1 ± 0.66 37.5 ± 0.72 56.7 ± 0.617.5 29.8 ± 0.63 29.9 ± 1.0 46.5 ± 0.90
11 33.4 ± 0.37 39.4 ± 0.64 64.3 ± 0.78
P. aeruginosa(MTCC7815)
2 28.7 ± 0.28 28.9 ± 0.56 57.1 ± 0.324 29.5 ± 0.04 34.1 ± 0.35 58.8 ± ± 0.646.5 29.7 ± 0.88 35.8 ± 0.64 57.6 ± 0.507.5 31.9 ± 0.43 36.2 ± 0.35 58.1 ± 0.53
11 33.2 ± 0.37 37.8 ± 0.24 48.1 ± 0.49
P. aeruginosa(MTCC7812)
2 31.8 ± 0.42 40.8 ± 0.62 67.0 ± 0.824 35.1 ± 0.21 50.5 ± 0.71 68.1 ± 0.296.5 39.4 ± 0.50 43.6 ± 0.69 61.2 ± 0.477.5 34.4 ± 0.40 48.1 ± 0.46 62.6 ± 1.43
11 36.5 ± 1.0 50.7 ± 0.56 68.1 ± 0.21
P. aeruginosa(MTCC7814)
2 30.9 ± 0.45 37.8 ± 0.96 65.1 ± 0.424 31.5 ± 0.42 36.5 ± 0.78 59.9 ± 0.296.5 31.1 ± 0.45 36.5 ± 0.69 52.1 ± 0.21
3.4 ±3.4 ±
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esults represented mean ± S.D. of three independent experiments.
aused release of crude oil from the column. The released crude
il was quantified and the data thus obtained are presented graph-cally in Fig. 1. The optimum temperature for all four strainselonging to the species P. aeruginosa was 37 ◦C, but they couldot tolerate temperature above 40 ◦C. whereas, the pure biosur-isoc
able 6ffect of high temperature (100 ◦C) on biosurfactant produced in 2% glycerol suppleMD−2 g l−1)
acterial isolates 100 ◦C temperature for (min)
. aeruginosaMTCC8165)
510203060
. aeruginosaMTCC7815)
510203060
. aeruginosaMTCC7812)
510203060
. aeruginosaMTCC7814)
510203060
esults represented mean ± S.D. of three independent experiments.
0.42 38.4 ± 0.64 59.8 ± 0.250.45 38.5 ± 0.58 68.9 ± 1.23
actants were stable when exposed to 100 ◦C for 60 min. That
s why, when a biosurfactant was injected into the crude oil-aturated sand pack column; it could reduce the surface tensionf oil molecules making them mobile. The released crude oil wasollected from the saturated sand pack column and quantified.mented medium cultured for 96 h and at critical micelle dilutions (CMD−1 and
Surface tension (mN/m) in
Culture media CMD−1 CMD−2
29.1 ± 0.4 29.5 ± 1.0 46.3 ± 0.631.1 ± 0.3 35.0 ± 0.4 50.2 ± 0.631.2 ± 0.2 35.0 ± 0.6 51.8 ± 0.831.2 ± 0.3 35.8 ± 0.2 63.7 ± 0.231.9 ± 1.0 34.6 ± 0.3 59.8 ± 0.2
31.7 ± 0.2 36.5 ± 0.4 48.0 ± 0.331.8 ± 0.2 37.0 ± 0.3 48.2 ± 0.331.5 ± 0.4 37.0 ± 0.4 48.1 ± 0.432.7 ± 0.6 34.2 ± 0.2 49.5 ± 0.832.4 ± 0.3 34.1 ± 0.3 48.5 ± 1.2
34.4 ± 0.2 41.5 ± 0.4 67.1 ± 0.233.3 ± 0.4 41.5 ± 0.3 66.8 ± 0.232.1 ± 0.2 40.1 ± 0.3 56.2 ± 0.434.3 ± 0.5 41.6 ± 0.5 58.2 ± 0.234.3 ± 0.7 41.6 ± 0.3 58.4 ± 0.5
33.1 ± 0.2 37.9 ± 0.3 61.5 ± 0.433.4 ± 0.2 37.8 ± 0.2 59.9 ± 0.432.7 ± 0.1 36.5 ± 0.4 58.9 ± 0.933.5 ± 0.6 40.6 ± 0.5 60.1 ± 0.233.2 ± 0.9 40.9 ± 0.4 59.1 ± 0.1
N.K. Bordoloi, B.K. Konwar / Colloids and Su
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ig. 1. Percent recovery of crude oil from the sand pack column at room tem-erature, 70 and 90 ◦C treated with biosurfactants of bacterial origin.
ontrary to this, when bacterial isolates along with the cultureedium was injected into the sand pack column; they not only
locked the pores of the column but also could not survive atigher temperatures of 70 and 100 ◦C.
Biosurfactants of the bacterial strains MTCC7815,TCC7812 and MTCC8165 could recover 49–54% of crude
il from the saturated sand pack column at room temperature:2–57% at 70 ◦C and 58–62% at 90 ◦C. The biosurfactantf MTCC7814 was found to be less efficient. In the case ofontrol (culture medium), very little recovery (5–8%) could bebtained in the temperature range. Inoculation of fresh mediumeparately with four bacterial strains could not increase therude oil recovery from the saturated sand packed columns compared to the control. The bacterial strains require aemperature of 30–37 ◦C for their growth and temperaturebove 40 ◦C causes death of the cells.
. Discussion
Biosurfactants are microbial metabolites with the typicalmphiphilic structure of a surfactant, where the hydrophobicoiety is either a long chain fatty acid, hydroxy fatty acid, or �-
lkyl-�-hydroxy fatty acid and the hydrophilic moeity could bearbohydrate, amino acid, cyclic peptide, phosphate, carboxyliccid alcohol, etc. Arino et al. [17] separated a mixture of rham-olipids by thin-layer chromatography in several fractions andydrolysed these fractions into their respective sugar and 3-ydroxy fatty acids. The sugar was determined to be rhamnose,nd they deduced fatty acids and rhamnolipids too.
Bosch et al. [18], Pajarron et al. [19] and Deziel et al. [20]denified glycolipids of bacterial (P. aeruginosa) origin to beha–C8–C10 and Rha–C10–C8, Syldatk et al. [21] obtainedydrophilic rhamnolipids Rha–C10 and Rha–Rha–C10; fromulture supernatants of resting Pseudomonas sp. DSM 2874 buthe quantity was low. Arino et al. [17] reported the rhamnolipidrofile of P. aeruginosa strain GL1 isolated from hydrocarbon-ontaminated soil. The bacterial strain was grown with glycerols the carbon source. They observed a variety of mono- andirhamnolipids containing one or two 3-hydroxy fatty acids andne or two rhamnoses represented 90% of all rhamnolipids. The
atty acids were predominantly C10 with some C8, C12:1 and12. De Koster et al. [22] observed the same saturated C8–C12ono- and dirhamnolipids. The predominant rhamnolipids wereha–C8–C10 and Rha–C10–C8. The most abundant 3-hydroxylesgo
rfaces B: Biointerfaces 63 (2008) 73–82 79
atty acids were also C8 and C10 observed in rhamnolipids. Thensaturated fatty acid is present at the terminal end of rhamno-ipids [20].
Majority of known biosurfactants are synthesized by microor-anisms grown on water-immiscible hydrocarbons, but some areroduced on water-soluble substrates such as glucose, glycerolnd ethanol [23,24,25]. In the present investigation, the bacterialsolate P. aeruginosa (MTCC8165) was cultured in glucose andlycerol (1%, w/v) for the maximum growth and reduction ofurface tension of the culture medium owing to the productionf biosurfactant. This reflected the difference in the choice ofarbon source for the growth and energy production by bacteriat the genus level.
The observation on glucose being the best carbon source forhe production of biosurfactant by the bacterial isolate P. aerugi-osa (MTCC7815) (31.1 mN m−1) was found to be in agreementith other workers like Nakano et al. [26], Sandrin et al. [27],oongaswang et al. [28] and Vater et al. [29]. They also reported,
accharose and fructose to be good carbon sources, but glyceroleverely decreased surfactin, a component mass of biosurfac-ant production. Makkar and Cameotra [30,31] described thebility of Bacillus strains to use starch and sucrose as the pre-erred carbon source for the maximum growth and biosurfactantroduction.
The bacterial isolate P. aeruginosa (MTCC7815) exhibitedhe maximum growth in glycerol containing medium and theiosurfactant produced by it lowered the surface tension of theulture medium (to 29.7 mN m−1). Glycerol was also used byther investigators like Turkovskaya et al. [32] for the produc-ion of biosurfactant. The biosurfactant produced by the bacterialsolate in glucose-supplemented medium caused the maximumecrease in the surface tension; but its emulsifying activity wasnly 40%, whereas glycerol was the best source for the syn-hesis of biosurfactant and it exhibited a better emulsifyingroperty of 60% [32]. On the basis of reduction of surface ten-ion of culture medium, bacterial isolates were also cultured inliphatic hydrocarbons (hexadecane and octadecane), aromaticydrocarbons (benzene and toluene) and polycyclic aromaticydrocarbons (pyrene and fluorene) supplemented medium.iosurfactant produced by the bacterial isolate P. aeruginosa
MTCC7815) exhibited a higher reduction in surface tension ofulture medium (29.1 mN m−1) irrespective of the carbon sourcesed. Glycerol was found to be the second best carbon sourceor the growth and biosurfactant production by the bacterial iso-ate P. aeruginosa (MTCC7815) which was in agreement withuerra-Santos et al. [24], Reiling et al. [33], Schenk et al. [34]
nd Turkovskaya et al. [32].Diversity exist among the biosurfactant producing microor-
anisms, suggesting that biosurfactant production is anmportant survival tool for the producing microbes and appearso have evolved in an independent yet parallel fashion [35]. Theajority of known biosurfactant are synthesized by microorgan-
sms grown on water-immiscible hydrocarbons in mesophilic
nvironments [31]. Though there are various reports on theynthesis of biosurfactant by hydrocarbon degrading microor-anisms, some biosurfactant have been reported to be producedn water-soluble compounds such as glucose, sucrose, glyc-
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rol or ethanol. Glycolipids, lipopeptides and lipoproteins, fattycids, polymeric surfactant and particulate biosurfactant arehe major classes of biosurfactant produced by microbes eitherxtracellularly or attached to parts of cells, predominantly duringrowth on water-immiscible substrates [14].
Screening of bacterial isolates, estimation of bisurfactant pro-uced by them as well as properties of biosurfactant produced byhese bacterial isolates from petroleum contaminated soils con-rmed that they were potential biosurfactant producing ones.resent study documented that the bacterial isolates P. aerugi-osa (MTCC7812), followed by P. aeruginosa (MTCC7815), P.eruginosa (MTCC7814) and P. aeruginosa (MTCC8165) pro-uced higher biosurfactant with dry biomass yield of 8.6, 6.4,.4 and 6.4 g l−1, respectively (Table 2). Desai and Banat [14]eported little biosurfactant production when cells were growingn a readily available carbon source; only when all the solublearbon was consumed and when water-immiscible hydrocar-on was made available, the production triggered. In regard toable 3, hexadecane was found to be the better carbon source asompared to glycerol and glucose for biosurfactant yield andiomass production of all four bacterial isolates. In hexade-ane and glucose supplemented media, the isolate P. aeruginosaMTCC7812) produced the best yield of biosurfactant of 5.4nd 4.0 g l−1 with biomass production of 8.6 and 5.4 g l−1,espectively. However, in glycerol supplemented medium theerformance of the isolate was low as compared to other threesolates.
In media supplemented with three carbon sources, biosur-actant produced by each bacterial isolate could lower the σ
alue significantly as against the control in four durations of4, 48, 72 and 96 h after inoculation. Biosurfactant producedy the isolate P. aeruginosa (MTCC7815) in glycerol and glu-ose supplemented media effected the lowest σ value of 29.7nd 31.9 mN m−1. But in the case of hexadecane-supplementededium the isolate P. aeruginosa (MTCC8165) could cause the
owest σ value of 34.4 mN m−1 (Table 4).The CMC value of P. aeruginosa (MTCC7815) biosurfactant
as 110 mg l−1, whereas 100 mg l−1 in each of P. aeruginosaMTCC7815), P. aeruginosa (MTCC8165) and P. aeruginosaMTCC7814) biosurfactant. The concentration of biosurfactantroduced in glucose, glycerol and hexadecane supplementededium during the culture period of 96 h was diluted 10
CMD−1) and 100 (CMD−2) times and then measured the σ
alue (Tables 5 and 6).Although the environmental factors and growth conditions
uch as pH, temperature, agitation, and oxygen availability alsonfluence biosurfactant production through their effects on cellu-ar growth and activity. The pH of the medium plays an importantole in sophorolipid production by Torulopsis bombicola [36].owalla et al. [37] showed that penta- and disaccharide lipidroduction in Nocardia corynebacteroides is unaffected in theH range of 6.5–8. In addition, σ and CMCs of a biosurfactantemained stable over a wide range of pH values. The efficacy
f biosurfactant produced by each of the bacterial isolates wasound to be independent of pH. Interaction between biosurfac-ant produced by the bacterial isolates and pH (2–11) had noignificant role in altering the σ values of biosurfactant producedtr
m
faces B: Biointerfaces 63 (2008) 73–82
y four bacterial isolate. The efficacy in reducing σ of cultureedium remained intact similar to that of normal concentra-
ion (Table 5). But, 100 times dilution (CMD−2) was ineffectives depicted by the σ values. Desai and Banat [14] observedhat heat treatment on some biosurfactant caused no appreciablehange in their properties such as lowering of σ and interfa-ial tension and the emulsification efficiency after autoclavingt 120 ◦C for 15 min. In the present investigation too biosurfac-ants remained unaffected with respect to the property of σ whenxposed to high temperature (100 ◦C) for different time periodsf 5–60 min. Analysis of data presented in Table 6 revealed thathe σ value of culture media having the production of biosurfac-ant and the ones with reduced dilutions CMD−1 and CMD−2
emained unchanged on exposure to 100 ◦C for 5, 10, 20, 30, and0 min. However, the low concentration CMD−1 was found toe effective as that of the normal concentration of biosurfactantn the culture medium.
Banat [2] injected on treatment of B. subtilis biosurfactanto crude oil saturated sand packed columns (2.5 cm diame-er × 28 cm length), a release of 35% residual oil, as comparedo 21% using the nutrient solution control. In the present investi-ation, exposure of biosurfactant isolated from bacterial isolateso high temperature of 70 and 90 ◦C caused higher yield of crudeil from saturated sand packed column. Biosurfactant on treat-ent to crude oil saturated sand packed column could release
0–45% (Fig. 1) more oil as compared to the treatment with theulture medium (control). Whereas, on subsequent exposure toigh temperature of 70 and 90 ◦C caused incremental releasef crude oil 2–10% and 8–12%, respectively, from the column.reatments of P. aeruginosa (MTCC7812) and P. aeruginosaMTCC8165) biosurfactant caused 50 and 48% crude oil release,espectively, at room temperature, 53% at 70 ◦C and 60% in bothases at 90 ◦C.
Laboratory studies on MEOR have typically utilized coreamples and columns containing the desired substrates. Theseubstrates have been utilized to demonstrate the usefulness ofiosurfactants in oil recovery from sand and limestone. Simi-arly, core samples have been used as model in the movementf microorganisms and nutrients through substrates to ascertainheir usefulness after injection into oil reservoir [38].
. Conclusion
The usefulness of biosurfactant in the reduction of viscos-ty of hydrocarbon mixtures through the reduction of σ waslearly demonstrated. Biosurfactant were not only effectiveut also added benefit of being biodegradable. Studies of oilr hydrocarbon-contaminated sand or soil also indicated thaticroorganisms, which produced biosurfactant, when stimu-
ated properly, could aid bioremediation. Biosurfactant alsoemonstrated their usefulness in the solubilization and removalf oil from contaminated soils (Bordoloi and Konwar, unpub-ished) and sludge in the oil storage tank. Therefore, in ecological
erms, the use of biosurfactant is obvious for closed systems butemains speculative in the open environment.The utility of MEOR has not been conclusively docu-ented in the field at this stage. Preliminary findings from
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N.K. Bordoloi, B.K. Konwar / Colloids a
he present investigation, however, seem promising. The pre-ise mechanism of enhanced oil recovery in situ is unclearue to the lack of controls in some cases, with the unfore-een difficulty usually encountered in situ and insufficientnalyses in other cases. It would appear that in certain cir-umstances MEOR could be a viable alternative, which, ifarefully applied, could prove to be an economically fea-ible method of enhancing incremental oil from reservoir.uture efforts in strain improvement with the aid of geneticngineering are expected to help progress in this emergingechnology.
cknowledgment
We acknowledge the receipt of financial support in the from ofesearch fellowship from the Oil and Natural Gas Corporation,ndia. We would also like to thank Prof. D. Konwer, Depart-ent of Energy, Tezpur University, Napaam for his valuable
uggestions during the course of the investigation.
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