World J Microbiol Biotechnol (2008) 24:1047–1057
DOI 10.1007/s11274-007-9574-5
ORIGINAL PAPER
Biosurfactant production and hydrocarbon-degradation
by halotolerant and thermotolerant Pseudomonas sp.
Manoj Kumar Æ Vladimir León Æ Angela De Sisto Materano Æ
Olaf A. Ilzins Æ Luis Luis
Received: 2 May 2007 / Accepted: 26 September 2007 / Published online: 27 October 2007
Ó Springer Science+Business Media B.V. 2007
Abstract A hydrocarbon degrading and biosurfactant
producing, strain DHT2, was isolated from oil-contami-
nated soil. The organism grew and produced biosurfactant
when cultured in variety of substrates at salinities up to
6 g l-1 and temperatures up to 45°C. It was capable of
utilizing crude oil, fuels, alkanes and PAHs as carbon
source across the wide range of temperature (30–45°C) and
salinity (0–6%). Over the range evaluated, the salinity and
temperature did not influence the degradation of hydro-
carbon and biosurfactant productions. Isolate DHT2 was
identified as Pseudomonas aeruginosa by analysis of 16S
rRNA sequences (100% homology) and biochemical
analysis. PCR and DNA hybridization studies revealed that
enzymes involved in PAH metabolism were related to the
naphthalene dioxygenase pathway. Observation of both
tensio-active and emulsifying activities indicated that bio-
surfactants were produced by DHT2 during growth on
both, water miscible and immiscible substrates, including
PAH. The biosurfactants lowered the surface tension of
medium from 54.9 to 30.2 dN/cm and formed a stable
emulsion. The biosurfactant produced by the organism
emulsified a range of hydrocarbons with hexadecane as
best substrate and toluene was the poorest. These findings
further indicate that the isolate could be useful for
M. Kumar
V. León
A. De Sisto Materano

O. A. Ilzins
L. Luis
Unidad de Biotecnologı́a del Petróleo, Centro de Biotecnologı́a,
Fundación Instituto de Estudios Avanzados (IDEA), Apartado
17606, Caracas 1015 A, Venezuela
M. Kumar (&)
Indian Oil Corporation, Research and Development Center,
Faridabad 121007, Haryana, India
e-mail: manojupreti@rediffmail.com
bioremediation and bio-refining application in petroleum
industry.
Keywords Pseudomonas sp.
Biosurfactant

Halotolernat
Themotolerant
Hydrocarbon

Biodegradation
Introduction
Biosurfactants are heterogeneous group of surface-active
chemical compounds produced by a wide variety of
microorganisms. Their superior properties, such as low
toxicity, high biodegradability, mild production condition
and environmental compatibility have prompted their tre-
mendous applications in several industries (Cameotra and
Makkar 2004).
Pseudomonads are the best-known bacteria capable of
utilizing hydrocarbons as carbon and energy sources and
producing biosurfactants (Beal and Betts 2000; Noordman
and Janssen 2002; Rahman et al. 2002). Among Pseudo-
monads, P. aeruginosa is widely studied for production of
glycolipid type biosurfactant. However, glycolipid type
biosurfactant are also reported from some other species like
P. putida and P. chlororaphis. The literature suggests that
biosurfactant productions by Pseudomonas sp. are exclu-
sively dealt with mesophiles. Only few reports refer to
biosurfactant production by thermotolerant or thermophilic
Pseudomonas sp. Recently, Perfumo et al. (2006) reported
rhamnolipid production by a novel thermophillic hydro-
carbon degrading Pseudomonas aeruginosa APO2-1, which
grow on variety of hydrocarbons and produces biosurfactant
during growth on water soluble and insoluble substrate at
45°C. Das and Mukherjee (2005) have mentioned biosur-
factant production at 45°C by mucoid and non-mucoid
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strains of Pseudomonas aeruginosa. To best of our knowl-
edge there is no report on biosurfactant production and
hydrocarbon degradation by halotolerant and thermotoler-
ant Pseudomonas aeruginosa. Thermotolerant and
halotolerant microorganisms, degrading hydrocarbon and
producing tensio-active emulsifying agent (biosurfactant),
would be very effective for bio-refining applications due to
the prevailing high salinity and temperature conditions in
petroleum reservoirs and processing (Margesin and Schin-
ner 2001; Leon and Kumar 2005; Borgne and Quintero
2003). These microbes would also be useful for the treat-
ment of oil-polluted ecosystems with high salinity or wide
salinity gradient at wide range of temperature.
In the present study, we describe the isolation and
characterization of a thermotolerant and halotolerant strain
of P. aeruginosa which is able to utilize hydrocarbons
ranging from n-alkanes to polycyclic aromatic hydrocar-
bons as a carbon source, and produces biosurfactants
during the growth on hydrocarbons and both water-misci-
ble and immiscible carbon substrates. In fact, this is the
first report describing biosurfactant production and hydro-
carbon degradation by thermotolerant and halotolerant
P. aeruginosa strains.
Materials and methods
Enrichment and isolation of bacterium
A standard enrichment technique was used to isolate
thermotolerant and halotoerant hydrocarbon-degrading
microorganisms from soil samples obtained from Gua-
noco Lake, Sucre State (10°170 N; 64°240 W) Venezuela. A
few grams of soil sample was transferred to 500 ml
Erlenmeyer flask containing 100 ml of basal salt medium
(BSM) (Kumar et al. 2007) with 2% (vv-1) of crude oil
as carbon source. Flasks were incubated at 50°C on a
rotary shaker (200 rev min-1) for 4 days. After 4 days
1.0 ml of the culture was transferred to fresh media
containing crude oil (2% wv-1) and re-incubated for
another 4 days. During enrichment salinity was main-
tained by adding NaCl (50 gl-1) in BSM. Following five
cycles of such enrichment, 1.0 ml of culture was diluted
and plated on BSM agar plates containing crude oil as
sole carbon source. The bacterial colonies obtained were
further purified on Luria–Bertani agar. The ability of the
microbes to grow at various temperatures and salinity was
determined by growing them in BSM containing crude oil
(2% ww-1). The strain DHT2 exhibiting excellent growth
at wide range of temperature and salinity conditions was
selected for further study. The strain was stored at -70°C
in BSM mixed with sterile glycerol at a final concentra-
tion of the 25% (vv-1).
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World J Microbiol Biotechnol (2008) 24:1047–1057
Microbial growth on hydrocarbons and biodegradation
The ability of the isolate to utilize crude oil, kerosene,
diesel, gas oil, alkanes and various PAHs as sole source of
carbon and energy was assessed by inoculating it into a 24
well micro-titer plate containing 500 ll of BSM in each
well and 0.02% (wv-1) of test hydrocarbon as sole carbon
source (van der Gast et al. 2002). Stock solutions of PAHs
were prepared in absolute ethanol. Each well was inocu-
lated with 10-ll pre-culture (Luria–Bertani broth grown
OD600 = 0.8, approximately 108 cells ml-1) and incubated
at temperature ranging 30–45°C and 200 rev min-1 at salt
concentration ranging from 0 to 6% (wv-1) of NaCl. The
plates were properly sealed with the lid and laboratory film
to prevent cross contamination or evaporation. An inocu-
lated control well devoid of carbon-source and another un-
inoculated control well only containing carbon source was
also prepared for each carbon source/bacteria, for com-
parison purpose under same experimental conditions.
Growth was followed for 1 week by measuring turbidity
for the increase in cell density in comparison to controls
and the cell viability counts (colony forming units; c.f.u.
ml-1) on nutrient agar plates. No difference in turbidity
and c.f.u. of the well and that of the controls were con-
sidered as no growth (-), whereas, increase in turbidity
and viable cell counts (at least 10-fold) were considered as
growth (+). Results of the micro-titer plate were verified by
growing bacteria in 500 ml Erlenmeyer flasks containing
100 ml of BSM and monitoring turbidity and the colony
forming units (c.f.u. ml-1).
The ability of strain DHT2 to utilize different hydro-
carbons was also evaluated in solid BSM media (2% agar).
PAHs (except naphthalene) were dissolved in 5% (wv-1)
diethyl ether and sprayed on the surface of BSM agar Petri
plates. Naphthalene (1.0 g) was provided as crystals
directly placed on the plate lid. Liquid hydrocarbon such as
crude oil was mixed with BSM-agar. Growth on hydro-
carbon in solid media was considered positive by the
formation of a clear zone around the growing colonies or
appearance of pigments and growth (Kiyohara et al. 1982).
Quantitative degradation of representative PAHs, i.e.,
phenanathrene, pyrene and dibenzothiophene (DBT) was
studied at 30 and 45°C with 0 and 6% (wv-1) NaCl. Bacte-
rium was grown in batch culture in 250 ml flask containing
50 ml of BSM supplemented with 200 mg l-1 of test PAH.
The degradation of complex mixture of hydrocarbon taking
diesel oil as representative was studied into 250 ml flask
containing 50 ml of BSM and 5% (vv-1) of diesel oil as sole
carbon source and incubated at 30 and 45°C with 0 and 6%
(wv-1) NaCl. Experimental flasks were inoculated with 2%
(vv-1) inoculum (105 c.f.u. ml-1) and incubated in dark on
rotatory shaker (200 rev min-1) for 3 weeks. Un-inoculated
flasks and flasks without hydrocarbon served as controls. The
World J Microbiol Biotechnol (2008) 24:1047–1057
ability of strain to remove PAH was analyzed by gas chro-
matography according to Kumar et al. (2006), while the
diesel degradation was studied according to Rahman et al.
(2002). Cell growth was measured by protein estimation
following Bradford assay using bovine serum albumin
(BSA) as a standard.
Amplification of nah gene and southern hybridization
Total DNA of bacteria was isolated according to Chen and
Kuo (1993). Primers used for the nahA and nahE were
based on the gene sequences reported for P. putida G7 and
P. putida NCIB 9816 (Habe and Omori 2003). PCR was
performed according to the PCR conditions and tempera-
ture program as described by Kumar et al. (2006) using
PCT-100TM thermal cycler unit (MJ Research, Inc., USA).
All PCR run included control reaction mixtures without
added DNA. PCR products were routinely visualized by
running 10 ll of PCR mixture on 1% agarose gels (Bio-
Rad, Richmond, CA) in 0.59 Tris–borate–EDTA (TBE)
buffer stained with ethidium bromide (0.0001%). The PCR
product fractionated by gel electrophoresis were blotted on
to Zeta probe GT (Bio-Rad) and non-radioactive labeling,
hybridizations and probe detection using the ECL direct
nucleic acid labeling and detection system (Amersham
Biosciences, NJ, USA) according to the supplier specifi-
cations. All experiments included control reaction mixtures
without added DNA. Prototype naphthalene bacteria
P. putida NCIB 9816-4 was taken as positive control while
E. coli DH5a was taken as negative control.
Analysis of cell surface hydrophobicity
The bacterial adhesion to hydrocarbons (BATH) assay was
used in order to test the hydrophobicity of the strain
(Rosenberg and Rosenberg 1985). Bacterial cells were
harvested from growth culture by centrifugation at 8,000g
for 10 min at 4°C and washed twice. The cells were then
re-suspended in a buffer–salts solution (pH 7.0) containing
16.9 g of K2HPO4, 7.3 g of KH2PO4, 1.8 g of urea, and
0.2 g of MgSO4
7H2O per liter to give an optical density
(OD) of 1.0 at 400 nm. Cells (4.0 ml) and 1.0 ml of dif-
ferent hydrocarbons were mixed in a screw-top test tube
and vortexed for 60 s. Hydrocarbon and aqueous phases
were allowed to separate for 45 min. The aqueous phase
was then carefully removed and turbidity was measured at
400 nm. Hydrophobicity is expressed as the percentage of
adherence to hydrocarbon, which is calculated as follows:
100 9 (1 - OD of the aqueous phase/OD of the cell sus-
pension). Percent increase in adhesion indicated the level
of hydrophobicity of the cell surface.
1049
Production and partial purification of biosurfactant
To study biosurfactant production and activity, bacterium
was either grown in YPG medium (g/l): Yeast extract—5;
Peptone—5; Glucose—15 or BSM containing water-solu-
ble and/or insoluble carbon sources (2% wv-1, final
concentration) at temperatures ranging from 30 to 45°C at
various NaCl concentrations (0–6% wv-1) for 2 weeks. At
the timed intervals, samples were withdrawn for protein
estimation and emulsification index (E24) determination.
Effect of various carbon sources on biosurfactant produc-
tion was also studied by amending BSM with any one of
the carbon sources.
For partial purification of surface-active component(s),
the supernatant was subjected to liquid–liquid extraction
following acidification with 1 N HCl to pH 2.0 (Rahman
et al. 2003). Supernatant fluid was mixed with an equal
volume of chloroform:methanol (2:1) mixture. The solvent
was evaporated and the material was used as crude bio-
surfactant. The crude product was dissolved in the dichlo-
romethane and filtered to remove any coarse material.
Solvent was removed with the aid of rotary evaporator and
the solid was washed with three volumes of hexane to
remove alkanes and free fatty acids. The solvent was
evaporated and the material was used as crude biosurfac-
tant and weighed to evaluate the yield.
Detection of biosurfactant activity
Drop test and oil spread test was carried out according to
Youssef et al. (2004). Emulsification index (E24) was
determined by the addition of hydrocarbon to the same
volume of cell free culture broth, mixed with a vortex for
2 min and left to stand for 24 h. The emulsification activity
was determined as the percentage of height of the emul-
sified layer [mm] divided by the total height of the liquid
column [mm]. To study the stability of emulsion, emulsi-
fied solutions were allowed to stand at room temperature
and emulsification index was analyzed at different time
intervals.
Bacterial strain was tested for hemolytic activity by
plating cells onto blood agar followed by incubation at
30/45°C for 48 h. Surface tension of biosurfactant was
measured by the ring method using a CSC-DuNouy Ten-
siometer at room temperature. The culture supernatant was
diluted with distilled water, and surface tension was mea-
sured at various concentrations. Blue agar plates containing
cetyltrimethylammonium bromide (CTAB) and methylene
blue were used to detect extracellular glycolipid production
(Siegmund and Wagner 1991). Biosurfactant production
was observed by the formation of dark blue halos around
the colonies.
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Effect of temperature, pH and NaCl concentration on
activity of biosurfactant was determined by measuring
emulsification index (Ilori et al. 2005). Foamability was
measured in terms of foam volume (FV) (Das et al. 1998).
Air at a constant rate (45 ml min-1) was passed by mea-
suring-cylinder containing 20 ml of the culture filtrate. The
FV estimated was the difference between the volumes
occupied by the liquid-plus foam and the volume of the
liquid at rest.
PAH solubilization effect of biosurfactant
PAH solubilization assay was carried out according to
Barkay et al. (1999). Briefly, 0.6 lg of anthracene, 6 lg of
phenanthrene, DBT or pyrene was distributed into glass
test tube and kept open inside the fume hood to allow the
solvent to evaporate, followed by the addition of 3.0 ml of
assay buffer (20 mM Tris–HCl, pH 7.0) and graded
amounts of crude biosurfactant (105–500 lg ml-1). Tubes
were incubated at 30°C with shaking (200 rpm) for 24 h.
Samples were filtered through 1.2 lm filters and 2.0 ml
filtrate was removed in a clean tube to which 2.0 ml of
hexane was added prior to the extraction by vortexing for
2 min. This emulsion was centrifuged at 10,000 rpm for
10 min to separate the aqueous and hexane phases. PAH in
the hexane extracts was measured spectrophotometrically
at 335, 252, 250 or 273 nm for DBT, phenanthrene, anth-
reacene or pyrene, respectively. From a calibration curve of
individual PAH (in hexane), the concentration of each PAH
was determined. Assay buffer with biosurfactants and
without PAH was extracted with hexane identically and
served as blank. Control was also run in parallel where no
biosurfactant was added to PAH before extraction with
hexane.
Carbohydrate, protein and lipid analysis
Determination of the carbohydrate content of the partially
purified biosurfactant was done by anthrone reagent
method using 620 nm (Spiro 1966). Protein was assayed by
Bradford (1976) method using BSA as a standard. Lipid
was analyzed as described by Ilori and Amund (2001).
Bacterium identification
The bacterial identification was carried out by determining
the gene sequence coding for 16S rRNA and biochemical
analysis. The PCR was carried out as conditions and
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World J Microbiol Biotechnol (2008) 24:1047–1057
temperature program described by Kumar et al. (2006).
The PCR product was separated by agarose gel electro-
phoresis and visualized by SYBR1 Green 1 staining
(Sigma, St. Louis, USA) and, finally purified by using a
Wizard PCR Preps Purification System (Promega Corp.,
Madison, USA) according to the manufacturer’s instruc-
tions. The purified DNA was sequenced as per procedure
described below. To identify the isolated bacterium, the
16S rDNA consensus sequence, obtained by analyzing with
DNAMAN version 5.2.9 (Lynnon BioSoft, Quebec, Can-
ada, was then compared with 16S rRNA gene sequences
from the public GenBank, EMBL, and DDBJ databases
using the advanced gapped n-BLAST program, version 2.1.
The program was run via Internet through the National
Center for Biotechnology Information website (http://
www.ncbi.nlm.nih.gov/blast/). Sequences with more than
98% identity with a GenBank sequence were considered to
be of the same species as the highest score-matching
sequence on the public sequence databases. The bio-
chemical reactions were also carried out according to
Bergey’s Manual of Determinative Bacteriology to identify
the bacterium.
DNA sequencing
DNA sequencing reaction was performed with an ABI
PRISM Big Dye Terminator Cycle Sequencing Kit
(Applied Biosystems, CA, USA) and the sequencing
products were separated by capillary electrophoresis by
using a 310 Sequencer (Perkin–Elmer Corp., Applied
Biosystems, USA).
Detection of rhl gene and sequencing
Primers used for rhlAB were based on the gene sequences
reported for Pseudomonas aeruginosa PAO 1 (Ochsner
et al. 1994a, b; Deziel et al. 2003; Soberon-Chavez et al.
2005). The PCR conditions and temperatures are given in
Table 1. The PCR product was separated by agarose gel
electrophoresis and visualized, purified and sequenced as
described earlier.
Nucleotide sequence accession number
The sequences obtained in this study were submitted in the
GenBank database. The gene bank accession numbers for
the 16S rRNA and rhlAB are EF140717 and EF140718,
respectively.
World J Microbiol Biotechnol (2008) 24:1047–1057 1051

and 2 min at 72°C, and (iii) a final extension

Results

(i) 5 min at 95°C; (ii) 30 cycles, with 1 cycle

consisting of 30 s at 95°C, 1 min at 50°C,
Isolation and characterization of bacterium

Several microbes with ability to degrade/modify the heavy

crude oil/bitumen from were isolated from Guanaco lake
by enrichment technique. The strain designated as DHT2

step of 10 min at 72°C

was selected for further study because of its ability to grow
Temperature program

on wide range of hydrocarbon in wide range of tempera-

ture and salinity conditions and producing biosurfactant.
DHT2 was an aerobic Gram-negative motile rod and was
able to grow between temperatures 30 and 45°C and
salinity 0–6% with out any detrimental effect of tempera-
ture and salinity. Growth inhibition was observed above
45°C and 6% NaCl. The bacterium did not grow at 10%
forward primer, 0.2 lM reverse primer, and 0.1 lg of template

NaCl and at 60°C. Taxonomical identification of this

triphosphate, 2 U Taq DNA polymerase (Invitrogen), 0.2 lM
50 mM KCl, 1.5 mM MgCl2, 200 lM each deoxynucleoside
50-ll reaction mixtures contained 20 mM Tris–HCl (pH 8.4),

organism, designated DHT2, was performed by amplifi-

cation and sequencing the 16S rRNA genes and comparing
them to the database of known 16S rRNA sequences.
Alignment of the 16S rRNA gene sequences of DHT2 with
sequences obtained by doing a Blast searching revealed
100% similarity to Pseudomonas aeruginosa. The bio-
PCR conditions and temperature program

chemical test and cultural characteristics also support that

the bacterium is Pseudomonas aeruginosa (Table 2). In
present paper this strain is designated as Pseudomonas sp.
strain DHT2.

Microbial growth on hydrocarbons and biodegradation

genomic DNA

Bacterial strain DHT2 was tested for its ability to grow on a

variety of hydrocarbon including various PAHs, n-alkanes
and complex hydrocarbon mixture at temperature ranging
Table 1 Primers and PCR conditions for the detection of rhlab gene

50 -CAGGCCGATGAAGGGAAATA-30
50 -AGGACGACGAGGTGGAAATC-30

Table 2 Toxiconomical properties of Pseudomonas sp. DHT2

Gram stain Negative
Shape Rod
Motility +
Spore formation -
VP test -
MR test -
Indole production -
Sequences

Reduction of nitrate +
Urease -
Gelatin liquification +
Starch hydrolysis -
Product size (bp) 777

Catalase test +
Rhlabf 1544–1563
RhlAbr 786–806
Primer/position

Oxidase test +
Utilization of glucose +
Utilization of galactose -
Production of pigment +

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from 30 to 45°C and salinity ranging from 0 to 60 g Table 3 PAHs and diesel oil degradation by strain DHT2 at different
NaCl l-1 by growing them in BSM, containing the test experimental conditions
hydrocarbon as carbon source. The bacterium was capable Substrate Degradation (% ww-1) after 2 weeks
of utilizing various PAHs, alkanes and complex hydrocar-
30°C 30°C with 45°C 45°C with Abiotic
bons mixture like crude oil and fuels, as carbon source. The without 6% NaCl without 6% NaCl control
result of hydrocarbon substrate specificity test revealed that NaCl NaCl
the organism could grow on crude oil, gas oil, diesel oil,
DBT 61 ± 2 57 ± 3 54 ± 2 66 ± 5 4 ± 1.2
hexadecane, naphthalene, dibenzothiophene, pyrene,
Pyrene 56 ± 3 52 ± 3 49 ± 2 50 ± 4 3 ± 0.4
phenanthrene, anthracene salicylate, catechol, kerosene, n-
Diesel 66 ± 3 73 ± 2 70 ± 2 73 ± 4 5±2
decane, gasoline, n-hexane, and octadecane while, there
was no growth in toluene, phenol, alpha methylnaphthalene, Anthracene 61 ± 3 56 ± 2 51 ± 3 57 ± 4 5±1
2-hydoxyquinoline and carbazole. Over the range evaluated Each value represents the mean of three samples
(30–45°C and 0–60 g NaCl l-1), the ability to grow on
hydrocarbon was not influenced by salinity and tempera- degradation was 73 ± 2% at 45°C and 6% salt concen-
ture. When the bacterium was grown on agar plates tration. Diesel degradation at 30°C was 66%. Results
containing hydrocarbon as carbon source, growth and/or further indicated that the ability to degrade hydrocarbon
zone of clearing appeared indicating hydrocarbon utiliza- was not influenced by salinity and temperature.
tion. This strain formed blue indigo pigmentation in the
presence of indole, indicating naphthalene dioxygenase
activity (Ensley and Gibson 1983). PCR amplification of nah gene and hybridization
Ability of this strain to utilize hydrocarbon was further
supported by an increase in bacterial growth concomitant When the primers nahAf and nahAr were used, a single
with a decrease in hydrocarbon concentration. Results PCR fragment of the expected size (3.4 kb) was amplified
indicated that the abiotic processes caused insignificant in DHT2 and Pseudomonas putida NCIB 9816-4. No
decrease in PAH concentrations. The bacteria could amplification was observed in the negative control and
remove around 43% of phenanthrene in 2 weeks (Fig. 1). control without DNA. Similarly, when the set of primers
Table 3 depicts the hydrocarbon degradation after 2 weeks nahE was used, a product of the expected size (990 bp) was
at varying temperature and salinity conditions. The bacte- produced in DHT2 and Pseudomonas putida NCIB 9816-4
rium could degrade 61 ± 3% DBT at 30°C, while DBT (Fig. 2). Since the nah genes of the isolated strains could
degradation was 66 ± 4% at 45°C and 6% salt concen- be amplified using primers designed from the control
tration. Strain DHT2 could grow in the diesel and the strains and the amplicons had the expected size, it further
suggested that both organisms have the same structural
organization of the nah gene. To assess the homology
Remaining phenanthrene (µg ml-1)

200 200
between prototype naphthalene degradative enzymes of
P. putida 9816-4 and DHT2, the nahA and nahE gene PCR
Protein ( µg ml-1)

150 150
product of strain P. putida 9816-4 was purified, non-
100 100 radioactively labeled and used as probe in southern blot-
ting. The strain DHT2 shows hybridization signals with all
50 50
of the probes in high stringency conditions (data not
0 0
shown). This indicates that the naphthalene dioxygenase
0 5 10 15 20 encoding gene of DHT2 and 9816 have some degree of
Time(days) homology. No hybridization was observed in the negative
Protein (µg/ml) at 30°C control. This is interesting to note here that the P. putida
Protein (µg/ml) at 45°C
9816-4 is a mesophilic bacterium, while the DHT2 is a
Protein (µg/ml) at 30°C & 6% NaCl
Protein (µg/ml) at 45°C & 6% NaCl
thermotolerant and halotoerant bacteria. However, both of
Remaining phenanthrene (µg/ml) 45°C them have same enzymatic machinery.
Remaining phenanthrene (µg/ml) 30°C
Remaining phenanthrene (µg/ml) 30°C & 6 % NaCl
Remaining phenanthrene (µg/ml) 45°C & 6 % NaCl Detection of biosurfactant production
Control phenanthrene (µg/ml)

DHT2 grew by forming colonies surrounded by halos of

Fig. 1 Phenanthrene degradation and growth of strain DHT2 at
various temperature and salt concentrations. Each value represents the hemolysis on blood agar plates. Initial biosurfactant pro-
mean of three samples duction and activity assessment was carried out by growing

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80 DHT 2
Media

70

60

Surface tension (dy/cm)

50

40

30

20

10

0
0 20 40 60 80
Concentration ( % v/v)

Fig. 3 Surface tension vs. concentration of the culture supernatant.

YPG is the medium used for biosurfactant production. Each value
Fig. 2 Agarose gel electrophoresis of PCR products. M, 1 kb DNA represents the mean of three readings with standard error \5%
ladder; 1. nahA DHT2; 2. nahA 9816-4; 3. nahE DHT2; 4. nahE
9816-4
Table 4 The emulsification activity (E24, with hexadecane) of cul-
bacteria in YPG. The culture drop of DHT2 caused oil to ture broth during growth on YPG and BSM containing different
carbon source
spread, thus forming a wide clear zone on the oil–water
surface, and completely collapsed over oil-covered slide Media E24
surface. These qualitative tests are indicative of the surface YPG 65 ± 2%
and wetting activities (Youssef et al. 2004). The strain BSM-Glucose 55 ± 2%
DHT2 was capable of the surface reduction of YPG media BSM-Glycerol 63 ± 2%
from 54.9 to 30.2 dN/cm. It was interesting to note that BSM-DBT 45 ± 3%
most of the surface active activity was confined to the BSM-Naphthalene 52 ± 2%
culture supernatant. The reduction of surface tension (more
BSM-Hexadecane 61 ± 3%
than 20 dN/cm) was comparable to the findings of Desai
BSM-Pyrene 53 ± 3%
and Banat (1997). Surface tension at various concentrations
BSM-Phenanthrene 45 ± 2%
following dilution of culture supernatant with distilled
BSM-Glucose + Hexadecane 78 ± 4%
water has been shown in Fig. 3. Results revealed that more
BSM-Kerosene 61 ± 3%
than 24% increase in concentration did not produce sig-
BSM-Diesel 44 ± 3%
nificant decrease in surface tension suggesting that
BSM-Gas oil 70 ± 4%
biosurfactant molecule began to aggregate (Cassidy and
Hudak 2001).
The strain DHT2 was capable to produce biosurfactant
when grew on various water soluble and insoluble carbon day. The highest cell density and peak of biosurfactant
sources (Table 4). Highest emulsification activity and production were obtained on 7th day (Fig. 4). Similar
biosurfactant production (7.05 g l-1) was obtained when emulsification index E24 was obtained when the bacterium
the bacterium was grown in BSM containing glucose was grown at various temperature (30–45°C) and salinity
(1% wv-1) and hexadecane (1% wv-1) in equimolar con- (0–6% wv-1) conditions. Table 5 depicts emulsification
centration (BSM-GH) as carbon source. Detection of index (E24) with various hydrocarbon for supernatant grown
biosurfactant in the BSM-GH occurred for first time in 4th on BSM-GH at 30 and at 45°C and 6% NaCl (wv-1).

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with NaCl concentrations greater than 10%. Emulsion

200 80
index was more or less same between pH 5.5–8.5, however,

Emulsification index (%)

60
decrease was observed in highly acidic or alkaline pH.
150
Autoclaving (121°C, 15 min, 15lbs) did not influence the
Protein

100 40 activity of biosurfactant. Increase in temperature from 25

to 70°C had no effect on the stability of emulsion. At
50 20 temperature higher than 70°C, the stability of the emulsion
was decreased. It is however, noteworthy to mention that
0 0
0 2 4 6 8 10
70% of the original activity of its emulsion index was
Days retained at of 100°C.
In the Siegmund and Wagner plate, the bacteria pro-
Protein (µg/ml) Emulsification index
duced blue halo around the colonies indicating rhamnolipid
Fig. 4 Growth and biosurfactant production in BSM-GH. Values are nature of the biosurfactant produced by the bacteria. This
average of three cultures was confirmed by amplifying rhl gene and by chemical
analysis. When the primers rhlABf and rhlABr were used, a
single PCR fragment of the expected size (777 bp) was
amplified in DHT2 (Fig. 5). We sequenced the PCR
Table 5 Emulsification index (E24) with various hydrocarbons for
supernatant grown in BSM-GH at various temperature and salinity product and blast search was carried out. Blast search of
conditions for a week rhlab gene of Pseudomonas aeruginosa DHT2 showed
more than 99% similarity with rhlAB gene of Pseudomo-
Hydrocarbon Emulsification index (E24)
nas aeruginosa PAO, a prototype bacteria known to
30°C 30°C 45°C 45°C produce rhamnolipid type of biosurfactant. The rhlAB gene
without with 10% without with 10%
NaCl NaCl NaCl NaCl
partial consensus sequence, obtained from the isolate
DHT2, was different only by three mismatches in 620
Hexadecane 78 ± 4 75 ± 3 73 ± 4 70 ± 4 bases with sequences of Pseudomonas aeruginosa PAO.
Hexane 41 ± 3 38 ± 2 35 ± 2 43 ± 4 No protein was detected in partially purified biosurfactant;
Gas oil 67 ± 3 73 ± 4 75 ± 3 72 ± 4 it however contained carbohydrate and lipid, indicating it
Gasoline 36 ± 4 40 ± 2 44 ± 4 47 ± 2 as a glycolipid. The results further revealed that the sur-
Kerosene 47 ± 5 56 ± 3 54 ± 2 50 ± 3 factant produced by the strain DHT2 is rhamanolipid.
Octadecane 70 ± 3 69 ± 4 65 ± 3 63 ± 4
Diesel oil 56 ± 3 60 ± 2 66 ± 4 60 ± 2
Toluene 07 ± 2 09 ± 2 10 ± 1 05 ± 1

Results indicated that among the hydrocarbons tested, as

substrate for emulsification by biosurfactant DHT2, the
maximum (78 ± 4%) emulsification activity was with n-
hexadecane while toluene was the poorest.
The emulsion was found stable at room temperature for
72 h with out any significant change in emulsification
index. The biosurfactant produced from growth on one
substrate was equally effective in emulsifying it as well as
other hydrocarbons. This suggested that the emulsification
activity of the biosurfactant was not substrate-specific. The
isolated biosurfactant from BSM-GH (45°C, 6% NaCl) was
re-dissolved in distilled water and effect of NaCl and pH
was studied. To study the effect of salinity the salt con-
centration of the solution was kept 0–20%. The emulsion
index was determined as previously stated with hexadec-
ane. The emulsion indices of the biosurfactant with NaCl
up to 10% and without NaCl were almost the same. Fig. 5 Agarose gel electrophoresis of PCR products of rhamnosyl
Decrease in emulsion index was observed in all other tests transferase gene M, 1 kb DNA ladder; 1. rhlab DHT2

123
World J Microbiol Biotechnol (2008) 24:1047–1057 1055

Table 6 Dose-dependent
Concentration of crude biosurfactant (lg/ Solubility of PAH (ng/ml)
solubilization of PAHs by crude
ml)
biosurfactant isolated from P. DBT Phenanthrene Pyrene Anthracene
Aeruginosa
0 300.0 ± 70.0 475.0 ± 70.0 124.0 ± 20.0 50.0 ± 7.0
100 370.0 ± 50.0 550.0 ± 30.0 500.0 ± 30.0 60.0 ± 10.0
200 500.0 ± 80.0 600.0 ± 50.0 700.0 ± 25.0 65.0 ± 5.0
300 700.0 ± 70.0 700.0 ± 40.0 850.0 ± 40.0 70.0 ± 6.0
400 1000.0 ± 100.0 900.0 ± 70.0 900.0 ± 30.0 100.0 ± 11.0
500 1200.0 ± 70.0 950.0 ± 80.0 950.0 ± 50.0 150.0 ± 10.0

PAH solubilization effect of biosurfactants Discussion

In general, the crude biosurfactants from strain DHT2 In search of crude oil modifying bacteria, soil samples
enhanced the apparent solubility of PAH in a dose- were collected from Guanoco Lake, Sucre State in Vene-
dependent manner (Table 6). However, solubilization of zuela. Guanoco Lake is one of the largest natural deposits
DBT by biosurfactants ([4 time higher compared to con- of asphalt and was used as a commercial source of asphalt
trol) was significantly higher when compared with the in past. The whole area is heavily contaminated with
phenanthrene, pyrene, anthracene solubilization effect of hydrocarbons and asphalt. We have been able to isolate
biosurfactants (2–3 times higher compared to control). bacteria with ability to degrade/modify the heavy crude oil/
bitumen from Guanaco lake (Kumar et al. 2007). The
present strain was selected for further study due to its
Analysis of cell surface hydrophobicity ability to grow on wide range of hydrocarbon in varied
range of temperature and salinity conditions and producing
The hydrophobicity of the bacterial cell surface was found extracellular effective biosurfactant. This isolate grew and
to be changed during growth (Fig. 6) on hydrocarbons produced biosurfactant when cultured in variety of sub-
suggesting that biosurfactant not only help in emulsifica- strates at salinities of up to 60 g l-1 and temperatures up to
tion but also play role in the change of the cell surface 45°C. Oil-degradation and biosurfactant-producing micro-
hydrophobicity and in turn may improve the affinity of bial investigations on Pseudomonas species mainly deals
microbial cells for the substrate to facilitate their bio- with mesophiles, while a few reports refer to thermophilic
availability. Significant increase in cell surface conditions (Cameotra and Makkar 1998). However, there is
hydrophobicity was observed towards aliphatic hydrocar- no report on biosurfactant production in halotolerant and
bons. Presumably, the degradation of different types of thermotolerant conditions. Thermophiles/thermotolerant
hydrocarbons by DHT2 was due to the solubilization, are of great interest for biotechnological applications, as
increased bioavailability of hydrocarbon as well as changes they can be used in most industrial processes that run at
in cell surface properties by extracellular biosurfactant. elevated temperatures (Niehaus et al. 1999). To the best of
our knowledge, Pseudomonas DHT2 is the first reported
80
thermotolerant and halotolerant biosurfactant producing
70 Pseudomonas strain.
% Adhered to hexadecane

60 In the present study microtiter plate-based assays was

50 used to evaluate the ability of microbe to utilize various
40
hydrocarbons and was found suitable alternate to the flask-
assays. Isolated DHT2 was capable of utilizing crude oil,
30
fuels, various pure alkanes and PAHs as a sole carbon and
20
energy source across the wide range of the temperature
10 (30–45°C) and salinity (0–6%). Over the range evaluated,
0 the degradation of hydrocarbon and biosurfactant produc-
tion was not influenced by salinity and temperature. The
se

ne

ne

l
oi

se
an

en

en

en
co

re

ie
as
ec

al

ph

os
th

Py
lu

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h

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ad

er
io
an
G

ht

effect of salinity and/or temperature on the degradation of

K
th
ex

ap

en

zo
H

Ph

en

hydrocarbon using single culture or microbial consortia has

ib
D

been studied by several investigators (Mille et al. 1991;

Fig. 6 Cell hydrophobicity during growth of Pseudomonas sp. DHT2
in different hydrocarbons. The bacterium was grown in BSM at 30°C
Bertrand et al. 1990; Diaz et al. 2002; Riss et al. 2003).
for 1 week, washed and assayed The bacterial consortia isolated by Bertrand et al. (1990)

123
1056
was incapable of utilizing hydrocarbons at salinities less
than 100 g/l, while other consortia isolated by the same
group was ineffective both in absence of NaCl and at
concentrations greater than 116 g/l. According to Diaz et
al. (2002), PAHs degradation by microbial consortia was
higher in low salinities with maximum at 40 g/l and
decreased with increasing salinity. In contrast to this, the
present bacterium, DHT2 could degrade PAHs to same
extent in across the wide range of salinity in varied range of
temperatures. Our findings are in good agreement with
observations of the Mille et al. (1991).
Bacterium DHT2, produces biosurfactant during growth
on various water soluble and insoluble carbon sources.
Pseudomonas strains have been reported to produce bio-
surfactants on hydrocarbons, water-immiscible substrates
(Benincasa et al. 2004; Rahman et al. 2002) and on
readily available carbon sources such as glucose (Bodour
et al. 2003), mannitol or glycerol (Arino et al. 1996). The
isolate also could produce biosurfactant during growth on
PAHs, which has rarely been reported. Deziel et al. (1996)
isolated a total of 23 PAH degrading bacterial strains from
petroleum-contaminated soils, and 10 of them were able to
produce biosurfactants. In one of the strains, Pseudomonas
aeruginosa 19SJ, the production of biosurfactant from
solid naphthalene was accompanied by an increase in the
aqueous concentration of the compound, thus suggesting a
role in promoting its solubilization. Highest emulsification
activity and biosurfactant production (7.05 g l-1) was
obtained when the bacterium DHT2 was grown in BSM
containing glucose and hexadecane in equimolar concen-
trations as carbon source. The emulsion was found stable
at room temperature for 72 h with out any significant
change in emulsification index. We found that biosurfac-
tant produced from growth on one substrate was equally
effective in emulsifying the same as well as other
hydrocarbons. It indicated that the emulsification activity
of the biosurfactant was not substrate-specific. However,
substrate-specific emulsification by biosurfactant has been
demonstrated by Falatko and Novak (1992), showing that
biosurfactants produced from growth on glucose or veg-
etable oil could not emulsify gasoline hydrocarbons, while
biosurfactants produced from growth on gasoline could
emulsify. Some microbes have been reported to be capa-
ble of producing surfactants with the ability to emulsify
various hydrocarbons, irrespective of the substrates used
as carbon source. Flavobacterium sp. DS5-73 and
Micrococcus sp. GS2-22 were able to produce surfactants,
which emulsified all the hydrocarbons tested (Rahman
et al. 2003). In the present study, the crude biosurfactant
also showed ability to solubilize PAHs in aqueous phase
indicating its possible role in increasing the bioavailability
of non-soluble organic compounds for bacterial
metabolism.
123
World J Microbiol Biotechnol (2008) 24:1047–1057
The chemical analysis revealed that the biosurfactant
produced by DHT2 contains carbohydrates and lipids. The
Siegmund and Wagner technique further indicated that
the biosurfactant produced is rhamnolipid in nature. The
production of rhamnolipids is a unique characteristic of
P. aeruginosa. Rhamnolipid synthesis proceeds by
sequential glycosyl transfer reactions, encoded by the
rhlAB operon, in which RhlA is involved in the synthesis
of the fatty acid dimer moiety of rhamnolipids while
rhamnosyl transferase I (rhlB) gene encode the catalytic
subunit of the rhamnosyltransferase (Ochsner et al. 1994a,
b). Detection of this gene in DNA extracts was used to
confirm ability of the microbe to produce rhamolipid type
of biosurfactant. DHT2 gave a single band of expected
size with the primers designed based on the rhlAb
sequences in data base. When the amplified fragment was
sequenced, it showed more than 99% similarity with well-
studied rhlAb gene of rhamnolipid producing bacteria
Pseudomonas aeruginosa PAO-1. These results indicate
that the DHT2 is P. aeruginosa and produces rhamnolipid
type of biosurfactants.
Conclusions
The bacterial isolate DHT2 was capable of utilizing
hydrocarbon and producing rhamnolipid type biosurfactant
across the wide range of temperature (30–45°C) and
salinity (0–6%). Over the range evaluated, the degradation
of hydrocarbon and biosurfactant production was not
influenced by salinity and temperature. This bacterium can
grow effectively in wide salinity and temperature gradients
hence can be used in fluctuating temperature and salinity
conditions. Facultative thermo and halotolerant microbes
may also be useful for resting cell condition (which is
considered near to reality for the development of bio-
process in petroleum industry) since they can be grown in
expense of less energy and materials before hydrocarbon
treatment.
Acknowledgment The authors wish to thanks to Dr R. K. Upreti,
ITRC, Lucknow for critical appraisal of the manuscript.
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