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Biosurfactant-Mediated Biodegradation of Polycyclic

Aromatic Hydrocarbons—Naphthalene
a a b a
Subramanyam Dasari , K. C. Venkata Subbaiah , Rajendra Wudayagiri & Lokanatha Valluru
a
Department of Biotechnology, Dravidian University, Kuppam, Andhra Pradesh, India
b
Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
Published online: 08 Aug 2014.

To cite this article: Subramanyam Dasari, K. C. Venkata Subbaiah, Rajendra Wudayagiri & Lokanatha Valluru (2014)
Biosurfactant-Mediated Biodegradation of Polycyclic Aromatic Hydrocarbons—Naphthalene, Bioremediation Journal, 18:3,
258-265, DOI: 10.1080/10889868.2014.933169

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 Bioremediation Journal, 18:258–265, 2014
Copyright Ó 2014 Taylor and Francis Group, LLC
ISSN: 1088-9868 print / 1547-6529 online
DOI: 10.1080/10889868.2014.933169
Biosurfactant-Mediated Biodegradation
of Polycyclic Aromatic
Hydrocarbons—Naphthalene
Subramanyam Dasari,1 K. C.
Downloaded by [Sri Venkateshwara University] at 21:34 13 August 2014
Venkata Subbaiah,1 Rajendra
Wudayagiri, 2 and Lokanatha
Valluru 1
1
Department of Biotechnology,
Dravidian University, Kuppam,
Andhra Pradesh, India
2
Department of Zoology,
Sri Venkateswara University,
Tirupati, Andhra Pradesh, India
Address correspondence to Lokanatha
Valluru, Department of Biotechnology,
Dravidian University, Kuppam 517426,
A.P, India. E-mail: lokanathav@yahoo.
co.in
Color versions of one or more of the
figures in the article can be found
online at www.tandfonline.com/bbrm.
ABSTRACT The present study is aimed at the naphthalene degradation with
and without biosurfactant produced from Pseudomonas aeruginosa isolated from
oil-contaminated soil. The present study was carried out to isolate the bacterial
strains for the naphthalene degradation and also for biosurfactant production.
The isolated strains were screened for their ability to degrade the naphthalene
by the methods of optimum growth rate test and for the production of
biosurfactants by cetyltrimethylammonium bromide, blood agar medium, and
thin-layer chromatography. The present study also focused on the effect of
biosurfactant for the degradation of naphthalene by isolate-1. Two bacterial
strains were isolated and screened, one for biodegradation and another for
biosurfactant production. The second organism was identified as Pseudomonas
aeruginosa by 16S rRNA analysis. The purified biosurfactant reduces the surface
tension of water and also forms stable emulsification with hexadecane and
kerosene. The end product of naphthalene degradation was estimated as
salicylic acid equivalent by spectrophotometric method. The results
demonstrated that Pseudomonas aeruginosa has the potential to produce
biosurfactant, which enhances the biodegradation of naphthalene. The study
reflects the potential use of biosurfactants for an effective bioremediation in the
management of contaminated soils.
KEYWORDS biodegradation, biosurfactant, emulsification, naphthalene, polycyclic
aromatic hydrocarbons (PAHs), Pseudomonas
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are one class of toxic pollu-
tants that have been accumulated in the environment by natural and
anthropogenic activities (Aronstein, Calvillo, Alexander 1991; Banat 1995).
They are mainly produced from incomplete combustion of organic mate-
rial, fossil fuels, spillage of petroleum products, and various industrial activ-
ities. Humans are exposed to PAHs by means of air, soil, food, water, and
occupational exposure (Box 1983; Brandt and Watson 2003). The adverse
health and environmental effects of PAH compounds are widely known,
258
 which include serious health problems in some
instances and genetic defects in humans (Chandrase-
karan and BeMiller 1980). Based on their ecotoxic-
ity, the US Environmental Protection Agency (US
EPA) has listed 16 PAHs as priority pollutants for
remediation (Bojes and Pope 2007).
The bioremediation of soils contaminated with
PAHs is limited by the bioavailability of these hydro-
phobic contaminants to microorganisms (Grimmer
1979). Surfactants were used for solubilization or emul-
sification and release hydrocarbons sorbed to soil
organic matter and increase the aqueous concentrations
of hydrocarbon compounds (Guerra-Santos, Kappeli,
and Fiechter 1984). However, recent studies indicate
that surfactants can enhance biodegradation of hydro-
carbons by increasing microbial accessibility to insolu-
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ble substrates (Haba et al. 2000). Several researchers
have investigated the addition of biosurfactants to
enhance the biodegradation of hydrocarbons.
The composition, structure, and properties of variety
of surfactants produced by bacteria, yeasts, and fungi
have been described (Hisatsuka, Nakahara, and Sano
Yamada 1971; Hommel et al. 1994). Among the best-
studied biosurfactants are the rhamnolipids of Pseudo-
monas aeruginosa. Most extracellular surface-active
compounds are synthesized by microorganisms grow-
ing on poorly soluble substrates (Hommel 1990). In
addition, biosurfactant enhances ability of microorgan-
isms to utilize hydrocarbons as substrates (Oberbremer,
Muller-Hurtig, and Wagner 1990). Koch et al. (1991)
have isolated a mutant of Pseudomonas aeruginosa
PG201 that had lost its capacity to grow on hexade-
cane, as it failed to secrete its extracellular rhamnolipids
(Oberbremer, Muller-Hurtig, and Wagner 1990).
The present study was undertaken to isolate the Pseu-
domonas sp. that degrades naphthalene, one of the com-
ponents present in diesel, from the oil-contaminated
soil. Besides, the studies also aimed to isolate the rham-
nolipid from Pseudomonas aeruginosa that can be used
as a biosurfactant in order to study the ability of Pseudo-
monas sp. in the biodegradation of naphthalene.
MATERIALS AND METHODS
Isolation of Naphthalene-Degrading
Microorganisms
Diesel-contaminated soil samples were collected
from the leakage channels in Indian oil tanks at
259
Surareddypalem near Ongole, in Andhra Pradesh,
India. The soil sample (1 g) is enriched over a time
period of 7–10 days with mineral salt medium (MSM)
containing 0.2% naphthalene dissolved in acetone.
The enriched soil samples are serially diluted and
plated on a Bushnell-Hass agar selective medium
(Himedia) contain naphthalene (0.2%) as a source of
carbon. After the incubation period, plates were enu-
merated and morphologically different bacteria were
selected for biodegradation.
Isolation of Biosurfactant-Producing
Bacteria
The same soil samples were used for the production
of biosurfactant. Soil samples are placed in sterile PYG
medium (peptone, 5.0 g; yeast extract, 5.0 g; glucose,
15.0 g; and distilled water 1 L) and kept at 37 C on
rotary shaker at 120 rpm for 72 h (Rocha et al. 1992).
The enriched soil samples are serially diluted and
plated on PYG medium. After the incubation period,
the plates were enumerated and morphologically differ-
ent bacteria were selected for biosurfactant production.
Screening for Biodegradation
of Naphthalene by Isolated Strains
The selected isolates were cultured in 100 ml Bushnal-
Hass (BH) medium containing 0.2% naphthalene
(dissolved in acetone) and incubated at 37 C and
100 rpm (Kiyohara, Nagao, and Yana 1982). Naphtha-
lene utilization of the selected isolates was initially
assessed by disappearance of naphthalene crystals, color
change, and increase in the optical density of the
medium at regular intervals.
Screening for Biosurfactant
Production
Isolated strain 2 was screened for the ability to
produce surfactants by cultivation in blue agar
plates containing cetyltrimethylammonium bromide
(C-TAB) (0.2 mg/ml) and methylene blue (5 mg/ml)
(Kastnar 2000) and was used to detect glycolipid
production with 2% dextrose or mannitol as a car-
bon source. Biosurfactants were observed by the for-
mation of dark blue hallows around the colonies.
Isolated strains were also screened on blood agar
Biosurfactant-Mediated Biodegradation of Naphthalene
 plates containing 5% (v/v) human blood and incu-
bated at 37 C for 24–48 h. Hemolytic activity was
detected as the occurrence of clear zone around the
colony (Carrillo et al. 1996).
Morphological and Biochemical Tests
for Identification of Isolated Strains
Isolates, grown on nutrient agar medium, were exam-
ined for Gram staining, cell morphology, and cultural
characteristics. The biochemical tests include indole,
methyl red Voges-Proskauer, and citrate utilization
(IMViC) tests, oxidase, catalase, and gelatin tests, along
with pigment production were studied according to
Bergey’s Manual of Determinative Bacteriology (Koneman
et al. 1997).
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Molecular Identification
of Biosurfactant-Producing Isolate
DNA Extraction and Cell Lysis
Total DNA was isolated from bacterial strain
(Sambrook, Fritsch, and Maniatis 1989). Heat-lysate
bacterial suspensions used in polymerase chain reac-
tion (PCR) analysis were prepared from cultures
grown on Luria agar for 24 h at 37 C. Two bacterial
colonies were suspended in 100 ml of lysate solu-
tion (0.05 mM NaOH, 0.25% sodium dodecyl sul-
fate [SDS]) and incubated for 15 min at 100 C.
The suspension was centrifuged for 1 min at
12,000 rpm and diluted 50-fold in sterile distilled
water. The PCR primers were designed (MWG, Bio-
tech, Eurofins, Bangalore, India) to amplify approxi-
mately 1300 bp of consensus 16S rRNA gene, with
forward primer 67 F (5’-CAG GCC TAA CAC ATG
CAA GTC-3’) and reverse primer 1387 R (5’-GGG
CGG WGT GTA CAA GGC-3’).
The PCR was performed in a 25-ml reaction mixture
containing 100 ng of template DNA, 1£ PCR buffer
(Fermantas, Ontario, Canada), 0.2 mM dNTPs,
2.5 mM MgCl2, 10 pmoles of each primer, and 1 U of
Taq DNA polymerase (Carbett Research, Sydney,
Australia). The reaction was initiated with a denatur-
ation step at 94 C for 5 min, followed by 30 cycles of
denaturation at 94 C for 30 S, annealing at 55 C for
1 min, and extension at 72 C for 2 min and the final
extension was at 72 C for 15 min. Amplified products
were separated by electrophoresis using 1% agarose gel
S. Dasari et al.
containing ethidium bromide (10 ng/ml) in 1£ TBE
buffer and visualized under ultraviolet light using an
Alpha Innotech Corp. (San Jose, CA, USA) gel docu-
mentation system (Figure 4).
Biosurfactant Production
and Purification
The medium containing high concentration (2%) of
mannitol was used as a substrate for biosurfactant pro-
duction. Growth was monitored by measuring the opti-
cal density at A600 nm. The concentration of
extracellular glycolipids was estimated in duplicates by
measuring the concentration of rhamnose with a modi-
fied orcinol assay (Mihelcic et al. 1993). The acidified
culture supernatant was extracted three times with
diethyl ether, and then the fractions were pooled,
dried, and resuspended in 0.05 M sodium bicarbonate.
A 200-ml sample was treated with 1.8 ml solution of
orcinol reagent and boiled for 20 min. After cooling at
room temperature for 15 min, the color intensity was
measured at 421 nm. Rhamnolipid concentrations
were calculated from standard curve prepared with L-
rhamnose and expressed as mg of rhamnose equivalents
(RE)/ml. The emulsifying activity of the dried powder
was estimated by adding 0.5 ml sample fluids and
0.5 ml hexadecane or kerosene to 4.0 ml of distilled
water. The contents were vortexed for 20 s and turbid-
ity of a stable emulsion was visually examined (Cooper
and Goldenberg 1987). The emulsification activity is
defined as the height of the emulsion layer divided by
the total height and expressed as percentage.
Naphthalene Degradation
by Isolate-1
The study on the degradation of naphthalene was
carried out in batch mode of operation. One hundred
milliliters of MSM containing 0.2% naphthalene (dis-
solved in acetone) as a sole carbon source were added
in each flask. These flasks were then inoculated with
0.1% (v/v) inoculum previously prepared, and the
flasks were kept in shaker at 120 rpm for a period of
10 days. The optical density (OD) of the broth was
measured at regular time intervals (24 h), and the same
broth was used to estimate the end product of biodeg-
radation in terms of mg of salicylic acid equivalents
(SE)/ml by the method of Box (1983).
260
 Effect of Biosurfactant (Rhamnolipid)
on Naphthalene Biodegradation
The degradation of naphthalene was carried out in
the presence of a glycolipid (biosurfactant). A 0.05%
sample of biosurfactant extracted from isolate-2 and
0.1 ml of overnight culture of isolate-1 were added to
100 ml of MSM containing 0.2% naphthalene. The
flasks were kept in shaker at 120 rpm for a period of
10 days or up to decreased optical density. The OD of
the broth was measured at regular intervals (24 h), and
the SE was estimated by the method of Box (1983).
RESULTS
Identification of Bacterial Isolates
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Table 1 shows the results of the morphological and
biochemical tests. The biodegrading bacterial cultures
were identified as genus of Pseudomonas, and the bio-
surfactant-producing strain (isolate-2) was identified as
Pseudomonas aeruginosa by 16S rRNA analyses
(sequence accession number: HQ324120).
Naphthalene Utilization
Thirteen bacterial colonies were grown on BH agar
plates containing 2% naphthalene. Out of 13 isolated
strains, only 4 strains have shown the ability to grow
TABLE 1 Microbiological and Biochemical Tests for Bacterial Strains Isolated from Diesel-Contaminated Soils
Sample no. Tests
1 Medium used for inocula
2 Colony character
3 Colony on nutrient medium
4 Gram staining
5 Motility
Biochemical tests
a Indole test
b Methyl red test
c VP test
d Citrate utilization test
e Oxidase test
f Nitrate test
g Catalase test
h Gelatin
i Growth on MacConky agar
j Growth on blood agar
k Oxidative fermentation
Note. BH medium D Bushnal-Hass medium; strain 1 D biodegrading bacteria; strain 2 D biosurfactant-producing bacteria; C D positive;
¡ D negative; NLF D nonlactose fermentor; Glu D glucose; Xyl D xylose.
261
on the plates with naphthalene as a sole carbon source.
This naphthalene utilization was initially assessed by
disappearance of naphthalene crystals and increase in
the OD of the medium (Figure 1). Out of four isolates,
only one strain was selected the degradation of naph-
thalene due to its optimum growth rate in 2% naphtha-
lene as a sole carbon source (Figure 1).
Screening for the Ability to Produce
Biosurfactant
When grown on PYG medium, morphologically
identical colonies with round, creamiest, and concave
features were observed. One strain was selected for bio-
surfactant production (Table 1). When grown on blue
agar plates containing C-TAB, methylene blue with 2%
glucose or mannitol, the isolated strain forms blue
color colonies with hallows (Figure 2A) and these iso-
lated strains have also exhibited clear zones on the
blood agar medium (Figure 2B).
Molecular Identification of
Biosurfactant-Producing Isolate
In PCR, a single band of expected size (»1300 bp)
using universal primer 67 F and reverse primer 1387 R
(Figure 3). The identity of the 1300-bp product was
confirmed by cloning and sequencing. The selected
Strain 1 Strain 2
BH C naphthalene BH C naphthalene
Round, yellow-whitish, convex Round, creamish, concave
White Yellow-white
Negative rods Negative rods
Motile Motile
C C
¡ ¡
¡ ¡
C C
C C
¡ ¡
C C
¡ ¡
NLF NLF
Clear zone Clear zone
Utilizes Glu, Xyl Nonutilizer
Biosurfactant-Mediated Biodegradation of Naphthalene
 FIGURE 3 PCR amplification of 16S rRNA from isolate-2.
FIGURE 1 Growth curves of four isolates on MSM with 2%
naphthalene as a sole carbon source.

Naphthalene Degradation
Naphthalene degradation products in supernatants
biosurfactant-producing strain (isolate-2) was
Downloaded by [Sri Venkateshwara University] at 21:34 13 August 2014

were accompanied by decrease in pH. The important

sequenced and deposited in NCBI GenBank and
metabolite in naphthalene degradation was salicylic
identified as Pseudomonas aeruginosa (accession no.
acid, which was determined by spectrophotometric
HQ324120).
method. The bacterial growth and the naphthalene
degradation gradually increased up to 10 days and
decreased after 10 days of incubation time, which indi-
Biosurfactant Production cates that the isolate-1 requires 10 days for the degrada-
and Purification tion of naphthalene (Figure 6).
The amount of biosurfactants produced was there- However, in the presence of the biosurfactant rham-
fore sufficient to reach the lowest possible surface ten- nolipid, the isolate-1 degraded naphthalene in 6 days
sion with critical micelle concentration (1.1 £ 10¡4 to (Figure 7) and the faster degradation was primarily due
1.5 £ 10¡3 M). The produced biosurfactants were iden- to the presence of rhamnolipid, which reduced the sur-
tified as rhamnolipid by orcinol assay (blue color) and face tension of the broth and subsequently increased
thin-layer chromatography (TLC) (yellow-colored spot, the bioavailability of naphthalene to the bacteria.
Figure 4). The maximum biosurfactant production was
seen at 18 h incubation time, and its concentration was
estimated at 3.1 mg/ml. The extracted rhamnolipid
forms stable emulsification with diesel and kerosene as
substrates compared with that of chemical surfactants
(Figure 5).

FIGURE 2 (A) Detection of glycolipids (biosurfactant) pro-

duced by isolated strain 2 with C-TAB agar medium shows
blue-colored colonies with hallow zone around the colony.
(B) Hemolytic activity of bacterial supernatant on blood agar FIGURE 4 TLC plate shows yellow-colored spots for rhamnoli-
medium showing clear zone around the bacterial streak. pid from the isolated Pseudomonas aeruginosa strain.

S. Dasari et al. 262

 FIGURE 5 Emulsification activity of isolate-2, SDS, Triton X-
100, and no surfactant (control).
DISCUSSION
During the past 30 years, different remediation tech-
nologies have been tested in efforts to remove the envi-
Downloaded by [Sri Venkateshwara University] at 21:34 13 August 2014
ronmental contaminants. Among them, bioremediation
is considered to be a safe and cost-effective option (Parra
et al. 1989; Prince 1997). In general, the mechanism of
aerobic degradation of naphthalene involves the incor-
poration of molecular oxygen into one of the aromatic
rings by naphthalene dioxygenase, leading to the forma-
tion of cis-1,2-naphthalene dihydrodiol. The latter
undergoes a number of further degradative steps and
finally gets metabolized to carbon dioxide through sali-
cylic acid. In the present study, the addition of biosurfac-
tant effectively enhanced the solubility of naphthalene.
This is mainly due to the amphiphilic nature of the bio-
surfactants, which tend to concentrate at interfaces and
to decrease interfacial tension. In addition, the biosurfac-
tant, at certain concentrations, enhances the formation
of stable aggregates of 10–200 molecules called micelles
that can increase the solubility of the hydrophobic
contaminants.
FIGURE 6 Naphthalene degradation by isolated strain 1, which
is estimated by the end product in salicylic acid equivalents.
263
FIGURE 7 Naphthalene degradation in the presence of biosur-
factant, which reduces the surface tension of the broth and
enhances the biodegradation of compound.
Bioremediation, in general, requires relatively long
time scale due to slower kinetics when compared with
other remediation options. In the case of PAHs, the
rate of in situ microbial metabolism is limited because
of lower metabolic capabilities due to low solubility of
these compounds, resulting in low bioavailability to
the organisms (Pritchard et al. 1995). In order to
enhance the solubility of PAHs, surface-active agents
such as mobilizing agents are being applied (Providenti
et al. 1995). Surfactant approaches to enhancing bio-
degradation often attempt to increase the solubility of
hydrophobic organic contaminants by the addition of
biosurfactants. Many studies of biosurfactant-enhanced
bioremediation have employed well-characterized bio-
surfactants such as rhamnolipid from Pseudomonas aeru-
ginosa (Rahaman et al. 2002), trehalose dimycolipids
from Rhodococcus sp. (Ramdahl 1985), sophorose
lipids from Candida apicola (Robert et al. 1989), lichen-
ysins from Bacillus sp. (Saraswathy and Hallberg 2002),
and surfactin from Bacillus subtilis (Seigmund and
Wagner 1991). These are potent surfactants, as they
reduce the surface tension from 72 to <30 dynes/cm.
In the present study, the isolated Pseudomonas aerugi-
nosa produces a biosurfactant that is identified as rham-
nolipid by orcinol method. The exact reason for the
production of surfactants by these microorganisms is
still not clear. However, some reports indicate that
these microorganisms facilitate their diffusion into the
cell by producing a variety of substances called biosur-
factants. Some bacteria excrete ionic surfactants, which
emulsify hydrocarbon substrates in the growth medium
(Guerra-Santos, Kappeli, and Fiechter 1984). Growth
conditions favorable for the production of surfactants
require an elevated C-to-N ratio and limiting iron
Biosurfactant-Mediated Biodegradation of Naphthalene
 concentration (Liu 2001). However, biosurfactant-
producing bacteria are found in higher concentrations
in hydrocarbon-contaminated soils. Accordingly, our
results also suggest that the capacity to secrete surface-
active agents is a common feature among PAH-
degrading soil bacteria (Stefen 2003). Haba et al.
isolated the strain Pseudomonas aeruginosa 47T2
NCIB40044, from 36 screened strains, which exhibits
capacity to produce 2.7 g/L of rhamnose in a nonaer-
ated culture with waste frying soybean oil (WFSO). Less
rhamnose production (1.41 g/L) had been achieved
using the Pseudomonas aeruginosa LBI strain in bioreactor
culture utilizing fresh soybean oil as the carbon source
(Ulric 2000). According to Rahaman et al. (2002), culti-
vation of the Pseudomonas aeruginosa GS 9-119 strain in
soybean and sunflower oil for 228 h resulted in maxi-
Downloaded by [Sri Venkateshwara University] at 21:34 13 August 2014
mum rhamnolipid production of 1.75 and 1.66 g/L,
respectively. Due to the presence of this biosurfactant,
naphthalene can be easily soluble in water or MSM and
is able to increase the bioavailability of the compound
to the organism, which facilitates faster degradation as
compared with the biodegradation without biosurfac-
tant. The Pseudomonas aeruginosa that is isolated in the
present study decreases the water surface tension up to
32 dynes/cm and also forms stable emulsification
against the kerosene and diesel.
Biosurfactant-mediated bioremediation have been
applied to clean up aromatic hydrocarbon contamina-
tion in various ecosystems and under a wide range of
environmental conditions. Their applications include
in situ and ex situ treatment of hydrocarbon-contami-
nated soil. Only limited numbers of pilot-scale and
field trials (Venosa et al. 1996, 2002) were effective in
bioremediation treatment. However, further field tests
are required to evaluate the effectiveness of this strat-
egy, particularly to determine whether the addition of
microbial cultures is necessary or effective in enhancing
biosurfactant-mediated biodegradation in soil.
The results obtained from these studies demonstrated
that the isolated Pseudomonas aeruginosa has the poten-
tial to produce biosurfactant. Under optimal condi-
tions, this process resulted in the production of 3.1 g/L
of rhamnolipid, with emulsification activity. The values
are greater than the most of the comparable data
reported earlier in Ochsner, Hembach, and Fiechter
(1996) (2 g/L) when using corn oil as a carbon source,
and Trummler, Effenberger, and Syldatk (2003) demon-
strated that Pseudomonas sp. (DSM 2874) produces
0.14 g/L of rhamnolipid. The extracted rhamnolipid
S. Dasari et al.
facilitates faster degradation of naphthalene as com-
pared with the biodegradation without rhamnolipid.
ACKNOWLEDGMENT
The authors are thankful to Head, Department of
Virology, Sri Venkateswara University, Tirupati, A.P,
India, for providing laboratory facilities and guidance.
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Biosurfactant-Mediated Biodegradation of Naphthalene