Isolation and Characterization of Naphthalene-Degrading Bacteria from Sediments ofCadiz Area (SW Spain)

D. Nair,1 F. J. Fernandez-Acero,2 E. Garcıa-Luque,1,3 I. Riba,1,3 T. A. Del Valls1,3

1Catedra UNESCO/UNITWIN/WiCop, Facultad de Ciencias del Mar y Ambientales,Universidad de Cadiz, Polıgono Rıo San Pedro s/n 11510 Puerto Real (Cadiz), Espana

2Laboratorio de Microbiologıa, Facultad de Ciencias del Mar y Ambientales, Universidad deCadiz, Polıgono Rıo San Pedro s/n 11510 Puerto Real (Cadiz), Espana

3Departamento de Quımica Fısica, Facultad de Ciencias del Mar y Ambientales, Universidadde Cadiz, Polıgono Rıo San Pedro s/n 11510 Puerto Real (Cadiz), Espana

Received 4 October 2007; revised 4 March 2008; accepted 16 April 2008

ABSTRACT: Petroleum hydrocarbon contamination of harbor sediments from shipping activity, fuel oilspills, and runoffs are becoming a great concern because of the toxicity and recalcitrance of many of thefuel components. Polycyclic aromatic hydrocarbons (PAHs) are of most concern due to their toxicity, lowvolatility, resistance to degradation, and high affinity for sediments. Microorganisms, especially bacteria,play an important role in the biodegradation of these hydrocarbons. The objective of the present studywas to characterize and isolate PAH-(naphthalene) degrading bacteria in the coastal sediments of Cadiz(SW Spain), since this area is mostly polluted by PAH occurrence. A total of 16 naphthalene-utilizing bac-teria were isolated from these sites. Introduction of bacteria isolated from contaminated sediments intomineral medium contributed to the increased rate of hydrocarbon utilization. The bacterial isolatesobtained from these sites are very potent in utilizing naphthalene and crude oil. It would be interesting toassess if the selected naphthalene-degrading isolates may degrade other compounds of similar structure.Hence these isolates could be very helpful in bioremediating the PAH-contaminated sites. Further pursueon this work might represent eco-friendly solution for oil contamination on sea surface and coastal area.# 2008 Wiley Periodicals, Inc. Environ Toxicol 23: 576–582, 2008.

Keywords: polycyclic aromatic hydrocarbons; naphthalene; biodegradation; bacteria; Cadiz

INTRODUCTION

The degradation of polycyclic aromatic hydrocarbons

(PAHs) by bacteria has been widely studied as many pure

cultures have been isolated and characterized for their

ability to grow on PAHs over many previous investigators

(Chadhain et al., 2006). The extensive investigation on

PAHs degradation still provide limited information about

the diversity of microbes involved in PAH degradation in

the environment. Bioremediation of PAH-polluted soil is

severely hampered by the low rate of degradation of the

higher PAHs, particularly the four- and five-ring PAH, due

to their very low water solubility and are often tightly

bound to soil particles. This results in very low bioavaila-

bility for bacterial degradation (Kotterman et al., 1998).

PAH concentrations in the environment vary widely,

depending on the proximity of the contaminated site to the

Correspondence to: E. G. Luque; e-mail: enrique.luque@uca.es

Contract grant sponsor: Spanish Ministry of Science and Education.

Contract grant numbers: CTM2005-07282-C03-C01/TECNO, VEM2003-

20563/INTER.

Contract grant sponsor: ERASMUS MUNDUS.

Published online 4 June 2008 in Wiley InterScience (www.interscience.

wiley.com). DOI 10.1002/tox.20408

�C 2008 Wiley Periodicals, Inc.

576

production source, the level of industrial development, and

the modes of PAH transport. Soil and sediment PAH con-

centrations at contaminated and uncontaminated sites rang-

ing from 1 lg kg21 to over 300 g kg21 have been reported

(Kanaly and Harayama, 2000). In the open oceans, PAHs

carried in the air are likely to be the main inputs. However,

other more localized inputs of PAHs include waterborne

industrial effluents (particularly from the metal and oil

industries), discharges of oil from shipping, and emissions

and flaring operations by the offshore oil and gas industry.

PAHs are always present as complex mixtures in PAH-

contaminated soils and sediments, and many organisms

isolated from contaminated systems have been found to

grow on a number of different PAHs as sole carbon sources

(Singleton et al., 2005). Several novel marine PAH

degraders, e.g., Cycloclasticus spp. and Neptunomonasnaphthovorans, have also been isolated from contaminated

sediments (Dyksterhouse et al., 1995; Geiselbrecht et al.,

1996; Hedlund et al., 1999). Marine contamination of water

and sediments has become a prime concern due to continu-

ous increase of maritime traffic and accidents in European

Atlantic waters. The English Channel and waters around

Galicia in Spain were the areas with most accidents. Recent

black tides from the Erika and Prestige vessels provided

new evidence for the high risk of accidents with serious ec-

ological impact, which is historically the most important oil

spill hotspot worldwide (Vieites et al., 2004). The interest in

the biodegradation mechanisms of PAHs is initiated because

of its wide ubiquitous distribution in marine ecosystem,

their low availability in soil, high hydrophobicity, where the

aqueous solubility of PAHs decreases almost logarithmi-

cally with increasing molecular mass, and solid–water ratio

consequences (Anders et al., 2005). The biodegradation of

PAHs has been reported to occur under anaerobic, sulfate

reducing and denitrifying conditions (Galushko et al., 1999;

Rockne et al., 2000; Karthikeyan and Bhandari, 2001). The

most important hydrocarbon-degrading bacteria in both ma-

rine and soil environments are Achromobacter spp., Acine-tobacter spp., Alcaligenes spp., Arthrobacter spp., Bacillusspp., Flavobacteriu spp., Nocardia spp. and Pseudomonasspp., and the coryneforms (Leahy and Colwell, 1990; Chung

and King, 2001). Daane et al. (2001) reported that wetlands

show a higher biological activity than many other ecosys-

tems and support enhanced biotransformation of toxic

chemicals. Considering the potential role of bacteria in

PAHs biodegradation, the present location was worth inves-

tigating as source of PAH-degrading bacteria.

Biological methods have an edge over the physicochem-

ical treatment regimes in removing spills since they offer

in situ biodegradation of PAH compounds and oil fractions

by the microorganisms. Thus, this research work explores

the isolation and characterization of isolates that could be

useful to degrade naphthalene by indigenous bacteria from

PAH-contaminated sediments of Cadiz and Algeciras areas

(South West of Spain).

MATERIALS AND METHODS

Isolation of Naphthalene Degrading Bacteria

Sampling for sediment and water was conducted during

September 2006. All the samples were aseptically collected

from different sites of Algeciras and Cadiz areas (SW

Spain) (Fig. 1).

Cadiz Area

Sampling stations: (i) ‘‘Cadiz-1’’ (Ca-1), a clean beach area,

considered as control site. (ii) ‘‘Cadiz-2’’ (Ca-2), a harbor

area, with Ro–Ro vessels, and fishing as main activities.

Algeciras Area

The port of Algeciras is one of the busiest ports in the world

with regard to maritime traffic (oil tankers, passengers’ fer-

ries, Ro/Ro vessels). Two rivers flow into the bay of Alge-

ciras, Palmones River and Guadarranque River. Sampling

stations: (i) ‘‘Palmones Estuary’’ (P-1) station is located in

the middle section of the Palmones River, (ii) ‘‘Guada-

rranque Estuary I’’ (GR30), and (iii) ‘‘Guadarranque Estuary

II’’ (GR4): both stations are located in the mouth of

Guadarranque estuary (which is polluted by industrial

effluents), close to Bay of Algeciras. The samples were

collected from the banks of opposite side of the river.

In all cases, water samples were transferred into polycar-

bonate bottles and the sediment samples were aseptically

transferred into sterile polythene bags and brought to the

laboratory for the study. The samples were stored in the

dark at 48C for further analysis. The pH and temperature of

the sediment samples were checked from the sampling site.

Salinity was measured with an induction salinometer

(Beckman, RS-10) in the laboratory.

Sediment samples were suspended in normal sterile sa-

line (0.85%) at the ratio of 1:100 (w/v) and incubated on the

shaker for 1 h at 150 rpm. The suspension was allowed to

settle down and 0.1 mL of clear supernatant was used to

carry out serial dilutions up to 105 for nutrient agar (NA) and

up to 102 for mineral salt medium (MSM agar: FeSO4, 0.12

Fig. 1. General areas sampled and locations of the five fieldsampling stations.

577NAPHTALENE-DEGRADING BACTERIA FROM CADIZ AREA

Environmental Toxicology DOI 10.1002/tox

3 g L21; K2HPO4, 12.60 g L21; KH2PO4, 3.64 g L21;

NH4NO3, 2.0 g L21; MgSO4�7H2O, 0.2 g L21; MnSO4,

0.0012 g L21; Na2MoO4�2H2O, 0.0012 g L21; CaCl2, 0.15 g

L21). Viable count was determined by spread-plating super-

natant directly as well as its dilutions on NA prepared with

artificial sea water for heterotrophic total counts and MSM

agar containing 0.01% naphthalene (Scharlau Chemie, Bar-

celona, Spain) as well as in MSM agar with 0.2 g of naphtha-

lene added to the lid of Petri plates, and then the plates were

inverted and sealed with parafilm so that the organisms could

utilize naphthalene in vapor phase for naphthalene-degra-

ding organisms (Guerin and Boyd, 1992). NA plates were

incubated at room temperature for 24 h and MSM plates

were incubated for 7 days at 228C. The distinct morphologi-

cal colonies from MSM media were picked, and the isolates

were further purified and maintained on MSM agar with

0.2% naphthalene for further studies.

Characterization and Identification of Isolates

After plating the samples, the isolates obtained on NA and

MSM agar plates were picked and transferred to new plates,

which were incubated at room temperature for 1 week. The

growth rate and morphological characteristics of the iso-

lates was observed. These selected isolates were further

characterized using different percentages of naphthalene.

The obtained isolates were plated on MSM agar with diffe-

rent concentrations of naphthalene, based on their growth

with high percentage of naphthalene (up to 1%) on agar

and their growth on broth with naphthalene was the criteria

used for the selection of the potent isolates.

Growth curve study was carried out by selecting all the

selected isolates and were inoculated in MSM broth with

1% naphthalene and incubated at 288C on the shaker at 150

rpm. The growth was measured after every 24 h at 600 nm

for 3 days. As negative control, the cultures from Spanish

Type Culture Collection (CECT, in Spanish initials): Baci-llus spp. CECT 40, Pseudomonas fluorescens CECT 378,

and E. coli CECT 101 were also inoculated.

Considering the importance of these isolates showing

interesting growth and utilization of higher percentage of

naphthalene, and to confirm their identity based on the mo-

lecular level, isolates were sent out for further identification

to Spanish Culture Collection Center (CECT). The cultures

were identified on the basis of 16S rRNA homology analysis.

RESULTS AND DISCUSSION

To check the total heterotrophic bacterial count of the sedi-

ment, the diluted sediment samples were plated on NA. It

was expected that different kinds of microorganisms would

grow, which gives a total viable count (TVC) of bacteria in

the sediment sample with its enormous diversity. The TVC

was ranging from 1.5 3 104 to 1.93 3 106, expressed in cfu

g21 of sediment. ‘‘GR3’’ site was found to be the most

microbiologically diverse location among the sampling

sites, which may be due to the very high fluctuations in

salinity, wherein all kinds of bacteria which are tolerant to

wide salinity range could grow and also due to the number

of industries situated around this area. As has been previ-

ously reported (Shiaris, 1989a) for ecosystems with low

salinity estuarine sediments subject to salt water intrusion,

it was observed that phenanthrene degraders are tolerant to

a wide range of salinities. As the first objective of the study

was to screen the PAH degrading bacteria, the same sedi-

ment samples were plated on MSM to check the TVC with

PAH (0.01% of naphthalene) and it was expected that only

those bacteria which can utilize PAH as carbon source

would grow. A significant number of bacteria were

observed growing solely on the selected PAH-containing

MSM agar. The ‘‘GR3’’ site was found to be most diversely

populated with PAH-tolerant bacteria, which in turn sub-

stantiate the fact that the estuarine soil is a rich source of

diverse microorganisms (Shiaris, 1989a,b). This work point

out clearly that the ability to utilize hydrocarbons is widely

distributed among diverse microbial populations, as direct

isolation method is used to isolate dominant members in a

microbial community (Zhuang et al., 2002). Hydrocarbons

are naturally occurring organic compounds and, therefore,

it is not surprising that microorganisms have evolved with

the ability to utilize these compounds. When natural eco-

systems are contaminated with petroleum hydrocarbons,

the indigenous microbial communities are likely to contain

microbial populations of differing taxonomic relationships,

which are capable of degrading the contaminating hydro-

carbons (Leahy and Colwell, 1990). The isolation of naph-

thalene-degrading bacteria was done by two methods, viz.,

incorporating naphthalene into medium and introducing

naphthalene vapors as carbon source. Although the method

of isolation was different, the results obtained (TVC)

appeared with minor differences. Thus, this result reinfor-

ces a paradigm of environmental microbiology that diffe-

rent isolation strategies can result in the isolation of different

bacteria with the same physiological characteristic of inter-

est. A total of 442 colonies were obtained from NA plates

and 189 colonies from MSM agar from all the sites. The

colonies with varied morphology were also obtained with

size ranging from pinpoint to over 3-mm diameter. Out of

this, only 16 colonies were selected for further studies since

many were identical. These cultures were subcultured later

and maintained on the MSM agar and NA.

The Gram and colony characters were noted. Although

the isolation methods could select for both Gram-positive

and Gram-negative bacteria, all candidate strains were Gram-

positive except for one, isolate 7. The dominance of Gram-

positive bacteria should not be surprising, since

Gram-positive bacteria have a stronger cell envelope than

Gram-negative bacteria that allows them to thrive in the

578 NAIR ET AL.

Environmental Toxicology DOI 10.1002/tox

highly variable intertidal sediment environment, where

sediment temperatures are high in the day and osmotic

pressures and nutrient supply may change periodically over

a daily cycle (Zhuang et al., 2003a). Many different species

of bacteria with the ability to degrade naphthalene and

other PAHs have been isolated, mostly from soil environ-

ments (Wilson and Jones, 1993). The majority of the PAH-

degrading bacteria were previously found to belong to the

genus Pseudomonas (Gram-negative) (Garcıa-Valdes et al.,

1988) but from the present study, most of the PAH-degrad-

ing isolates were found to be Gram-positive bacteria.

Many studies have shown that the number of hydrocar-

bon-utilizing microorganisms and their proportion in the het-

erotrophic community increases with exposure to petroleum

or other hydrocarbon pollutants and the levels of hydrocarbon

utilizing microorganisms generally reflect the degree of

contamination of the ecosystem (Leahy and Colwell, 1990;

Wilson and Jones, 1993). The addition of hydrocarbons to an

ecosystem may result in a selective increase in microorga-

nisms capable of utilizing the hydrocarbons and those that are

capable of utilizing metabolites produced by the hydrocar-

bon-degrading bacteria (Venkateswaran et al., 1995; Ferrari

et al., 1996). The enhancement or reduction will depend on

the chemical composition of the contaminating hydrocarbons

and on the species of microorganisms present within the mi-

crobial community of the particular ecosystem (Atlas, 1995).

Several studies have shown a rise in populations of hydrocar-

bon-utilizing microorganisms after oil spills (Atlas, 1981;

Regina et al., 2006). The bacterial cultures that were consis-

tent in growth on the MSM agar with 0.01% naphthalene

medium were selected for further study.

Characterization of the Bacteria with Respectto Utilization of Various NaphthaleneConcentrations

The selected 16 isolates were grown in different concentra-

tion of naphthalene with and without yeast extract

(0.005%) as nutritional supplement. The concentrations of

naphthalene used ranged between 0.01% and 1% in MSM

agar. Most of the isolates showed almost identical pattern

of growth in the assayed conditions. A previous study has

shown that yeast extract supplement initiates the growth of

the bacterial isolates in MSM (Law and Teo, 1997). How-

ever, this study indicated that the presence or absence of

yeast extract did not produce significant effect on the

growth of isolates. For this reason, further studies were

done without the addition of yeast extract.

Growth behavior of all the selected isolates was observed

in 0.01% naphthalene in MSM medium. After 7 days of

incubation, the optical density (OD) was observed and di-

fference in OD was measured as rate of growth in 7 days.

The isolates 4, 5, 9, 10, 11, and 12 grow in much higher rate

than the rest of the isolates (Table I), which could be related

to a potent degradation of naphthalene. Growth characteris-

tics of these isolates also emphasize the fact that these iso-

lates can grow in minimal medium utilizing naphthalene as

sole carbon source. Further increase in concentration of

naphthalene would eliminate most of the loosely resistant

bacteria and screen for naphthalene-degrading bacteria.

Since the isolates showed growth in 1% naphthalene show-

ing high OD they were selected for further study. The rest of

the isolates showed negligible growth.

The isolates which showed a very good growth with

highest percentage of naphthalene on solid media (1%

naphthalene) and the isolates which showed faster growth

in liquid media (0.01% naphthalene) were inoculated in

MSM broth with 1% naphthalene. The OD was assessed

after 7 days of incubation at 288C on the shaker at 150 rpm.

The viability of these isolates was observed with the due

course of incubation with increasing OD. The increasing

number of viable bacterial counts and increasing OD of the

broth clearly indicated that the culture can grow, tolerate,

and utilize up to 1% of naphthalene as carbon source.

The selected seven isolates were sent to the Spanish Cul-

ture Collection Center (CECT) to confirm their identity

TABLE I. The growth of different isolates in 0.5% and 1.0% of naphthalene

Isolates

Concentration of

Naphthalene (%)

OD at 600 nm

(0 h)

TVC at 0 h

(cfu mL21)

OD at 600 nm

(after 7 days)

TVC after 7 days

(cfu mL21)

4 0.5 0.006 2.1 3 102 1.018 8.7 3 105

1.0 0.010 2.0 3 102 0.677 4.3 3 104

5 0.5 0.008 1.8 3 102 0.964 4.9 3 105

1.0 0.010 1.6 3 102 0.248 3.8 3 104

9 0.5 0.004 1.3 3 102 0.045 2.3 3 103

1.0 0.018 1.0 3 102 0.028 1.2 3 102

10 0.5 0.002 1.5 3 102 0.642 3.8 3 104

1.0 0.014 1.2 3 102 0.453 2.7 3 104

11 0.5 0.004 3.1 3 103 1.235 8.9 3 106

1.0 0.013 2.2 3 103 1.062 6.8 3 104

12 0.5 0.003 3.0 3 103 1.156 7.8 3 106

1.0 0.011 2.7 3 103 0.836 5.9 3 104

579NAPHTALENE-DEGRADING BACTERIA FROM CADIZ AREA

Environmental Toxicology DOI 10.1002/tox

based on the molecular level. The cultures were identified

on the basis of 16S rRNA homology analysis. The analysis

of sequences was done at NCBI server (http://

www.ncbi.nlm.nih.gov/BLAST). Sequence of the isolates

representing different name but having identical sequences

was considered as single representative.

Isolates 4, 11, and 12 showed 99.9% sequence homology

with sequence AJ628743.1 in BLAST analysis, which indi-

cated that these isolates are almost genetically identical to

Bacillus simplex, although the initial characterization was

unlikely to the molecular identification. The sequence analy-

sis of 16S rRNA gene confirmed their identity. Out of the

six isolates selected as potent isolates, three isolates were

identified as Bacillus simplex. These isolates has been

named as Bacillus simplex strain 4 (isolate 4), Bacillus sim-plex strain 11 (isolate 11), and Bacillus simplex strain 12

(isolate 12), and showed better growth than the other iso-

lates (Fig. 2). These potent isolates were also grown on

agar plate to confirm their viability on solid media. The

consistent good growth in both media reconfirms the fact

that these isolates have constitutive biochemical setup to

degrade naphthalene.

The isolate Bacillus simplex strain 11 showed a remark-

able increase in growth with 1% naphthalene in MSM broth

when compared with other isolates, followed by Bacillussimplex strain 4 and Bacillus simplex strain 12. Among the

rest of the isolates, the isolate 10 was identified as Bacillusindicus and showed higher growth in 1% naphthalene when

compared with the others (isolates 5 and 9) (Fig. 2). It is

interesting to note that most of the isolates were still in the

log phase of their growth up to 72 h except isolate 9, which

clearly indicates that these isolates can utilize naphthalene

as sole carbon source and grow. As negative controls, the

growth of these naphthalene users were compared with

standard cultures (Bacillus spp. CECT 40, P. fluorescensCECT 378, and E. coli CECT 101) procured from CECT.

None of these bacteria showed a growth rate comparable

with the isolates from sediments and, hence, proves that the

selected potent isolates are capable of degrading PAHs

from any contaminated sites. This work also suggests that

Gram-positive bacteria may play a key role in PAH degra-

dation on contaminated sediments with similar characteris-

tics than Cadiz area. Bacillus spp. was found to be dominat-

ing in this study, which could be due to the ability to pro-

duce endospores as an adaptation mechanism that may

allow surviving in the highly variable environmental condi-

tions of the intertidal marine sediments from which it was

isolated (Zhuang et al., 2002). Sporogenesis enables mem-

bers of Bacillus, Paenibacillus, and related organisms to

withstand environmentally harsh conditions, allowing long-

term survival (Setlow, 1994). A batch study about marine

sediments enriched with naphthalene showed that cells of

the Bacillus genus grew to become dominant members of

the microbial community (Zhuang et al., 2002). These

physiological and growth studies are useful in assessing the

potential of these indigenous isolates for in situ or ex situnaphthalene-pollutant bioremediation in tropical marine

environments (Zhuang et al., 2003b).

An interesting point in this study was the isolation of a

probable new species of Paenibacillus (isolate 5), which

could degrade naphthalene to a higher extent (up to 1%)

and, hence, could play an important role in bioremediation.

The isolate 5 showed only 96.7% sequence similarity with

the available AY960748.1 sequence of database. It would

confirm that this isolate could be a novel species of its kind

from the selected study area, since when sequence similari-

ty is lesser that 97% it could be considered that isolate

could be a novel species (CECT personal communication).

Paenibacillus spp. was reported to be present in heavily

contaminated soil, rhizosphere, etc. (Hosoda et al., 2003).

There was no data available stating it as a naphthalene

degrader and, hence, this report may be the first of its kind.

There are species that clean hydrocarbon-contaminated

soils and others that remove sulfur groups reducing acid

rain production. Some species of Paenibacillus use polyaro-

matic hydrocarbons as a carbon source. Thus, the nutri-

tional requirements among the species of Paenibacillusvary widely (Uetanabaro et al., 2003). Perhaps naphthalene

or other PAHs are required to keep the PAH degradation

enzymes induced. Hence, it could be concluded that the

naphthalene-degrading bacteria are isolated from PAH-con-

taminated sites, which contributed to increase the rate of

utilization of hydrocarbons.

Since there was an increase in the TVC from the begin-

ning of the assay to day 7, it can be concluded that the con-

centration used for the experiment (1% naphthalene) is not

Fig. 2. Growth curve of the potent isolates in 1% naphtha-lene. Legend: isolate 4: Bacillus simplex strain 4; isolate 5:Paenibacillus sp.; isolate 9; isolate 10: Bacillus indicus;isolate 11: Bacillus simplex strain 11; isolate 12: Bacillussimplex strain 12.

580 NAIR ET AL.

Environmental Toxicology DOI 10.1002/tox

toxic for the bacteria isolated from PAH-contaminated sedi-

ments. A similar result was also reported by Maleszak et al.

(2004) with crude oil. Bacterial degradation of naphthalene

was not directly quantified because of its volatility and uti-

lization of very high concentrations. Naphthalene solubility

(32 mg L21) (Liu et al., 1995) was high enough to allow

unrestricted growth. However, at high cell density, the

availability of poorly soluble substrates becomes limiting,

because PAHs are utilized only in the dissolved state

(Deziel et al., 1996). The three isolates reported in this

study extend our knowledge of the range of naphthalene-

degrading bacteria found in marine and estuarine

environments.

The isolate 7, identified as Pseudomonas pohangensis,showed 97% similarity with the available sequence. Weong

et al. (2006) have reported P. pohangensis as a hydrocar-

bon-degrading bacteria from Korean soils, but identifying

this species as a naphthalene degrader from Cadiz harbor

has been done for the first time. The isolate 8, identified as

Paenibacillus agaridevorans, showed 97.5% similarity

with the available sequence. P. agaridevorans which was

first reported by Uetanabaro et al. (2003) and Hosoda

(2003) in sediments and rhizosphere was identified in this

study. The isolate 10 was identified as Bacillus indicus with

99% homology with database sequence. It is the first time

that this bacterium is reported as a naphthalene degrader. It

was first isolated and reported by Suresh et al. (2004) from

sand of an arsenic-contaminated aquifer in West Bengal,

India.

This study gives a comparative analysis of genetic and

enzymological evidences based identification of natural iso-

lates obtained from the marine sediments. It also confirmed

the fact that the analysis of conserved sequences is a tool

for bacterial identification although the biochemical method

can also be considered as a basic tool to characterize the

isolates.

CONCLUSIONS

The isolation and characterization of several strains of

naphthalene-degrading bacteria obtained from PAH-conta-

minated marine and estuarine sediments from Cadiz area

has been carried out, which is the first report from this area.

The introduction of bacteria isolated from contaminated

sediments into mineral medium contributed to increased

rate of hydrocarbon utilization and thus any other easily

available carbon source is not required for the growth of

these isolates.

Gram-positive bacteria were found to be predominant in

this study, out of which Bacillus was found to be the pre-

dominant genus as a naphthalene utilizer.

The cultures can grow, tolerate, and utilize up to 1% of

naphthalene as carbon source. This high concentration of

naphthalene is not toxic for the bacteria isolated.

Hence, these isolates can be very helpful in bioremedia-

ting the PAH-contaminated sites and oil spills thus preser-

ving the ecosystem in a natural way. Further pursue on this

work might represent eco-friendly solution for oil contami-

nation on sea surface and coastal area.

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