Environ Sci Pollut Res (2010) 17:383391

DOI 10.1007/s11356-009-0099-3

AREA 5 ENVIRONMENTAL MICROBIOLOGY RESEARCH ARTICLE

A microbiological study of the self-cleaning potential

of oily Arabian Gulf coasts
Huda Mahmoud & Redha Al-Hasan & Majida Khanafer &
Samir Radwan

Received: 10 August 2008 / Accepted: 17 December 2008 / Published online: 14 February 2009
# Springer-Verlag 2009

Abstract
Background, aim, and scope Due to the active production
and transport of crude oil in the Arabian Gulf region, the
Arabian Gulf coasts are routinely polluted with oil.
Therefore, such coasts have been subject of studies aiming
at assessing the roles of indigenous microbial consortia in
cleaning these environments. In the present study, epilithic
microbial communities along Kuwait coasts were studied
for their oil degradation potential.
Materials and methods Gravel particles coated with deep
green biofilms were collected from four coastal sites in
autumn, winter, and spring. Phototrophs in these consortia
were determined in terms of their chlorophyll a contents
and identified by their morphological characteristics. Total
bacteria were counted microscopically and cultivable
bacteria by the dilution plating method on nutrient agar as
well as on inorganic medium containing oil as a sole source
of carbon and energy. The bacterial community structures
were also characterized and compared by denaturing
gradient gel electrophoresis (DGGE).
Results Epilithic biomass samples from the four sites in the
three seasons were rich in diatoms and picocyanobacteria as
well as total bacteria. Direct counting gave bacterial
numbers per square centimeter gravel surface of 2 to 6
107 cells depending on the sampling site and season.
Cultivable bacterial numbers on nutrient agar and crude
oil as a sole source of carbon were 3103 to 8104 and 1

Responsible editor: Hailong Wang

H. Mahmoud : R. Al-Hasan : M. Khanafer : S. Radwan (*)
Faculty of Science, Department of Biological Sciences,
Kuwait University,
P. O. Box 5969, 13060 Safat, Kuwait
e-mail: samir.radwan@ku.edu.kw

103 to 7103 cells/cm2 gravel surface, respectively. The

DGGE profiles of epilithon biomass samples revealed
major 16S rDNA bands that matched bands of pure oilutilizing bacterial isolates.
Discussion The microbial communities showed a degree of
consistency in all sites and seasons.
Conclusions The microbial consortia coating gravel particles are potentially suitable tools for self-cleaning of oily
Gulf coasts. They are rich in oil-utilizing bacteria whose
activities are probably enhanced by oxygen produced by
the phototrophic partners in the consortia.
Recommendations and perspectives The combination of
conventional microbiological analysis with molecular
approaches gives an enhanced idea about natural microbial
communities especially those with environmental application
potential.
Keywords Arabian Gulf . Bioremediation . DGGE .
Epilithon consortia . Oil utilization .
Phototrophic microorganisms

1 Background, aim, and scope

The microbial degradation of crude oil, hydrocarbons, and
hydrocarbon derivatives in terrestrial and aquatic environments (e.g., Isikhuemhen et al. 2003; Jones et al. 2004;
Antic et al. 2006; Pu and Cutright 2007) has become the
basis of a promising biotechnology for self-cleaning and
bioremediation of such environments. More than one half
of the total world marine transported oil is produced and
transported in the Arabian Gulf region (British Petroleum
Co. 1979; Hunter 1982). Therefore, the Arabian Gulf water
body is one of those that are most heavily charged with oil

384

worldwide. Water samples from that water body were found

to contain up to 546g/ml oil, compared to only up to
66.8g/ml water from the Gulf of Mexico (Sen Gupta and
Kureishy 1981; Marchand et al. 1982; El Samra et al.
1986). Such marine ecosystems should depend mainly on
the indigenous oil-utilizing microflora for self-cleaning.
Results from our laboratory revealed that indigenous oilutilizing bacteria occur in the Arabian Gulf mainly in
association with animate (Sorkhoh et al. 1992; Radwan et
al. 2002, 2005, 2007) and inanimate (Radwan et al. 1999)
coastal materials, rather than free in water (for a recent
review see Radwan 2008). Among the interesting coastal
consortia were those coating gravel particles collected from
the north which, in laboratory experiments, exhibited oil
attenuation potential for oily waste (Radwan and Al-Hasan
2001). Repeated trips along the Kuwait coast of the Gulf
showed that these biofilm-coated gravel particles were
frequent most of the year in the intertidal zone along the
whole coast. Dynamic interaction between the Gulf water
and those immobilized coastal biofilms were observed
during wave and tidal movements. Those observations
directed our attention towards the need for surveying the
microbiology of these consortia through the whole coast,
putting emphasis on their contents of oil-utilizing
bacteria. Due to the complex nature of such consortia,
both culture-based and culture-independent microbiological techniques were combined in this work, which aimed
mainly at studying the self-cleaning potential of the
Kuwait coast as a representative sector of the entire Gulf
coasts.

2 Materials and methods

2.1 Sampling
Gravel particles coated with deep green epilithic growth
were collected from the intertidal zones of four different
sites, two inside the Kuwait bay (a shallow extension of
the Gulf, a few meters deep; Doha and Kuwait Towers)
and two outside the Kuwait bay (reaching about 30 m
maximum depth; Plajat and Anjefa). Sampling was done
in January (winter), April (spring), July (summer), and
October (autumn) 2006 using sterile polyethylene bags
and transported to the laboratory to be processed the same
day. The four sampling sites were rather close to our
laboratory, thus allowing to avoid long storage and
probable artifacts in samples from remote sites. To
determine the gravel total surface area, it was wrapped
in aluminum foil, and after trimming off excess foil, the
foil coat was weighed and its area was determined by
comparison with the weight of a foil sheet of known
surface area (Chappell 1994).

Environ Sci Pollut Res (2010) 17:383391

2.2 Biofilm (epilithon) suspensions

For microbiological and molecular analyses, total microbial
consortia coating gravel particles were brought in suspension in membrane filtered seawater. The total biofilm was
scrapped off using a sterile toothbrush (by autoclaving at
120C for 20 min) in known water volumes and the
suspension was homogenized. Such biofilm samples will
also be referred to in the text as epilithon samples.
2.3 Total microbial numbers
Phototrophic microorganisms in the epilithon suspensions
were determined in terms of the chlorophyll a content. Total
chlorophylls were extracted with 95% ethanol at 72C for
5 min (Ainsworth and Goulder 1998), and chlorophyll a in
the extract was determined spectrophotometrically at 665
and 750 nm (Jesperson and Christoffersen 1987). The
predominant phototrophs were also examined and identified
by their morphological characteristics using an epifluorescence microscope (Zeiss, Germany).
For microscopic counting of total bacteria in the
suspensions (Yu et al. 1995), 10 ml aliquots were
preserved at 4C for 14 days using 0.2 m neutral
formaldehyde. A fluorescent dye was prepared by dissolving 0.01 g 4-6-diamidino-2-phenylindole (DAPI) in
10 ml membrane filtered seawater. Cells in the epilithon
suspensions were stained with 0.1 ml of DAPI solution for
40 min in the dark. Aliquots of 0.5 ml of the stained
suspensions were treated with 5 ml aliquots of membrane
filtered sweater, and the mixtures were filtered through
0.2 m polycarbonate membrane filters. Bacteria on the
filters were counted using epifluorescent microscope, with
membrane filtered seawater as a blank. Total bacterial
numbers per square centimeter of gravel surface were
calculated. Ten replicate gravels for each site and season
were counted.
2.4 Cultivable bacterial numbers
The standard dilution plate method using conventional
nutrient agar was adopted to determine the total cultivable bacteria in the epilithon suspensions. The same method
was used to determine the numbers of oil-utilizing bacteria;
the medium used was an inorganic medium containing 1%
crude oil as a sole source of carbon (Sorkhoh at al. 1990).
Also, here, ten individual gravel particles were separately
used for each counting. Numbers per square centimeter
gravel surface were calculated.
The countable plates (50200 colonies) containing
colonies of oil-utilizing bacteria from every ten parallel
gravel particles were pooled, and colonies were differentiated according to their morphology (sizes, colors, shapes,

Environ Sci Pollut Res (2010) 17:383391

margins) and cell-shaped as well as to the Gram stain

reactions. Representative colonies were subcultured for
each bacterial strain purified and maintained on slants for
further study.
2.5 Molecular analysis
Total genomic DNA in the epilithon suspensions and in
cells of pure cultures of all the representative oil-utilizing
strains were extracted, purified, amplified, and subjected to
denaturing gradient gel electrophoresis (DGGE). Biomass
suspensions of every ten individual gravel particles for each
site and season were also analyzed separately. Cell pellets
in suspensions and liquid cultures were prepared by
centrifugation and washed in phosphate-buffered saline
(Sambrook et al. 1989). Genomic DNA was extracted from
each pellet by the bead-beating method (Kuske et al. 1998).
After centrifugation, the aqueous phase was purified from
proteins which were precipitated with phenol/chloroform/
isoamylalcohol (Wawer and Muyzer 1995). The DNA in
solution was precipitated with ice-cold ethanol, dissolved in
TAE buffer (Sambrook et al. 1989), and subjected to PCR
amplification of 16S rDNA genes using the universal
eubacteria primer combination GM5F-GC clamp and
S907R (Santegoeds et al. 1998). The PCR products were
concentrated (Teske at al. 1996) and analyzed by parallel
DGGE; the denaturants concentrations were 3070%. For
DGGE, the Dcode Universal Mutation Detection System
(Bio-Rad, CA, USA) was used and processed with constant
voltage of 200 V at 60C for 4.30 h. Each gel was incubated
in 500 ml of 1 TAE buffer with 50l of ethidium bromide
for 30 min and examined using a UV-transilluminator. The
bands were transformed into binary matrix; the presence of
bands was given the weight of (1) and their absence (0).
The binary matrix produced was analyzed using cluster
analysis and dendrograms were plotted.

3 Results
Two successive trips in November and December 2005
along the entire Kuwait coast from the extreme north to the
extreme south, a total distance of about 250 km, revealed
that the intertidal zone in most sectors was rich in gravel
particles coated with deep green biomass. Their frequencies
varied from one site to another, yet the gravel free sectors
of the coast occupied only short distances. These particles
usually occurred in rather shallow aggregates, one to a few
meters wide, which routinely became totally submerged
with water only during high tide. Figure 1 is a map showing
the four sampling sites. Table 1 presents relevant environmental parameters in the different sampling sites at the
three seasons. During the hot summer, all gravel particles in

385

all sites were visually free of the green biomass; therefore,

they were not analyzed. Some factors, like the water pH
values and salinities, were rather consistent in the different
sites and seasons. On the other hand, higher water temperatures associated with lower dissolved oxygen contents in
all sites were recorded in the spring as compared with the
winter and autumn. The highest water conductivity values
in all sites were in the autumn, but the water turbidity
values were higher in Anjefa and Kuwait Towers than in
Doha and Plajat in the three seasons. The chlorophyll a
content values were higher in the autumn and spring
samples in all sites except Anjefa than in the winter
samples. There were large variations in the chlorophyll
contents among every parallel ten gravel particles collected
from the sites and seasons.
Microscopic examination of epilithon suspensions
showed that diatoms and picocyanobacteria were the
predominant phototrophic microorganisms in these biofilms. The following genera of diatoms occurred in
descending order of frequencies: Navicula, Nitzchia, Fragillaria, Fallacia, Pinnularia, Licmophora, Acanthes,
Stouroneis, Diplonies, Bacillaria, Encyonema, Mestogolia,
Gomphonema, Melosira, and Cymbella. The most dominant picocyanobacterium was Synechococcus. The composition of the phototrophic microflora in biofilms on gravel
particles collected from the four sites during the three
seasons was rather consistent.
The bacterial numbers determined by the direct microscopic method of counting per square centimeter of the
surface of gravel particles collected from the four sites
during the three seasons were 2 106 to 6 107 cells
(Table 2). The numbers of cultivable bacteria were 1103
to 8104 cells/cm2 gravel surface. With all methods of
counting, there were considerable variations in numbers of
bacteria among the individual gravel particles collected
from the same site at the same season. However, there were
no dramatic differences in the mean numbers due to site
and season variations. As expected, the numbers counted
on nutrient agar were more than those counted on crude oil
as a sole source of carbon. Yet, the latter numbers were
always still considerably high as compared with the former
numbers. Totally, 121 strains of oil-utilizing bacteria were
isolated from all samples and purified, and their genomic
DNA was extracted and amplified. The DGGE analysis for
individual samples collected from the four sites in the three
seasons revealed distinguishable bands, six to 14 per track
(sample). The average value of total band numbers in the
three seasons of samples from Doha, Kuwait Towers,
Plajat, and Anjefa were 10, 9, 9, and 11, respectively. Each
track comprised only one to four strong (dominant) bands;
the remaining were correspondingly weaker (minor). As a
rule, the dominant bands were similar for every ten parallel
gravel particles from the same site in the same season; some

386

Environ Sci Pollut Res (2010) 17:383391

Fig. 1 Map of Kuwait coast

showing the locations of the two
bay stations (Kuwait Towers and
Doha) and the two non-bay
stations (Plajat and Anjefa)

differences, if at all, involved the minor bands only (Fig. 2

is a typical DGGE profile). The least degree of similarity
in the DGGE profiles among the minor bands was for
the parallel gravel particles collected from Kuwait Towers
in the spring (Fig. 3). To make a comprehensive
comparison of the DGGE profiles of samples collected
from the four sites in the three seasons, the biofilm
samples of every ten parallel gravel particles were pooled
prior to nucleic acid extraction and analysis. The DGGE
profiles of the pooled samples (Fig. 4) were similar to
those of the individual stones from the same site. In most
cases, these profiles revealed even the same dominant
bands in pooled samples collected from the four sites in the
three seasons.
The comparison of the DGGE profiles of the pooled
samples from the four sites with those of pure oil-utilizing
bacterial isolates revealed that the bands of one or more of

the oil utilizers matched at least one major band of the

pooled samples (Fig. 5, a representative example). In
addition, several oil-utilizing bacterial bands matched
several of the minor bands in the pooled samples. However,
many of the oil-utilizing isolates from the four sites and in
the three seasons had bands that did not match any in the
DGGE profiles of the epilithon samples (see Fig. 5 and
Table 3).
Figure 6 illustrates the results of the cluster analysis
using Euclidean distances. In general, there was only little
tendency for samples from each of the four sites in the three
seasons to cluster together. However, Plajat and Doha
samples collected in the winter and spring exhibited higher
similarities to each other, i.e., were clustered together. On
the other hand, Kuwait Towers and Anjefa samples
collected in the winter and autumn showed a tendency to
cluster together separately.

Table 1 Chlorophyll a contents of gravel biofilms and environmental parameters of coastal waters from the sampling sites
Season

Site

g chlorophyll a/cm2 gravel

surface (minimummaximum)

pH

Dissolved
oxygen (mg/l)

Conductivity
(mS/cm)

Turbidity
(NTU)

Temperature
(C)

Salinity
(%)

Autumn

Doha
Kuwait Towers
Plajat
Anjefa
Doha
Kuwait Towers
Plajat
Anjefa
Doha
Kuwait Towers
Plajat
Anjefa

2.6
1.7
3.4
1.4
1.2
1.2
1.2
1.6
2.2
1.0
1.2
3.1

7.6
7.5
7.3
7.4
7.7
7.3
7.6
7.4
7.3
7.1
7.2
7.3

6.4
6.7
5.0
8.9
6.6
6.5
5.2
5.0
5.0
4.9
ND
4.1

6.6
6.5
6.4
6.5
4.2
5.4
4.4
5.2
4.0
5.1
5.1
5.0

10.0
30.0
18.1
36.2
13.0
22.3
9.2
30.0
10.4
32.6
19.6
48.7

18.0
15.0
18.1
18.3
17.9
11.5
13.8
15.0
24.5
20.2
20.7
25.8

2.6
3.2
3.1
3.3
2.7
3.4
2.8
3.4
2.6
3.3
3.3
3.2

Winter

Spring

(0.17.9)
(1.02.5)
(0.86.6)
(1.13.8)
(0.82.4)
(0.62.0)
(0.51.6)
(0.83.0)
(0.75.0)
(0.41.3)
(0.52.1)
(0.86.2)

Gravel particles collected in the summer were visually free from blue green biofilms
ND not determined

Environ Sci Pollut Res (2010) 17:383391

387

Table 2 Numbers of total and cultivable bacteria in biofilms coating gravel particles from the Arabian Gulf coast
Site

Mean (minimummaximum) bacterial numbers/cm2 of particle surface

Autumn
Totala

Winter
Cultivableb
On nutrient
agar

Doha
Kuwait
Towers
Plajat
Anjefa

Total
On crude
oil

Spring
Cultivable
On nutrient
agar

Total
On crude oil

Cultivable
On nutrient
agar

6 (39)107 3 (16)104 2 (12)104 6 (314)107 9 (419)103 1 (15)103 6 (310)107 3 (28)104

2 (14)107 3 (25)103 1 (12)103 3 (25)107 8 (420)103 6 (313)103 4 (25)107 1 (12)104
2 (13)107 1 (12)104 4 (25)103 2 (15)107
4 (26)107 5 (38)103 1 (12)103 4 (26)107

2 (12)104
6 (49)103

2 (13)103
1 (12)103

On crude
oil
1 (12)104
4 (15)103

5 (110)107 4 (19)104 3 (24)103

5 (110)107 8 (116)104 7 (311)103

Values are the means of ten parallel determinations each

a

Determined microscopically

Determined by the conventional dilution plating method

Several natural microbial consortia rich in oil-utilizing

bacteria were recorded along the coasts of the Arabian Gulf,
and their potential role in cleaning oily areas was discussed
(for review, see Radwan 2008). One such consortium was
that of microbial mats over oil sediments on Saudi Arabian
coasts that comprised filamentous cyanobacteria and oil-

utilizing bacteria (Sorkhoh et al. 1992). In this paper, another

interesting coastal microbial association that has a potential
for cleaning oily areas is described. This consortium appears
yearly in the form of blue green epilithic growth on coastal
gravel particles during autumn, winter, and spring and
disappears during the hot summer (ambient coastal temperature >55C). Our previous studies showed that summer
collected particles still did host surviving forms of algae and

Fig. 2 Between stone variation in the epilithic bacterial community

on stones from Anjefa, spring. a DGGE gel: lane E Escherichia coli
(positive control); lanes 110 stones epilithon. b Cluster analysis
using Euclidean distances; numbers of stones indicated as 110

Fig. 3 Between stone variation in the epilithic bacterial community

on stones from Kuwait Towers, spring. a DGGE gel: lane E E. coli
(positive control); lanes 18 stones epilithon. b Cluster analysis using
Euclidean distances; numbers of stones indicated as 18

4 Discussion

388

Environ Sci Pollut Res (2010) 17:383391

Table 3 Numbers of dominant and minor bands in pooled biomass
samples matching bands of oil-utilizing bacterial isolates
Site
Season

Number
of oilutilizing
isolates

Plajat
Winter
10
Autumn
5
Spring
6
Doha
Winter
6
Autumn
11
Spring
5
Kuwait Towers
Winter
7
Fig. 4 Seasonal variation in the epilithic bacterial community at the
four sample sites. a DGGE gel: lane E E. coli (positive control); lane
1 Kuwait Towers, winter; lane 2 Kuwait Towers, spring; lane 3
Kuwait Towers, autumn; lane 4 Plajat, winter; lane 5 Plajat, spring;
lane 6, Plajat, autumn; lane 7 Doha, winter; lane 8 Doha, spring; lane
9 Doha, autumn; lane 10 Anjefa, winter; lane 11 Anjefa, spring; lane
10 Anjefa, autumn. b Cluster analysis using Euclidean distances;
numbers are the same as in DGGE profiles

bacteria; by wetting with nitrate rich water and cooling down

to about 20C in the laboratory, they turned green after a few
weeks (Al-Awadhi et al. 2003). The fact that those biomass
coated gravel particles are frequent through the entire
Kuwaiti coast most of the year underlines their potential as
oil-cleaning tools. Similar to the microbial mats (Sorkhoh et
al. 1992), the epilithic consortia comprise both phototrophic
partners and oil-utilizing bacteria. Evidence of oil and
hydrocarbon attenuation by bacterial cultures isolated from
these consortia has been provided in a previous study

Fig. 5 DGGE gel comparing genomic DNA of oil-utilizing bacterial

isolates from Anjefa in winter with the total genomic DNA of the
epilithon from which these isolates where obtained. Some of the
isolates were found matching strong bands, e.g., lane 3; others were
found matching faint bands, e.g., lane 2; and some did not match any
bands in the epilithon sample, e.g., lane 16

Autumn
Spring
Anjefa
Winter
Autumn
Spring

Number
of DGGE
bands

Number of
isolates
matching a
strong band

Number of
isolates
matching a
weak band

5
6
4

1
1
1

1
0
0

4
9
4

1
4
0

1
4
1

2
5

6
5

0
1

1
0

6
3
5

5
10
5

0
1
1

1
2
1

(Radwan and Al-Hasan 2001). However, unlike those of

the mats, the phototrophs in the epilithic consortia are mainly
unicellular eukaryotes, namely diatoms. The frequency of
such phototrophs, as expressed in terms of chlorophyll a
contents, was rather consistent in the different sites and
seasons. This was true, although dramatic variations in
chlorophyll a contents of individual gravel particles from one
and the same site were recorded. There was a tendency in
some sites toward enhanced growth of the phototrophs in the
autumn and spring where the water temperature was warmer
than in the winter. The interesting role that phototrophs may
play in this epilithic consortium is providing oxygen through
photosynthesis, which oil-utilizing bacteria need for the
initial step of attack on hydrocarbons (Rehm and Reiff 1981;
Singer and Finnerty 1984). The epilithic biomass samples
were also rich in bacteria. Numbers determined by direct
microscopic counting were in the magnitude of ten million
cells per square centimeter of the gravel particle surface.
Such rather high numbers were also consistent in the
different sites and seasons, although considerable differences
were recorded among individual replicate samples (see
Table 2). As expected, the numbers of cultivable bacteria
were much lower than those determined by direct counting,
obviously due to the selective conditions associated with the
plating technique. Nevertheless, the numbers determined on
crude oil as a sole source of carbon and energy were not at
all much less than those determined on the conventional
nutrient agar medium (both in the magnitude of thousands to

Environ Sci Pollut Res (2010) 17:383391

389

Fig. 6 Cluster analysis using Euclidean distances showing between stone variation in the epilithic bacterial community on stones collected from
Doha in a winter, b spring, and c autumn; Kuwait Towers in d winter, e spring, f autumn; Plajat in g winter, h spring, i autumn; Anjefa in j winter,
k spring, l autumn

ten thousands per square centimeter). This fact allows one to

expect that oil-utilizing bacteria probably also make up a
considerable proportion of the total numbers determined by
the direct microscopic counting. In fact, the molecular
analysis of the epilithic biomass samples confirmed and

consolidated this conclusion. Thus, the DGGE fingerprinting

revealed that one or more of the dominant 16S rDNA bands
in every epilithic biomass sample matched the 16S rDNA
band of an oil-utilizing isolate. Other dominant bands in the
biomass samples could also be uncultivable oil-utilizing

390

bacteria; however, they could also be of other conventional

bacteria. In other words, at least some of the predominant
bands most likely represent dominant oil-utilizing bacteria in
all epilithic consortia. That many of our oil-utilizing isolates
had bands that did not match any band of the epilithon
samples should not shed doubt in the occurrence of such
isolates in the epilithon consortia. The detection limit of the
DGGE analysis is approximately 1% of the total nucleic acid
(Muyzer and Smalla 1998). Isolates whose bands were
absent in the epilithon profiles probably represent just minor
inhabitants that were underrepresented in PCR amplification
(Bernard et al. 2000). Such minor oil-utilizing bacteria may
flourish under alternative conditions. In this context, coastal
areas studied in this work were visually pristine, i.e., free of
oil sediments. It should be expected that epilithic oil-utilizing
bacteria described here would be considerably enriched after
any potential oil slick.

5 Conclusions
Members of the epilithon microbial consortia along the
Kuwaiti coasts of the Arabian Gulf have the capability of
utilizing oil pollutants. Firstly, these consortia flourish on
the coasts through three seasons of the year. Secondly,
epilithic microbial consortia are immobilized naturally on
the gravel particles; we watched how the green biomass
was protected from being washed out in the open water.
Thirdly, the associated phototrophic microorganisms probably provide the oil-utilizing bacterial partners with
oxygen, a by-product of photosynthesis, needed for the
first step of attack on the hydrocarbon substrates. Fourthly,
the consortia are rather rich in oil-utilizing bacteria as the
results of counting and molecular fingerprinting revealed.
The microbial consortia coating gravel particles are, thus,
potentially suitable tools for self-cleaning of oily Gulf
coasts.

6 Recommendation and perspectives

The combination of conventional microbiological analysis
with molecular approaches gives an enhanced idea about
natural microbial communities, especially those with
environmental application potential. There is need for
further research to elucidate the degradation abilities of
isolates found to have the capability to use crude oil as sole
carbon source.

Acknowledgments This work has been supported by the Kuwait

University, Research Grant RS 02/04. Thanks are due to Amar Habib
for technical assistance.

Environ Sci Pollut Res (2010) 17:383391

References
Ainsworth AM, Goulder R (1998) Microbial organic-nitrogen transformations along the Swale-Ouse river system, Northern England. Sci Total Environ 210(211):329355
Al-Awadhi H, Al-Hasan RH, Sorkhoh NA, Salamah S, Radwan SS
(2003) Establishing oil-degrading biofilms on gravel particles
and glass plates. Int Biodeter Biodeg 51:181185
Antic MP, Jovancicevic B, Ilic M, Vrvic MM, Schwarzbouer J (2006)
Petroleum pollutant degradation by surface water microorganisms. Environ Sci Pollut Res 13:320327
Bernard L, Schafer H, Joux F, Courties C, Muyzer G, Lebaron P
(2000) Genetic diversity of total, active and culturable marine
bacteria in coastal seawater. Aquat Microb Ecol 22:111
British Petroleum Co (1979) BP Statistical Review of the World Oil
Industry. British Petroleum, London
Chappell KR (1994) Extracellular Enzyme Activity in Freshwaters.
PhD thesis. UK: University of Hull
El Samra MI, Emara HI, Shunbo F (1986) Dissolved petroleum hydrocarbons in the Northwestern Arabian Gulf. Mar Pollut Bull 17:6568
Hunter JR (1982) The physical oceanography of the Arabian Gulf: a
review and theoretical interpretation of previous observations. In:
Halwagy R, Clayton D, Behbehani M (eds) The First Arabian Gulf
Conference on Environment and Pollution. Kuwait University,
Faculty of Science, Kuwait, pp 123
Isikhuemhen O, Anollefo G, Oghale O (2003) Bioremedaition of
crude oil polluted soil by the white rot fungus, Pleurotus
tuberregium (Fr.) Sing. Environ Sci Pollut Res 10:108112
Jesperson AM, Christoffersen K (1987) Measurements of chlorophyll
a from phytoplankton using ethanol as extraction solvent. Arch
Hydrobiol 109:445454
Jones R, Sun W, Tang C-S, Robert F (2004) Phytoremediation of
petroleum hydrocarbons in tropical coastal soilsIImicrobial
response to plant roots and contaminants. Environ Sci Pollut Res
11:340346
Kuske CR, Banton KL, Adorada DL, Strak PC, Hill KK, Jackson PJ
(1998) Small scale DNA sample preparation method for field
PCR detection of microbial cells and spores in soil. Appl Environ
Microbiol 64:24632472
Marchand M, Monfort JP, Rubio AC (1982) Distribution of hydrocarbons in water and marine sediments after the Amoco Cadez
and Istoc. 1 oil spills. In: Keith L (ed) Energy and Environmental
Chemistry. Arbor Science, MI, vol 1, pp 487509
Muyzer G, Smalla K (1998) Application of denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Anton Leeuw Int J 73:127141
Pu X, Cutright T (2007) Degradation of pentachlorophenol by pure
and mixed cultures in two different soils. Environ Sci Pollut Res
14:244250
Radwan S (2008) Microbiology of oil-contaminated desert soils and
coastal areas in the Arabian Gulf region. In: Dion P, Nautiyal CS
(eds) Microbiology of Extreme Soils, Soil Biology 13. Springer,
Berlin, pp 275298
Radwan SS, Al-Hasan RH (2001) Potential application of coastal
biofilm-coated gravel particles for treating oily waste. Aqu
Microb Ecol 23:113117
Radwan SS, Al-Hasan RH, Al-Awadhi H, Salamah S, Abdullah HM
(1999) Higher oil biodegradation potential at the Arabian Gulf
coast than in the water body. Mar Biol 135:741745
Radwan SS, Al-Hasan RH, Mahmoud HM, Eliyas M (2007) Oil
Utilizing bacteria associated with fish from the Arabian Gulf. J
Appl Microbiol 103:21602167
Radwan SS, Al-Hasan RH, Salamah S, Al-Dabbous S (2002)
Bioremediation of oily sea water by bacteria immobilized in
biofilms coating macroalgae. Int Biodeter Biodeg 50:5559

Environ Sci Pollut Res (2010) 17:383391

Radwan SS, Al-Hasan RH, Salamah S, Khanafer M (2005) Oilconsuming microbial consortia floating in the Arabian Gulf. Int
Biodeter Biodeg 56:2833
Rehm H-J, Reiff I (1981) Mechanisms and occurrence of microbial
oxidation of long-chain alkanes. Adv Biochem Eng 19:175216
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, New York
Santegoeds CM, Ferdelman TG, Muyzer G, Beer DD (1998) Structural
and functional dynamics of sulfate-reducing populations in
bacterial biofilms. Appl Environ Microbiol 64:37313739
Sen Gupta R, Kureishy TW (1981) Present stage of oil pollution in the
northern Indian Ocean. Mar Pollut Bull 12:295301
Singer ME, Finnerty WR (1984) Microbial metabolism of straightchain and branched alkanes. In: Atlas RM (ed) Petroleum
Microbiology. Macmillan, New York, pp 159
Sorkhoh N, Al-Hasan RH, Radwan S, Hpner T (1992) Self-cleaning
of the Gulf. Nature 359:109

391
Sorkhoh NA, Ghannoum MA, Ibrahim AS, Stretton RJ, Radwan SS
(1990) Crude oil and hydrocarbon degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments
in Kuwait. Environ Pollut 65:117
Teske A, Wawer C, Muyzer G, Ramsing NB (1996) Distribution of
sulfate-reducing bacteria in a stratified Fjord (Maiager Fjord,
Denmark) as evaluated by most-probable-number counts and
denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appl Environ Microbiol 62:14051415
Wawer C, Muyzer G (1995) Genetic diversity of Desulfovibrio spp. in
environmental samples analysed by denaturing gradient gel
electrophoresis of [NiFe] hydrogenase gene fragments. Appl
Environ Microbiol 62:22032210
Yu W, Dodds WK, Banks K, Skalsky J, Staruss E (1995) Optimal
staining and sample storage time for direct microscopic enumeration of total and active bacteria in soil with two fluorescent
dyes. Appl Environ Microbiol 61:33673372