Bio Remediation of Crude Oil Pollution in the Kuwaiti Desert the Role of Adherent Microorganisms
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Transcript of Bio Remediation of Crude Oil Pollution in the Kuwaiti Desert the Role of Adherent Microorganisms
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Pergamon
PI1 SO160-4120(98)00074-9
Environment International, Vol. 24, No. 8, 823-834, 1998p.
Copyright 01998 Elsevier Science Ltd
Printed in the USA. All rights reserved
0160-4120/98 $19.00+.00
BIOREMEDIATION OF CRU DE OIL POLLU TION INTHE KUWAITI DESERT: THE ROLE OF ADHERENT
MICROORGANISMS
C.O. Obuekwe and S.S. Al-Zarban
Department of Biological Sciences (Microbiology Division), Faculty of Science, Khaldiya Campus,
Kuwait University, Kuwait
EI 9710-203 M (Received 21 October 1997; accepted 28 February 1998)
Pieces of stones and other solid m aterials found in the oil lake sites of the Kuwaiti desert ap peare d
clean, providing indications of surface-a ssociated enhanced crude oil degradatio n. Scanning
electron m icroscop e studies revealed tha t such surfaces were colonized by active microbial
populations. The colonization of the stone surfaces w as concentrated within crevices. When
enriched from washed pieces of stones from the oil lake, the resulting mixed population ofadherent
microorganisms degraded much m ore crude oil (44.4%) in the presence of inert carrier materials
(Styrof oam chips) in laboratory cultures, than in the absence of the inert materials (21.8 %). T he
inert materials wer e found to be extensively colonized by microorg anisms just as was observe d w ith
the stone and other solid samples from the oil lake. 0199 8 Elsevier Science Ltd
INTRODUCTION
With the Iraqi invasion, more than 60 million barrels
(9.4 Tg) of crude oil were released in the Kuwaiti
desert over an area of about 49 km’, forming numerous
large pools referred to as oil lakes (Al-Gounaim et al.
1995 ). Several studies on crude oil pollution of the
Kuw aiti desert and coastal soils have revealed the
varying capacity of the environm ent to cleanse itself ofoil pollution, depen ding on the structure of the in-
digenous microbial comm unities (Al-Gounaim et al.
1995; Sorkhoh et al. 1995; Al-Hassan et al. 1995;
Radw an et al. 1995). The information provided on the
structure of the microbial community has been re-
stricted to the numbers and types of organisms in-
volved in oil degradation, and there were no studies on
the physical interactions between the organisms and
their desert environment relevant to oil degrad ation.
Because of the hot, dry climate, degradation and re-
cycling of organic matter are generally slow. Studie s
on the interaction between the organisms and their
environment should provide better understanding ofthe
structure and function of the microb ial comm unity.
During studies of an oil lake in the Kuw aiti desert, it
was observed that pieces of stone/pebbles, and other
solid mate rials, whe n lifted from the oil-soaked soils ofthe oil lake, exhibited clean surface s, even at their
undersides. This observation would suggest active
crude oil-removing activities associated with such sur-
faces. Such active oil-removal activities wou ld possibly
arise from oil-degrading microorganisms attached to
the surfaces. Surfaces are important in the degradation
of contaminants in nature and general m icrobial acti-
vities (Van Loosdrecht 1990). Davis and Westlake
(1978) suggested that filamentous fungi in soils might
provide increased contact surfaces for enhanc ed hydro-
823
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82 4 CO. Obuekwe and S.S. Al-Zarban
carbon degradation by bacteria. S urface-associated
microorganisms are important in waste treatment
(Characklis and Marshall 1990) and are applicable in
oil degradation in laboratory studies (Wilson and
Bradley 1996).
The need for studies on the physical interactions be-tween microorganisms and their physical environment
for crude oil degradation in the Kuw aiti desert became
necessary with the report of Radw an et al. (1995) that
plants growing within the oil lake sites, when pulled
out, show ed clean roots (lacking any visible signs of oil
smears). This observation again suggested enhanced
root surface-associate d crude oil pollution rem ediation
activities.
The aim of the investigation being reported here was,
therefore, to study the surfaces of the more com monly
distributed solid ma terials (providers of the surfaces)found in the oil lake, for evidence of surface-associa ted
microorganisms active in crude oil degradation, and
determine also the effect of inert surfaces on the ability
of such recovered surface-associa ted microflora to
degrade crude oil.
MATERIAL AND METHODS
Samples
Samp les obtained for this work consisted of small
pieces of stones and pebbles, broken tw igs, and
feathers obtained from be ds of an oil lake (NE of
Kuw ait City) in the Kuw aiti desert, on Jahra-Al-
Subbian Road. S amples were obtained in the months of
November and December, were collected aseptically,
transported in plastic bags previously rinsed in 70%
ethanol, and dried in a sterile air-flow chamber under
UV overnight.
When not used immediately for microbial isolations,
all samples were stored at 4°C in a refrigerator.
Preparation of Styrofoam carrier materials
Carriers or surfaces for the adherence of the enriched
adherent microorganisms were prepared from styro-
foam chip s cut into irregular cubes of approxim ately
5 mm dimensions. The Styrofoam chips were sterilized
by immersion in methanol (Analar grade) for a period
of 30 min, and then dried overnight in sterile air
draught under U V light in a laminar flow chamber.
The Styrofoam chips so treated failed to support any
growth when implanted in nutrient agar or potato
dextrose agar and incubated for up to 48 h at 28°C and
were, therefore, adjud ged sterile.
Enrichment of culture for adherent microorganisms
The rationale for the selective enrichm ent of adheren t
microorganisms is based on the fact that organisms
whic h are sufficiently strongly attached to carrier
surfaces will not be easily dislodged when such carriers
are agitated, while organisms which are loosely asso-
ciated with such surfaces would.
In this work, microorganisms adherent to the samples
collected were enriched by initially ag itating the sam -
ples (pieces of stones, peb bles, twig s, and feathers) in
sterile 100 mL of mineral salts solution contained in
250 mL Erlenmeyer flasks to wash off non-adherent
microorganisms. The mineral salts solution (to be used
as mineral salts medium) contained (gL_‘): K,HPO ,,
0.5; Na,SO,, 2.0; NH&L, 1 O; CaCl,.2H,O, 0.15;
Mg S0,.7H,O, 0.1; and FeS0,.6H,O, 0.02; final pH
adjusted to 7.2.The agitation to dislodge non-adherent microorga-
nisms from the samples was at 300 rpm for 10 min at
room temperature. Subsequently, the agitated samples
were rinsed in the fresh mineral salts medium before
being transferred to 100 mL volumes of the same fresh
mineral salts solution contained in a 250 mL Erlen-
meyer flask, as the enrichment culture flask. Each en-
richme nt culture flask contained each of the agitated
feather, stone, and twig samples, supplemented with
1 mL weathered Kuw aiti crude oil.
Incubation was in a Control Environment IncubatorShaker (New Brunsw ick Scientific, NJ) at 30°C and an
agitation rate of 200 rpm for up to 7 d. After 7 d of
incubation, 1 mL of each culture was transferred se-
quentially three times to fresh medium .
Any organism not dislodged by the initial agitation
process aimed at removing non-adherent organisms,
and able to grow in the enrichment culture, was
deemed to have adhered strongly to the samples ab
initio. Thus, the resultant culture was considered an
enrichment culture for adherent microorganisms colo-
nizing the surfaces of the samples, and able to grow on
crude oil as sole carbon/energy source. Th ese were
used for subsequent experiments, and will be referred
to as “the enriched culture”.
Isolations from the enriched culture were made by
streaking loopfuls of the culture on Czapek Dox Agar
(CDA ) plates containing streptomycin and penicillin
(250 ug mL_‘) for isolation of fungi, andN utrient Agar
(NA) containing nystatin (250 pg mL“ ) for isolation of
bacteria. Following a 3-d incubation, discrete colonies
were transferred to fresh potato Dextrose Agar and
Nutrien t Aga r lacking the antibiotics for the isolation
of the fungal an d bacterial organ isms, respectively.
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Adherent microorganisms for bioremediation 82 5
Effect of Styrofoam carriers on crude oil degradation
by the enriched adherent cultures
To determ ine the effect of carriers (inert surfaces) on
the degrad ation of crude oil by the enriched cultures,
0.1 g of sterile styrofoam chips (~75 particle s 0.1 g-‘)
wa s added into one of the two sets of replicate, sterile
fresh culture media dispensed in 100 mL aliquots in
250 mL Erlenmeyer flasks, and inoculated with 1 mL
of the enriched culture. This culture w as first
maintained for 30 min at 30°C and 200 rpm, to allow
possible adherence of the organisms on the carriers,
before the addition of 1 mL weathered Kuw aiti crude
oil as the sole carbon/energy source. U ninoculate d
cultures with or withou t Styrofoam carriers served as
controls. The cultures were maintained at the above
growth conditions for up to 18 d in a New Brunswick
Control Environment Incubator Shaker.Replicate cultures were sampled aseptically and con-
tinually for pH changes, which were determined using
an Orion R esearch digital pH meter.
Crude oil analysis
Each whole flask culture (with or without styro-
foam ) was extracted in four changings (3x30 mL and
10 mL) of hexane, totaling 100 mL and the extracts
pooled together. The pooled hexa ne extract from each
culture was reduced to a final volume of 75 mL by
evaporation with N,, and 1 uL aliquots analyzed by gas
liquid chromatography in Chrompack CP9000 ; in-
jection temperature 300°C.
Resolution was on fused silica capillary column
(capillary wide bore, 0.53 mm by 10 m) maintained at
a temperature range of 60-3 10 “C and programmed to
change at the rate of 10 C min. The stationary phase
was CP-Sil5CB . The carrier gas was N2 at a flow rate
of 10 mL m in-‘, while detection was by means of flame
ionization detector (temp. 3 15 “ C). Identification and
quantification of comp onent hydrocarbons were by
comparison of their peak areas with those of standard
hydrocarbons of known concentrations, and by
MO SAIC data acquisition system, respectively.
Electron microscopy
Attachment of microorganisms to surfaces was visu-
alized by scanning electron microscopy (SEM ). All
specimens for SEM were fixed in 0.25 M glutar-
aldehyde for 3 h, washed for 10 min each in three
changings of Millonig’s (1961) phosphate buffer
(pH 7.3), and post-fixed in 0.04 M osmium tetraoxide
for 1 l/2 h. Subsequently, post-fixed samples were
again washed in Millonig’s buffer before being dried in
an acetone gradient of 4.07, 5.43, 6.78, 8.14, 9.50,
10.86, 12.21, and 13.57 M concentrations for periods
of 10 min at each concentration. How ever, Styrofoam
samples were dried in an ethanol gradient, as itdissolves in acetone.
The specimens were finally dried in a Balzer CPD -
030 critical point dryer using acetone/liquid CO, (or
ethanol/liquid CO, for styrofoam sam ples), a s
desiccant.
The dried specimens were mounted and sputter-
coated in gold for 30 s in Balzer SCD -050 sputter
coater, before being observed in a JOEL ISM-6300
scanning electron microscope at an accelerating voltage
of 20 kv, or less for Styrofoam sam ples.
RESULTS
SEM of solid samples from oil lake
SEM showed that all samples of stones/pebbles and
feathers obtained from the oil lake show ed extensive
surface colonization. How ever, there wa s no evidence
of surface co lonization on stone sam ples picked from
a non-oil lake area.
Figures 1a- 1c represent the scannin g electron micro-
graphs of stones and pebbles obtained from the oil
lake, while Figs. 2a-2c are the electron micrographs of
feather samples.
The distribution of microorganisms on the stone sur-
faces was heterogeneous (Figs. 1a- 1c). The organisms
occurred singly or in microcolonies of relatively few
cells (Fig. lc) or as dense continuous matrices where
individual cells were massed on one another and em-
bedded in amorphous material (Figs. la and lb). In
others, sam ples were covered by a lace-work of highly
branched tilamentous forms. Diverse morphological
forms were evident on the stone samples and included
coccoid, bac illoid (includ ing vibroid and spiral), and
filamentous forms.
As evident from Figs. 1a- 1c, the pattern of coloni-
zation of the stone substrata varied. The occurrence of
individual cells was sparse and appeared restricted to
plain (flat), exposed stone surfaces (Fig. lc). These
singly occurring cells possesse d thick cords or strand s
by which they were attached to the substratum. On the
other hand, microcolonies and dense masses of growth
occurred at the edges of and within crevices (or not-
ches) on the stone substrata (Figs. 1a and 1b). This am-
plification of growth in crevices may be of consider-
able ecological importance in the arid environment.
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82 6 C.O. Obuekwe and S.S. Al-Zarban
Fig. l(a and b). SEM showing colonization of crevices exposed flat surfaces of different pieces of stone in the oil lake.
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Adherent microorganisms for bioremediation 82 7
Fig. lc. SE M showing coloniza tion of crevices exposed flat surfaces of different pieces o f stone in the oil lake.
Fig. 2a. SEM showing colonization of typical feather specimens Tom an oil lake at low magnification.
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82 8 CO. Obuekw e and S.S. Al-Zarban
Fig. 2b. SEM showing colonization of typical feather specimens from an oil lake at colononization by mixed m icrobial population.
Fig. 2c. SEM showing colonization of typical feather specimens from an oil lake by yeast-like organisms (2~).
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Adherent microorganisms for bioremediation 82 9
As seen in Figs. 2a-2c, feather surfaces found in the
oil lake were also colonized by microorganisms of
diverse morph ological forms. How ever, the microflora
observed on the feather samples appeared more limited
than those seen on stone samples, as massed growths
were few and were not restricted to crevices. Also, nospiral or vibrio morphological forms were observed in
all feather specimens examined.
Crude oil degradation in laboratory cultures
Crude oil was degraded by mixed cultures of micro-
organisms enriched from washed stone and other solid
specimens obtained from the lake. Most of the orga-
nisms isolated from the mixed culture were identified
as Pseudomonas spp, Candida spp, and Aspergillus
SPP.Because the organisms were obtained from enrich-
ment of washed specimens, such organisms were ad-
justed to be adherent.
Figure 3 shows the gas chromatographic profiles of
the crude oil fraction extracted from cultures grow n in
the presence and absence of a carrier (cell support) -
Styrofoam particles. Crude oil degradation by mixed
culture occurred whether styrofoam particles were
incorporated or not incorporated in the culture. H ow-
ever, a s evident from F ig. 3, the presence of styrofoam
particles, as a cell support system, enhanced crude oil
degradation compared to the cultures without styro-foam. This is shown by the greater decline in the con-
centration (GC profile) of crude oil comp onents in
cultures containing styrofoam.
In the absence of the organisms (controls), no such
decline in crude oil components was evident, even
whe n Styrofoam wa s incorporated. Therefore, the ob-
served losses in comp onents of the crude oil were not
ascribab le to the presence of styrofoam. Table 1 show s
the percent degradation of crude oil and some major
aliphatic com ponents. In the presence of the styrofoam
carriers, the amount of total hydrocarbon degraded wastwice (44.4%) as much as was observed in the absence
of the carriers (22.8%), and losses in some individual
n-alkane components were more than 90%.
During the degradation of crude oil by the mixed
cultures, the culture pH declined continuously during
incubation. However, the pH of the cultures remained
unchanged in uninoculated controls (Fig. 4). The de-
cline in pH of cultures containing Styrofoam particles
was faster and greater, suggesting the production of
more acidic products. This is consistent with increased
degrad ation of crude o il by cultures in the presence of
carrier p articles (styrofoam), as shown by gas chroma-
tographic analysis.
Scanning electron m icroscope studies showed that
the Styrofoam particles incorporated into the crude oil
cultures were extensively colonized. Figures 5a and 5b
are the electron micro graphs of Styrofoam particles inthe mixed culture of crude oil-degrading organisms
enriched from stones/peb bles collected from the oil
lake. No such colonization is evident in uninoculated
cultures (Fig. 5~). The organisms colonizing the sur-
faces of the Styrofoam particles appeare d to be predo-
minately the filamentous forms, including Aspergihs
sp (Fig. 5d), although a few non-filamentous forms
were also observed. The dominance of fungi on the
Styrofoam chips was probably due to the resulting
higher acidity (pH 3.6) by the end of the incubatio n.
DISCUSSION
To dem onstrate the possible existence of active
crude oil-degrading and adherent microorganisms on
the surfaces of the materials found in the oil lake, the
gravels and other solid materials sampled from the oil
lake were subjected to scanning electron microscope
studies. Electron micrographs obtained showed a
variety of microb ial forms colonizing such surfaces.
That the microorganisms, especially bacteria, were
active under the existing environm ental conditions in
the oil lake wa s indicated by the occurrence of severalbacterial cells at different stages of cell division. If the
organisms were dormant, no such indications of cell
division would be evident. Thus, the scanning electron
microscope studies indicated the existence of active
microb ial cultures adheren t on the surfaces of the soil
mate rials found in the oil lake. These active, adheren t
populatio ns w ere, therefore, considere d responsible for
the removal of crude oil from the contam inated sur-
faces.
Am ongst the solid samples exam ined microscopi-
cally were the gravels and feathers. These two sampletypes constituted the commonest solid materials pre-
sent in the oil lake environment. While the stone pieces
are indigen ous of the site, the feathers represent extra-
neous solid materials introduced post-pollution and
should provide indications of what may happen if other
non-indigenous materials were deliberately introduced.
Radw an et al. (1995) also associated crude oil degra-
dation in the Kuw aiti desert w ith plant roots. These
worke rs noted that plants pulled out from the soil of the
oil lake exhibited clean root surfaces, even though the
soil was heavily contaminated with crude oil.
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83 0 C.O. Obuekwe and S.S. Al-Zarban
II0 2 4 8 8 lb li lh l ' s $8 2b 21 2
Retention time ( mn )
Fig. 3a. GC profiles of alkane com ponents of crude oil from laboratory cultures of microorganisms in the presence or absence of styrofoam
carrier after 18 d. A, uninoculated control + Styrofoam; B, uninoculated control without Styrofoam; C , inoculated without Styrofoam; and D,
inoculated + Styrofoam.
\.
02
ICl 4
t
i
IQ 2 4 8 8 1 0 1 2 1 4 1 8 1 8 2 0 2 2 2 4
Retention time ( min)
Fig. 3b. Profiles of alkanes standards.
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Adherent microorganisms for bioremediation 83 1
Table 1. Biodegradation of Kuwa it crude oil by enriched (mixed) culture of adherent microorganisms in the absence (free) and presence
of Styrofoam carriers, after 18 d of incubation.
Crude oil biodegradation (%)*
Culturesystem
Total n-alkane components
hydrocarbon Total(crude oil) aliphatics CU CM CM CM CU CM CM Cl0 CU Czz Cl3 CZ4 CZj C,,
N o
Styrofoam 21.8 32.0 30.9 32.0 48.2 48.7 54.5 47.2 39.8 34.4 35.3 30.6 35.4 32.3 30.2 35.7
Plus
Styrofoam 44.4 85.2 75.3 65.9 77.2 80.3 89.6 99.0 78.3 70.2 96.4 99.3 74.8 98.7 66.1 91.7
* Each value is the average of duplicate samples after adjustment for volatilization and extraction losses.
6-
In 5-
4-
3-
2-,.
6 3 6 9 12 15 18
Incubation Period (days)
Fig. 4. pH changes in crude-oil degrading cultures of adherent micro-
organisms in the presence (.), absence (m) of Styrofoam carriers, and
uninoculated controls (0), respectively.
Although SEM showed that the solid sample materials
were generally colonized, it was also observed that the
attached microorganisms, especially bacteria, were
mostly concentrated within nooks and crevices where
they formed dense masses or biofilms. Also, the flat
smoo th portions of the stone surfaces were found to be
scantily colonized with cells occurring mainly singly,
when present, and generally smaller in size than those
found in the crevices. No such information on the inter-
action of microorganisms with solid surfaces in oil
remediation in the desert environment appears to have
been reported, althoug h concentration in crevices is
common in flowing streams (W eise and Rheinheimer
1978; Geesey and Costerton 1979; Beeftink and
Staugaard 1986), where it is thought to confer pro-
tection from hydrodynamic shear stress. In the Kuw aiti
desert, intense solar radiation and dryness are the pre-
vailing environmental conditions, and the concentra-
tion ofthe microb ial grow th in crevices of stones foundin the oil lake migh t constitute an ecological response
to the inclement environment. Apart from protection
from solar radiation s and trap for moisture , the crevices
might also serve as traps for substrates when compared
with the flat, exposed surfaces. It might be assumed
that the substrate -trapping function of the crevices wa s
not paramount in the oil lake situation, since ample
substrate (crude oil) was available. However, poor
deposition of substrate s on the flat surfaces should not
be completely discounted , since electron mic rographs
showed that the bacteria on the flat, exposed portions
of the surfaces were sm aller in size than those found in
the crevices. Morita (1982) noted that cell miniaturi-
zation is an adaptive survival strategy during starva-
tion. Thus, the flat, exposed surfaces on the solid speci-
me ns mig ht be less favorable for the deposition of
substrate m aterials, especially on stones. Moreover,
these solid materials found in the oil lake simply pro-
vided large surface are as for oil degrad ation by the
microbes.
In the work being reported here, non-adherent micro-
bial populations on the stone surfaces were removed by
washing, and the subsequent culturing of the washed
stones/grav els, therefore, provided a mixed microb ial
culture w hich should possess the ability to adhere to
surfaces. Such washing procedure was used in differ-
ential determ ination of adheren t (biofilm-forming) and
non-adherent (non-biofilm) microorganisms on sand
and gravel particles in a water treatment system (El-
Masry et al. 1995). Although the organisms left on the
stone sam ples used in this work were sufficiently
tenaciously attached , subseq uent introduction of the
washed stone samples led to the establishment of a sus-
pension of mixed popula tions of organ isms potentially
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83 2 CO. Obuekw e and S.S. Al-Zarban
ia. SEM of Styrofoam carriers from mixed cultures of enriched adherent microorganisms showing colonization by predom
filamentous forms.
linantly
Fig. 5b. SEM of Styrofoam carriers from mixed cultures of enriched adherent m icroorganisms showing colonization by typical tight network of
filamentous colonizers.
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Adherent microorganisms for bioremediation 83 3
Fig. 5c. SEM of Styrofoam carriers from cultures of uninoculated control.
Fig. 5d. SEM of Styrofoam carriers from mixed cultures of enriched ad herent microorganisms showing colonization by Aspergillus sp.
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83 4 CO. Obuekw e an d S.S. Al-Zarban
capable of adhering to surfaces. Detachment of micro- CONCLUSIONS
organisms from biofilms because of hydrodynamic
shear had been reported (R&m an 1989). It was alsoActive crude oil degrad ation activities in the oil-pol-
such a suspension of potential adherent microorgan-luted Kuw aiti desert are associated with the adherent
isms recovered from washed stone samples and en-microb ial population which preferentially developed in
riched for oil degrad ation that wa s employed for the
crevices of stones and other solid ma terials dispersed
laboratory aspects of this investigation .in the polluted environment.
In the laboratory studies, the introduction of inert Acknowledgment-The authors gratefully acknowledge the services of
support m aterial like styrofoam chips was aimed at
simulating the natural situation in the Kuw aiti desert,
where stone pieces a nd other solid materials were
found in the oil lake. It, therefore, provided the me ans
of assessing the importance of the adherent organisms
associated with such surfaces in the bioremediation of
crude oil contamination.
Results obtained from cultures containing Styrofoam
chips, and crude oil as the sole carbon and energysource, June 24,199s demonstrated that solid surfaces
(Styrofoam) enhanc ed crude oil degrada tion by the
mixed m icrobial populations from the oil lake envi-
ronme nt. E lectron microscopy revealed tha t the styro-
foam chips were extensively colonized by the micro-
organisms of the culture, just as it was observed on
surfaces of stones and other solid sub strata in the oil
lake.
Data available from this study show that, in the
natural environment of the Kuw aiti desert, greater
growth and activities of microorganisms appeared to be
restricted to the cracks and depressions on solid ma-
terials, especially gravels, found in the oil lakes. These
crevices perhap s serve as essential niches. T his essen-
tiality is perha ps characteristic of an arid environm ent,
where there is need for protection of active m icrobial
growth from intense solar radiation, wind, and their
drying effects. If this were so, it is an essential strategy
that an oil-polluted environment in an arid environment
like the Kuw aiti desert should be provided with solid
support m aterials possessin g crevices for the estab-
lishm ent of an adheren t (biofilm-forming ), active oil-
degrada tion flora for effecting crude oil pollution re-
med iation. The results obtained in the laboratory
studies in which stvrofoam chins were used as SUDDOI?
Mr. Mohamm ed Ratiq of the Electron M icroscope Unit for technical
assistance. We thank D r. Mulder for introduction to the site and Ms.
Magda Kanafer for GC analyses. Gratitude for the work goes to the
Research Adm inistration of Kuwait University, which supports the
project by the grant S O 070.
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