Pergamon

Environment

International, Vol. 24, No. 8, pp. 823-834, 1998 Copyright 01998 Elsevier Science Ltd Printed in the USA. All rights reserved 0160-4120/98 $19.00+.00

PI1 SO160-4120(98)00074-9

BIOREMEDIATION OF CRUDE OIL POLLUTION IN THE KUWAITI DESERT: THE ROLE OF ADHERENT MICROORGANISMS
C.O. Obuekwe and S.S. Al-Zarban
Department of Biological Sciences (Microbiology Kuwait University, Kuwait Division), Faculty of Science, Khaldiya Campus,

EI 9710-203 M (Received 21 October 1997; accepted 28 February 1998)

Pieces of stones and other solid materials found in the oil lake sites of the Kuwaiti desert appeared clean, providing indications of surface-associated enhanced crude oil degradation. Scanning electron microscope studies revealed that such surfaces were colonized by active microbial populations. The colonization of the stone surfaces was concentrated within crevices. When enriched from washed pieces of stones from the oil lake, the resulting mixed population ofadherent microorganisms degraded much more crude oil (44.4%) in the presence of inert carrier materials (Styrofoam chips) in laboratory cultures, than in the absence of the inert materials (21.8%). The inert materials were found to be extensively colonized by microorganisms just as was observed with the stone and other solid samples from the oil lake. 01998 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 Kuwaiti desert and coastal soils have revealed the varying capacity of the environment to cleanse itself of oil pollution, depending on the structure of the indigenous microbial communities (Al-Gounaim et al. 1995; Sorkhoh et al. 1995; Al-Hassan et al. 1995; Radwan et al. 1995). The information provided on the structure of the microbial community has been restricted to the numbers and types of organisms involved in oil degradation, and there were no studies on the physical interactions between the organisms and their desert environment relevant to oil degradation. Because of the hot, dry climate, degradation and re823

cycling of organic matter are generally slow. Studies on the interaction between the organisms and their environment should provide better understanding ofthe structure and function of the microbial community. During studies of an oil lake in the Kuwaiti desert, it was observed that pieces of stone/pebbles, and other solid materials, when lifted from the oil-soaked soils of the oil lake, exhibited clean surfaces, even at their undersides. This observation would suggest active crude oil-removing activities associated with such surfaces. Such active oil-removal activities would possibly arise from oil-degrading microorganisms attached to the surfaces. Surfaces are important in the degradation of contaminants in nature and general microbial activities (Van Loosdrecht 1990). Davis and Westlake (1978) suggested that filamentous fungi in soils might provide increased contact surfaces for enhanced hydro-

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CO. Obuekwe and S.S. Al-Zarban

carbon degradation by bacteria. Surface-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 between microorganisms and their physical environment for crude oil degradation in the Kuwaiti desert became necessary with the report of Radwan et al. (1995) that plants growing within the oil lake sites, when pulled out, showed clean roots (lacking any visible signs of oil smears). This observation again suggested enhanced root surface-associated crude oil pollution remediation activities. The aim of the investigation being reported here was, therefore, to study the surfaces of the more commonly distributed solid materials (providers of the surfaces) found in the oil lake, for evidence of surface-associated microorganisms active in crude oil degradation, and determine also the effect of inert surfaces on the ability of such recovered surface-associated microflora to degrade crude oil. MATERIAL AND METHODS Samples Samples obtained for this work consisted of small pieces of stones and pebbles, broken twigs, and feathers obtained from beds of an oil lake (NE of Kuwait City) in the Kuwaiti desert, on Jahra-AlSubbian Road. Samples 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 4C in a refrigerator. Preparation of Styrofoam carrier materials Carriers or surfaces for the adherence of the enriched adherent microorganisms were prepared from styrofoam chips cut into irregular cubes of approximately 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 UV 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 28C and were, therefore, adjudged sterile.

Enrichment of culture for adherent microorganisms The rationale for the selective enrichment of adherent microorganisms is based on the fact that organisms which are sufficiently strongly attached to carrier surfaces will not be easily dislodged when such carriers are agitated, while organisms which are loosely associated with such surfaces would. In this work, microorganisms adherent to the samples collected were enriched by initially agitating the samples (pieces of stones, pebbles, twigs, 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; MgS0,.7H,O, 0.1; and FeS0,.6H,O, 0.02; final pH adjusted to 7.2. The agitation to dislodge non-adherent microorganisms 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 Erlenmeyer flask, as the enrichment culture flask. Each enrichment culture flask contained each of the agitated feather, stone, and twig samples, supplemented with 1 mL weathered Kuwaiti crude oil. Incubation was in a Control Environment Incubator Shaker (New Brunswick Scientific, NJ) at 30C and an agitation rate of 200 rpm for up to 7 d. After 7 d of incubation, 1 mL of each culture was transferred sequentially 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 colonizing the surfaces of the samples, and able to grow on crude oil as sole carbon/energy source. These 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, andNutrient 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 Nutrient Agar lacking the antibiotics for the isolation of the fungal and bacterial organisms, respectively.

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Effect of Styrofoam carriers on crude oil degradation by the enriched adherent cultures To determine the effect of carriers (inert surfaces) on the degradation of crude oil by the enriched cultures, 0.1 g of sterile styrofoam chips (~75 particles 0.1 g- ) was 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 was first maintained for 30 min at 30C and 200 rpm, to allow possible adherence of the organisms on the carriers, before the addition of 1 mL weathered Kuwaiti crude oil as the sole carbon/energy source. Uninoculated cultures with or without 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 continually for pH changes, which were determined using an Orion Research digital pH meter. Crude oil analysis Each whole flask culture (with or without styrofoam ) was extracted in four changings (3x30 mL and 10 mL) of hexane, totaling 100 mL and the extracts pooled together. The pooled hexane 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; injection temperature 300C. 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 min- while detection was by means of flame , ionization detector (temp. 3 15 C). Identification and quantification of component hydrocarbons were by comparison of their peak areas with those of standard hydrocarbons of known concentrations, and by MOSAIC data acquisition system, respectively. Electron microscopy Attachment of microorganisms to surfaces was visualized by scanning electron microscopy (SEM). All specimens for SEM were fixed in 0.25 M glutaraldehyde 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 buffer before being dried in s 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. However, Styrofoam samples were dried in an ethanol gradient, as it dissolves in acetone. The specimens were finally dried in a Balzer CPD030 critical point dryer using acetone/liquid CO, (or ethanol/liquid CO, for styrofoam samples), as desiccant. The dried specimens were mounted and sputtercoated 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 samples.
RESULTS

SEM of solid samples from oil lake SEM showed that all samples of stones/pebbles and feathers obtained from the oil lake showed extensive surface colonization. However, there was no evidence of surface colonization on stone samples picked from a non-oil lake area. Figures 1a- 1c represent the scanning electron micrographs 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 surfaces 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 embedded in amorphous material (Figs. la and lb). In others, samples were covered by a lace-work of highly branched tilamentous forms. Diverse morphological forms were evident on the stone samples and included coccoid, bacilloid (including vibroid and spiral), and filamentous forms. As evident from Figs. 1a- 1c, the pattern of colonization 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 possessed thick cords or strands 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 notches) on the stone substrata (Figs. 1a and 1b). This amplification of growth in crevices may be of considerable ecological importance in the arid environment.

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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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Fig. lc. SEM showing colonization of crevices exposed flat surfaces of different pieces of 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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CO. Obuekwe and S.S. Al-Zarban

Fig. 2b. SEM showing colonization of typical feather specimens from an oil lake at colononization by mixed microbial population.

Fig. 2c. SEM showing colonization of typical feather specimens from an oil lake by yeast-like organisms (2~).

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As seen in Figs. 2a-2c, feather surfaces found in the oil lake were also colonized by microorganisms of diverse morphological forms. However, 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, no spiral 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 microorganisms enriched from washed stone and other solid specimens obtained from the lake. Most of the organisms isolated from the mixed culture were identified as Pseudomonas spp, Candida spp, and Aspergillus SPP. Because the organisms were obtained from enrichment of washed specimens, such organisms were adjusted to be adherent. Figure 3 shows the gas chromatographic profiles of the crude oil fraction extracted from cultures grown 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. However, as evident from Fig. 3, the presence of styrofoam particles, as a cell support system, enhanced crude oil degradation compared to the cultures without styrofoam. This is shown by the greater decline in the concentration (GC profile) of crude oil components in cultures containing styrofoam. In the absence of the organisms (controls), no such decline in crude oil components was evident, even when Styrofoam was incorporated. Therefore, the observed losses in components of the crude oil were not ascribable to the presence of styrofoam. Table 1 shows the percent degradation of crude oil and some major aliphatic components. In the presence of the styrofoam carriers, the amount of total hydrocarbon degraded was twice (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 decline in pH of cultures containing Styrofoam particles was faster and greater, suggesting the production of more acidic products. This is consistent with increased degradation of crude oil by cultures in the presence of

carrier particles (styrofoam), as shown by gas chromatographic analysis. Scanning electron microscope studies showed that the Styrofoam particles incorporated into the crude oil cultures were extensively colonized. Figures 5a and 5b are the electron micrographs of Styrofoam particles in the mixed culture of crude oil-degrading organisms enriched from stones/pebbles collected from the oil lake. No such colonization is evident in uninoculated cultures (Fig. 5~). The organisms colonizing the surfaces of the Styrofoam particles appeared to be predominately 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 incubation.
DISCUSSION

To demonstrate 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 microbial forms colonizing such surfaces. That the microorganisms, especially bacteria, were active under the existing environmental conditions in the oil lake was indicated by the occurrence of several bacterial 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 microbial cultures adherent on the surfaces of the soil materials found in the oil lake. These active, adherent populations were, therefore, considered responsible for the removal of crude oil from the contaminated surfaces. Amongst the solid samples examined microscopically were the gravels and feathers. These two sample types constituted the commonest solid materials present in the oil lake environment. While the stone pieces are indigenous of the site, the feathers represent extraneous solid materials introduced post-pollution and should provide indications of what may happen if other non-indigenous materials were deliberately introduced. Radwan et al. (1995) also associated crude oil degradation in the Kuwaiti desert with plant roots. These workers 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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I
0
I

lb

li
time

lh

l's

$8

2b

212

Retention

( mn )

Fig. 3a. GC profiles of alkane components 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

Cl4 t

.
2 4

I
i
8

10

12

14

18

18

20

22

24

Retention

time

( min)

Fig. 3b. Profiles of alkanes standards.

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Table 1. Biodegradation

of Kuwait crude oil by enriched (mixed) culture of adherent microorganisms of Styrofoam carriers, after 18 d of incubation. Crude oil biodegradation (%)*

in the absence (free) and presence

Culture system No Styrofoam Plus Styrofoam

Total hydrocarbon (crude oil) 21.8

n-alkane components Total aliphatics 32.0 CU 30.9 CM CM CM 48.7 CU CM CM 39.8 Cl0 34.4 CU 35.3 Czz 30.6 Cl3 35.4 CZ4 32.3 CZj 30.2 C,, 35.7

32.0 48.2

54.5 47.2

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.

6I n 5432-, .

3 I

6 I

9 I

12 I

15 I

18 1

Incubation

Period (days)

Fig. 4. pH changes in crude-oil degrading cultures of adherent microorganisms 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 smooth 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 interaction of microorganisms with solid surfaces in oil remediation in the desert environment appears to have been reported, although concentration in crevices is common in flowing streams (Weise and Rheinheimer 1978; Geesey and Costerton 1979; Beeftink and Staugaard 1986), where it is thought to confer protection from hydrodynamic shear stress. In the Kuwaiti

desert, intense solar radiation and dryness are the prevailing environmental conditions, and the concentration ofthe microbial growth in crevices of stones found in the oil lake might constitute an ecological response to the inclement environment. Apart from protection from solar radiations 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 was not paramount in the oil lake situation, since ample substrate (crude oil) was available. However, poor deposition of substrates on the flat surfaces should not be completely discounted, since electron micrographs showed that the bacteria on the flat, exposed portions of the surfaces were smaller in size than those found in the crevices. Morita (1982) noted that cell miniaturization is an adaptive survival strategy during starvation. Thus, the flat, exposed surfaces on the solid specimens might be less favorable for the deposition of substrate materials, especially on stones. Moreover, these solid materials found in the oil lake simply provided large surface areas for oil degradation by the microbes. In the work being reported here, non-adherent microbial populations on the stone surfaces were removed by washing, and the subsequent culturing of the washed stones/gravels, therefore, provided a mixed microbial culture which should possess the ability to adhere to surfaces. Such washing procedure was used in differential determination of adherent (biofilm-forming) and non-adherent (non-biofilm) microorganisms on sand and gravel particles in a water treatment system (ElMasry et al. 1995). Although the organisms left on the stone samples used in this work were sufficiently tenaciously attached, subsequent introduction of the washed stone samples led to the establishment of a suspension of mixed populations of organisms potentially

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CO. Obuekwe and S.S. Al-Zarban

ia. SEM of Styrofoam carriers from mixed cultures of enriched adherent microorganisms filamentous forms.

showing colonization by predom linantly

Fig. 5b. SEM of Styrofoam carriers from mixed cultures of enriched adherent microorganisms showing colonization by typical tight network of filamentous colonizers.

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Fig. 5c. SEM of Styrofoam carriers from cultures of uninoculated control.

Fig. 5d. SEM of Styrofoam carriers from mixed cultures of enriched adherent microorganisms

showing colonization by Aspergillus sp.

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CO. Obuekwe and S.S. Al-Zarban

capable of adhering to surfaces. Detachment of microorganisms from biofilms because of hydrodynamic shear had been reported (R&man 1989). It was also such a suspension of potential adherent microorganisms recovered from washed stone samples and enriched for oil degradation that was employed for the laboratory aspects of this investigation. In the laboratory studies, the introduction of inert support material like styrofoam chips was aimed at simulating the natural situation in the Kuwaiti desert, where stone pieces and other solid materials were found in the oil lake. It, therefore, provided the means 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 energy source, June 24,199s demonstrated that solid surfaces (Styrofoam) enhanced crude oil degradation by the mixed microbial populations from the oil lake environment. Electron microscopy revealed that the styrofoam chips were extensively colonized by the microorganisms of the culture, just as it was observed on surfaces of stones and other solid substrata in the oil lake. Data available from this study show that, in the natural environment of the Kuwaiti desert, greater growth and activities of microorganisms appeared to be restricted to the cracks and depressions on solid materials, especially gravels, found in the oil lakes. These crevices perhaps serve as essential niches. This essentiality is perhaps characteristic of an arid environment, where there is need for protection of active microbial 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 Kuwaiti desert should be provided with solid support materials possessing crevices for the establishment of an adherent (biofilm-forming), active oildegradation flora for effecting crude oil pollution remediation. The results obtained in the laboratory studies in ..______ stvrofoam ___.____ which _-,__---.--- chins were used as SUDDOI? ___ .---r~~ -._ materials tend to support this strategy. The styrofoam chips, with their extensive crevices, encouraged the establishment of a biofilm population which degraded crude oil more, as reflected by the greater decreases in the components of crude oil and increased culture acidity. The increased culture acidity arose from the dissolved acidic degradation products of the oxidation of hydrocarbons.

CONCLUSIONS

Active crude oil degradation activities in the oil-polluted Kuwaiti desert are associated with the adherent microbial population which preferentially developed in crevices of stones and other solid materials dispersed in the polluted environment.
Acknowledgment-The authors gratefully acknowledge the services of Mr. Mohammed Ratiq of the Electron Microscope Unit for technical assistance. We thank Dr. Mulder for introduction to the site and Ms. Magda Kanafer for GC analyses. Gratitude for the work goes to the Research Administration of Kuwait University, which supports the project by the grant SO 070.

REFERENCES
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