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



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.



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.



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.




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.



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~).




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.



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.




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



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.




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.



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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