A feasibility study on seeding as a ... - Wiley Online Library

Journal of Applied Microbiology 1997, 83, 353–358

A feasibility study on seeding as a bioremediation practice for the oily Kuwaiti desert S.S. Radwan, N.A. Sorkhoh, I.M. El-Nemr and A.F. El-Desouky Department of Botany and Microbiology, Faculty of Science, Kuwait University, Kuwait 5946/10/96: received 9 October 1996, revised 17 January 1997 and accepted 25 January 1997 S .S . R A DW AN , N. A. S OR KH O H, I. M . E L- N EM R A N D A .F . EL -D E SO UK Y . 1997. Immediately after a simulated oil spill, and for 28 weeks, Kuwaiti desert samples became steadily enriched with one specific, indigenous, oil-degrading Arthrobacter strain, KCC 201. Other indigenous oil degraders, including other Arthrobacter strains, either remained unchanged at low numbers or steadily disappeared. The partial hydrocarbon degradation in the polluted samples was primarily due to the indigenous, actively propagating Arthrobacter strain. Seeding the 28-week-old polluted samples with local or foreign oildegrading isolates did not lead to enhancement of hydrocarbon degradation and resulted in dramatic decreases in the numbers of the predominant, indigenous, oil-degrading Arthrobacter strain, KCC 201. Some of the seeded organisms, particularly the foreign isolates, failed to establish themselves in the polluted samples, apparently because of microbial competition.

INTRODUCTION 2

Since the 1991 Gulf war, about 50 km of the Kuwaiti desert has been left heavily polluted with oil sediments. This area represents the bottoms of the so-called oil lakes, in which the crude oil had penetrated to depths reaching about 50 cm, thus polluting about 4×107 tons of soil. Bioremediation appears to be the only feasible technology for cleaning such a huge amount of soil. This technology involves the seeding of soil with hydrocarbon-degrading micro-organisms and soil management, e.g. fertilization, to enhance already existing oil degraders (U.S. Congress, Office of Technology Assessment 1991 ; Sorkhoh et al. 1995). It is tentatively assumed that seeding of oil-polluted environments with exogenous oil-degrading micro-organisms should enhance the cleaning process. Professional companies offer microbial cocktails claimed to be useful for this purpose (Aldhous 1991), even after storage for up to 3 years (Applied Biotreatment Association 1990). The few earlier experiments on seeding of oily aquatic and terrestrial ecosystems gave mixed, with more negative than positive, results (for review see Leahy and Colwell 1990). Atlas and Busdosh (1976) recorded increased oil degradation in an Arctic pond after seeding with an oil-degrading PseudoCorrespondence to : Dr S.S. Radwan, Department of Botany and Microbiology, Faculty of Science, Kuwait University, PO Box 5969, Safat 13060, Kuwait. © 1997 The Society for Applied Bacteriology

monas sp. On the other hand, Tagger et al. (1983) found no increase in oil degradation in sea water after inoculation with a mixture of hydrocarbon degraders. Similarly, seeding oily beaches resulting from the Exxon Valdez spill with two oil degraders was of no significant effect although reportedly fertilization enhanced oil biodegradation (U.S. Congress, Office of Technology Assessment 1991). Seeding oil-polluted soils also did not seem to enhance the cleaning process (Jobson et al. 1972 ; Lehtomaki and Niemela 1975 ; Verstraete et al. 1976 ; Atlas 1977 ; Barles et al. 1979). In none of the above studies was the effect of the seeded micro-organisms on the indigenous oil-degrading microflora investigated. We have found previously that oil-polluted Kuwaiti desert samples were naturally rich in oil-degrading micro-organisms, and that watering and fertilization with nitrates enhanced the cleaning process (Radwan et al. 1995b). However, since there was no access to the polluted areas after the war, those earlier experiments were conducted 18–24 months after the spill. To study the immediate changes in the microbial and chemical make-up of the desert we simulated an oil spill under open conditions and conducted the analysis immediately. Twenty-eight weeks after the spill, the changes in the numbers of oil-degrading local and foreign microorganisms, after their inoculation into the polluted samples, and their effects on the hydrocarbon contents of those samples were investigated for another 20 weeks. The results of this study are presented in this paper.

354 S .S . R A DW AN E T A L .

M AT ER I AL S A N D M ET H OD S

Hydrocarbon analysis

The desert samples

Sand cores, 40×40×30 cm, from a clean desert area at AlKhaldiyah, Kuwait, were transported to the Botanical Garden of Kuwait University. There, the cores were located into soil holes of the same dimensions lined with plastic sheets, and kept permanently under open field conditions. Some of the cores were artificially polluted by mixing the sand with 20% (w/w) of weathered crude oil ; others were left untreated as a control. Cores were irrigated once weekly at a rate of 1 l water/10 kg soil. Samples were collected weekly for microbiological and chemical analysis.

The residual extractable hydrocarbons in 5 g soil were recovered by extraction three times with hexane. After volatilizing the solvent the residue was weighed and redissolved in 10 ml of hexane and 2 ml solution was analysed by gas–liquid chromatography using a Chrompack (NJ, USA) CP-9000 instrument equipped with a flame ionization detector, a WCOT fused silica CP-Sil-SCB capillary column and a temperature programme of 45–250°C, raising the temperature 16°C min−1. The total peak areas as well as those of individual compounds were thus a quantitative measure of the relative concentrations of the total and individual extractable alkanes. To calculate the actual concentrations of the extractable alkanes a solution of n-octadecane was used as an external standard.

Seeding experiments

Microbiological analysis

Micro-organisms capable of utilizing crude oil were counted using the standard plate method. We used a solid inorganic medium supplemented with 1% (w/v) weathered crude oil as a sole source of carbon and energy. The inorganic medium had the following composition (g l−1) : NaNO3, 0·85 ; KH2PO4, 0·56 ; Na2HPO4, 0·86 ; K2SO4, 0·17 ; MgSO4 . 7H2O, 0·37 ; CaCl2 . H2O, 0·007 ; Fe (III) EDTA, 0·004, and trace element solution, 0·25 ml consisting of (g/l) : ZnSO4 . 7H2O, 2·32 ; MnSO4 . 4H2O, 1·78 ; H3BO3, 0·56 ; CuSO4 . 5H2O, 1·0 ; Na2MoO4 . 2H2O, 0·39 ; KI, 0·66 ; EDTA, 1·0 ; FeSO4 . 7H2O, 0·4 ; NiCl2 . 6H2O, 0·004 ; agar, 15 ; pH 7. Cultures were incubated aerobically at 30°C. After counting the total and individual organisms, representative strains were isolated on the same medium, purified and identified by consulting pertinent keys and comparing them with previously identified strains in our collection. Bacteria growing in liquid cultures were also counted directly under a phase-contrast microscope using a haemocytometer.

RESULTS AND D ISCUSSION

The clean sand was found to contain 1·8 (20·09)×104 total oil-utilizing micro-organisms g−1. Apparently, oil degraders are natural inhabitants of most soils, where they utilize hydrocarbons naturally occurring in decaying organs and microorganisms. The oil-utilizing microflora of the studied sample consisted of two Arthrobacter strains, one, Kuwaiti Culture Collection (KCC) 201 with white, and the other, KCC 202, with orange colonies, and one strain each of Pseudomonas KCC 203, Rhodococcus KCC 204, and Streptomyces KCC 205. The five strains occurred in nearly equal proportions. The total numbers of oil degraders steadily increased immediately after the addition of crude oil to reach, after 28 weeks, 2·5 (20·1)×106 cells g−1 soil. However, this increase was due solely to the white strain of Arthrobacter KCC 201 ; the orange strain KCC 202 as well as the remaining three bacterial genera either remained constant in number or even disappeared. Figure 1 presents, for simplicity, the changes in numbers of only the white (KCC 201) and orange (KCC 202) Arthrobacter

Numbers × 1000 per g soil

Twenty-eight weeks after the simulated spill, samples in the individual holes were inoculated with individual oil-degrading isolates and with mixtures of isolates suspended in tap water. The seeded organisms included two local isolates, Arthrobacter nicotianae and Candida parapsilosis, that were originally isolated from thick blue-green mats currently covering oily coasts of the Arabian Gulf (Sorkhoh et al. 1992 ; Al-Hasan et al. 1994). In addition, five Arthrobacter strains were isolated from an oil-polluted soil sample obtained from Mu¨nster/Westf, Germany. The individual Arthrobacter strains were differentiated by their colony colours and shapes. The concentration of each inoculated organism was such as to give an initial count of 2·8×106 g−1 sand sample. Sand samples were collected weekly for microbial and chemical analysis for another 20 weeks.

3000 2500 2000 1500 1000 500 100 0

0

4

8 12 16 20 Incubation period (weeks)

24

Fig. 1 Changes in numbers of two indigenous oil-utilizing strains

of Arthrobacter in oil-polluted sand. Ž, White strain KCC 201 ; , orange strain KCC 202

© 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 353–358

S EE DI N G F OR B IO RE M ED IA T IO N F O R T HE O IL Y K U WA IT I DE SE R T 355

the orange strain, i.e. the white strain appears to be the strongest competitor for oil and therefore it predominated after the oil spill. Associated with the above-described change in the microbial make-up of the polluted sand there occurred a parallel decrease in its oil content. Thus, the amount of total extractable oil decreased after 28 weeks to reach only 36(21·6)% of the original value. Figures 3 and 4 show 80

mg per 5 g soil

strains. In this context, it may be noted that oil-degrading bacteria associated with roots of plants growing in the polluted desert were also found to belong to the genus Arthrobacter (Radwan et al. 1995a). This, however, should not necessarily imply that only Arthrobacter spp. predominate in all polluted localities of the Kuwaiti desert. In fact, previous studies in this laboratory showed that other, but always few, genera, viz. Pseudomonas, Bacillus and nocardioforms, are predominant in the polluted sand (Radwan et al. 1995b). Apparently, the specific combination of conditions in each desert locality determines which oil degraders ultimately predominate. To understand why, in this experiment, the white Arthrobacter KCC 201 strain predominated over all other strains after the oil spill, its growth kinetics were compared with those of the orange Arthrobacter KCC 202 strain in media containing various carbon sources. The results showed that both strains followed similar growth patterns with time, but only when conventional carbon sources, e.g. glucose, were available (Fig. 2). In the presence of n-octadecane (an aliphatic hydrocarbon) or phenanthrene (an aromatic hydrocarbon) as sole carbon sources, the white strain grew much faster than

60

40

20

0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 Incubation period (wees)

Fig. 3 Changes in concentrations of alkanes of varying chain

lengths in oil-polluted sand with time. Ž, Alkanes longer than C18 ; , C18 ; , C17 ; ž, C16 ; R, alkanes shorter than C16

300 × 106 6

250 × 10

C16

6

200 × 10

C18

6

Cell number per ml medium

150 × 10

C12

6

100 × 10

C22

6

50 × 10

C30

28 × 106 24 × 106 20 × 106 6

16 × 10

6

12 × 10

6

8 × 10

6

4 × 10

1 × 106 0

4 8 12 16 20 24 28 Incubation period (h)

0

2

4

Fig. 2 Growth kinetics of two indigenous Arthrobacter strains in

a medium containing different carbon sources. R, ž, Ž, White strain KCC 201 ; r, , , orange strain KCC 202 ; R, r, glucose as a sole carbon source ; ž, , n-octadecane as a sole carbon source ; Ž. , phenanthrene as a sole carbon source

6

8 10 12 14 16 Retention time (min)

18

20

22

Fig. 4 Typical gas–liquid chromatography profiles of residual alkanes extracted from polluted sand samples at zero time (top profile), 12 weeks (middle profile) and 28 weeks (bottom profile) after the simulated oil spill



–—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Organism Colony colour Colony shape Colony diameter (mm) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Indigenous strain KCC 201* White Circular 2–3 Inoculated strains Local strains Arthrobacter nicotianae Pale yellow Irregular 5–6 Candida parapsilosis White Punctiform 1 and less Foreign strains Arthrobacter KCCG 351 Yellow Punctiform 1 and less Arthrobacter KCCG 352 Pale orange Irregular 2–3 Arthrobacter KCCG 353 Orange Circular 2–3 Arthrobacter KCCG 354 Creamy Irregular 5–6 Arthrobacter KCCG 355 Pale orange Punctiform 1 and less –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — * The strong competitive Arthrobacter strain predominating after the simulated oil spill

further that C18 (n-octadecane) and shorter alkanes were more readily eliminated from the soil than longer alkanes. The seeding experiments were started 28 weeks after the simulated spill. For this study, only bacterial strains whose colonies exhibited easily determinable morphological differences on solid media were selected. Table 1 summarizes the colony morphological features that were used for differentiating and recognizing the individual strains. The results in Table 2 show that the highest hydrocarbon consumption continued to occur in the control non-inoculated soil which also contained by far the highest numbers of the strongest indigenous competitor strain (the white Arthrobacter KCC 201 strain). Seeded local strains isolated from the Gulf coast established themselves rather easily in the polluted sand. After 20 weeks, their numbers increased 11 to 27–fold but, simultaneously, the numbers of the indigenous white Arthrobacter KCC 201 strain were reduced by 2–3 fold. On the other hand, the German Arthrobacter strains (KCCG 351–355) faced difficulty in establishing themselves. Two strains, KCCG 351 and KCCG 353, did not survive until week 20 ; another two, KCCG 352 and KCCG 354, decreased in number by about 3-fold and only one, strain KCCG 355, survived but hardly doubled its numbers in 20 weeks. Seeding with all German strains resulted in dramatic decreases in the numbers of the indigenous white Arthrobacter KCC 201 strains. Seeding with a mixture of the local strains and another mixture of the German strains gave similar results. The highest hydrocarbon consumption was in the control noninoculated sand samples. Although generally seeding did not cause dramatic inhibition of hydrocarbon consumption, which means that seeded strains were also active in hydro-

Table 1 Distinctive morphological

features of the studied organisms (see Table 2)

carbon degradation, it was obvious that inoculation with the German strains gave the lowest consumption values. However, after a total of 48 weeks, the duration of the study, all samples had already lost more than 90% of the total extractable alkanes, i.e. the oily sand was bioremediated to a promising extent. This was confirmed by the observation that the samples at the end of the 1-year experiment became richly inhabited with insects and worms, which were missing during the first 30 weeks. That the 50 km2 polluted desert area of Kuwait did not reach any satisfactory degree of recovery after 5 years is obviously mainly due to the lack of water essential for the indigenous microflora. In conclusion, terrestrial oil spills appear to be naturally associated with enriching the soils with the strongest indigenous microbial competitors for hydrocarbons. Such an indigenous microflora is best suited for bioremediation (Radwan 1991), providing that proper management, especially watering in dry habitats, is conducted to enhance their activities. Seeding with cocktails irrespective of their origin appears to be a useless or even harmful practice particularly if conducted immediately after the spill, because the inoculated organisms compete with the indigenous strains.

ACKNOWLEDGEMENTS

This work was supported by Kuwait University, Research Grant No. S0049. Thanks are due to A. Khan and MarieNoelle Grand-Alyagout for technical assistance.



2·4 2·4 2·4 2·4 2·4 2·4 2·4 2·4

2·8 2·8 2·8 2·8 2·8 2·8 2·8

Indigenous strain

—

Inoculated strain

64 64 64 64 64

64 64

64

0 20 2 15 3

70 30

—

Inoculated strain

2 2 7 1 6

230 670

280

Indigenous strain

No. (×106 g−1)

No. (×106 g−1) % of alkanes consumed

10

0

Weeks after inoculation

90 91 91 90 89

91 90

92

% of alkanes consumed

0 1 0 1 5

75 32

—

Inoculated strain

2 2 3 5 6

350 560

1025

Indigenous strain

No. (×106 g−1)

20

91 91 91 91 91

94 94

95

% of alkanes consumed

The local (isolated from oil sediments along the Arabian Gulf) and foreign (German) strains were seeded into sand samples that had already been mixed with oil for 28 weeks (see text). At zero time of inoculation the indigenous micro-organisms had already eliminated 64% of the extractable alkanes. The data in the table are means of three readings each, and the standard deviation did not exceed 4·2% of the mean values

Control (not inoculated) Local strains Arthrobacter nicotianae Candida parapsilosis Foreign strains Arthrobacter KCCG 351 Arthrobacter KCCG 352 Arthrobacter KCCG 353 Arthrobacter KCCG 354 Arthrobacter KCCG 355

Inoculated strains

in the polluted sand

Table 2. Establishment of seeded strains and their effects on the numbers of the strongest indigenous competitor (white Arthrobacter strain) and oil consumption

S EE DI N G F OR B IO RE M ED IA T IO N F O R T HE O IL Y K U WA IT I DE SE R T 357


REFERENCES Aldhous, P. (1991) Gulf oil spill. Nature 349, 447. Al-Hasan, R.H., Sorkhoh, N.A., Al-Bader, D. and Radwan, S.S. (1994) Utilization of hydrocarbons by cyanobacteria from microbial mats on oily coasts of the Gulf. Applied Microbiology and Biotechnology 41, 615–619. Applied Biotreatment Association (1990) The Role of Biotreatment of Oil Spills, 2nd review pp. 13–14. Atlas, R.M. (1977) Stimulated petroleum biodegradation. Critical Reviews of Microbiology 5, 371–386. Atlas, R.M. and Busdosh, M. (1976) Microbial degradation of petroleum in Arctic. In Proceedings of the 3rd International Biodegradation Symposium ed. Sharpley, J.M. and Kaplan, A.M. pp. 79–86. London : Applied Science Publishers. Barles, R.W., Daughton, C.C. and Hsieh, D.P.H. (1979) Accelerated parathion degradation in soil inoculated with acclimated bacteria under field conditions. Archives of Contamination and Toxicology 8, 647–660. Jobson, A., Cook, F.D. and Westlake, D.W.S. (1972) Microbial utilization of crude oil. Applied Microbiology 23, 1082–1089. Leahy, J.G. and Colwell, R.R. (1990) Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54, 305–315. Lehtomaki, M. and Niemela, S. (1975) Improving microbial degradation of oil in soil. Ambio 4, 126–129. Radwan, S.S. (1991) Gulf oil spill. Nature 350, 456.

Radwan, S.S., Sorkhoh, N.A. and El-Nemr, I. (1995a) Oil biodegradation around roots. Nature 376, 302. Radwan, S.S., Sorkhoh, N.A., Fardoun, F. and Al-Hasan, R.H. (1995b) Soil management enhancing hydrocarbon biodegradation in the polluted Kuwaiti desert. Applied Microbiology and Biotechnology 44, 265–270. Sorkhoh, N.A., Al-Hasan, R.H., Khanafer, M. and Radwan, S.S. (1995) Establishment of oil-degrading bacteria associated with cyanobacteria in oil-polluted soil. Journal of Applied Bacteriology 78, 194–199. Sorkhoh, N., Al-Hasan, R.H., Radwan, S.S. and Hoepner, Th. (1992) Self-cleaning of the Gulf. Nature 359, 109. Tagger, S., Bianchi, A., Julliard, M., Le Petit, J. and Roux, B. (1983) Effect of microbial seeding of crude oil in sea water in a model system. Marine Biology (Berlin) 78, 13–20. U.S. Congress, Office of Technology Assessment (1991) Bioremediation for Marine Oil Spills. Background Paper, OTA-BPO-70, Washington, DC : U.S. Government Printing Office. Verstraete, W., Vanloocke, R., De Borger, R. and Verlinde, A. (1976) Modeling of the breakdown and the mobilization of hydrocarbons in unsaturated oil layers. In Proceedings of the 3rd International Biodegradation Symposium ed. Sharpley, J.M. and Kaplan, A.M. pp. 99–112. London : Applied Science Publishers.
