BIOTECHNOLOGY LETTERS

Volume 15 No.6 (June 1993) pp.637-640

Receivedas revised 4th May

N-ALKANE BIODEGRADATION BY A MARINE BACTERIUM IN THE

PRESENCE OF AN OLEOPHILIC NUTRIMENT.

Laurent RIVET, Gilbert MILLE

Fact&C des Sciences et Techniques de St Jerome
Chimie Analytique de I’Environnement. C.N.R.S. - URA 1409
13397 Marseille Cedex 20. France

Anne BASSERES, Alain LADOUSSE

SocittC Nationale Elf Aquitaine, Artix. France

Claude GERIN, Monique ACQUAVIVA

and Jean-Claude BERTRAND
Centre d’Octanologie de Marseille (OS(I) URA 41
Fact& des Sciences de Luminy. Case 901
13288 Marseille Cedex 9, France
SUMMARY
Hexadecane biodegradation by a marine bacterium has been investigated in
the presence of an oleophilic nutriment (INIPOL EAP 22). Hydrocarbon attack was
only observed after metabolism of the fatty acids present in the fertilizer. The
bacterium used up 95 % fatty acids in the first 24 hours. Hexadecane biodegradation
took place after 50 h incubation and reached 40 % after 360 h.

The ability of some micro-organisms to grow on hydrocarbons as the sole

carbon and energy source has been known for many years. In natural
environments, these hydrocarbonoclastic micro-organisms play a major role in the
hydrocarbon biodegrading processes. Biodegradation depends on various factors,
either biotic or abiotic (physical and chemical composition of petroleum,
temperature, 02 concentration, composition and concentration of inorganic
compounds and nutriments, salinity, pressure). Making use of this ability offers an
appealing prospect to fight oil pollution generated by oil spills (Atlas, 1981; Bartha,
1986; Leahy and Colwell. 1990). In this perspective, several techniques have been
considered : the first one consists in introducing in the polluted area,
hydrocarbonoclastic bacterial strains of natural origin or derived from genetic
engineering (Chakrabarty, 1985). Till now, results established in the laboratory
have not been confirmed under natural conditions. In the second approach,
biodegrading natural processes are stimulated by supplying nutriments to the
polluted medium. Indeed. in the case of an oil spill, the microflora has access to a
large carbon source but nitrogen and phosphorus sources are limited. Therefore it
is necessary to supply nutriments in order to restore C/N and C/P ratios sustaining
the highest biodegradation. After several years of investigation, two kinds of
additives have been worked out : hydrosoluble and oleophilic nutriments (Atlas and
Bartha, 1973; Ladousse and Tramier, 1991; Olivieri er al., 1978). Oleophilic nutriments
turned out to be more efficient because they stay in contact with the oil layer.
After the pollution of the Alaska coast caused by the Exxon Valdez wreck
(March 1989), several products were tested to clean beaches polluted by crude oil.
These products had been previously tested in the laboratory to determine their
possible toxicity and efficiency (Safferman, 1991; Tabak et al.. 1991). Among thcsc
products, INIPOL EAP 22 (Safferman, 1991) was selected for its ability to accelerate
in situ biodegrading processes. This product is a micro-emulsion of inverse type,
containing urea, lauryl phosphate and oleic acid. Upon utilisation, oleic acid would
work as a starter and would induce a fast increase of the bacterial biomass. Though

637
the efficiency of this additive has been demonstrated in situ and in vitro (Pritchard,
I99I), its working mechanisms are still largely unknown. In order to understand
the effect of this oleophilic fertilizer on micro-organisms, we have studied the
biodegradation of an alkane (n-hexadecane) by a marine bacterial strain in the
presence of INIPOL EAP 22.

MATERIALS AND METHODS

Bacterial strain,
The strain formerly referred to as Aiteromonas sp. 17 (AlMallah et al.,
1990) has been recently described as a new species, Ma rino bat te r
hydrocarbonoclasticus ATCC 49840 (Gauthier et al., 1992).
Culture media.
“Rich medium” (RM) : synthetic seawater as described by AlMallah et al.
(1990), except ammonium which was absent, and enriched with 5 g. 1-l yeast extract
(Difco) and 5 g. 1-l bactopeptone (Difco).
“Hexadecane medium” (HM) : synthetic seawater containing 22 mg. l- *
nitrogen (as NHqCl), 1.73 mg. 1-l phosphorus (as K2HP04) and 0.74 mg. 1-l iron and
supplemented with 1 g. 1-l n-hexadecane. In HM + CI 8:1, oleic acid was added to HM
at a concentration analogous to that found in INIPOL EAP 22.
“Hexadecane-INIPOL EAP 22 medium” (HIM) : synthetic seawater (without
ammonium) supplemented with 1 g. 1-l hexadecane and 0.3 g. 1-l INIPOL EAP 22.
Growth conditions
Culture conditions are illustrated in Figure 1. Bacteria were grown at 30°C
in 250 ml inverted T-shaped tubes (specially designed for vigourous shaking and
aeration) and containing 60 ml culture medium. Aeration was maintained by
agitation on a reciprocal shaker (96 rpm).

PL,
1 ml H. I. M.

(JL-1 m; (J$l m; t-fL~ 1 ml C-‘-J

R. M. H. M. H. M. H. M.

1 ml
t &I
H. M.-Cl&l

Figure 1, Growth conditions for the strain Marinobacter hydrocarbonoclasticus on

HM and HIM. During the last step, the number of seeded inverted T-shaped tubes
corresponded to the studied incubation periods :
- 9 tubes for growth on HIM (5, 10 , 15, 24, 50, 100, 170, 360, 500 h)
- 8 tubes for growth on HM + C18:1 (5, 10 , 16, 24, 30, 35, 48, 132 h)
- 4 tubes for growth on HM (5, 16, 24, 48 h)

Scanning electron microscoDv : performed as described by Bertrand et al. (1990).

Proteins : determined by the method of LOWE et al. (1951).
yr_esl : titrated according to Amino and Kerouel (1982).
Hvdrocarbons and faltv acids analvsis : performed as described by Millc et al (1992).

638
RESULTS AND DISCUSSION.
We analysed a sample of INIPOL EAP 22 and found that oleic acid represented
60 % of all fatty acids. The others main fatty acids were as follows : C]4:0 = 7%. Cl6:O =
8%. C]6:1 = 8%. C17:l = 2%, C1g:l = 1.5% and C2O:l = 1.5%.
After 15 h of growth on HIM, 60% of all fatty acids and 40% of urea were
used up. After 24 h, whereas fatty acids were largely metabolized (90%). hcxadecane
was not yet biodegraded. After 50, 170 and 360 h incubation, the percentages of
hexadecane degradation were respectively 10, 32 and 40%. The bacterial biomass
increased accordingly : 130. 191 and 230 mg. I- 1 respectively. Beyond 360 h
incubation, the residual amount of hydrocarbon remained unchanged (Figure 2)
and the urea content was very low (6~ eq. at.g. l-l).

Time (hours)
&urc 2 ; Growth of Morinobocrcr hydrocarbonoclotticus on ii&f
Oleic acid (w) , n-hcxadccane (H) , proteins (w).

We have separately investigated cell growth on n-hexadecane in the

presence of oleic acid (HM + Cl 8:l). Under these conditions, after 15 h growth, 47%
oleic acid and 10% n-hexadecane were metabolized. After 25 h incubation, these
percentages rose respectively to 55.4 and 18% and reached 60 and 40% after 48 h.
These results are analogous to that observed with INIPOL EAP 22 (HIM). In the
absence of oleic acid (HM), n-hexadecane biodegradation remained undetectable for
the first 24 hours and reached at the end of the growth a percentage close to the
one determined in the presence of oleic acid. Thus oleic acid increased the rate of n-
hexadecane metabolism and has no effect on the final percentage of
biodegradation.
So during growth of Marinobacter hydrocarbonoclasticus on “HM + Cl 8 : I ”
we observed a simultaneous attack of fatty acid and hydrocarbon, in contrast with
the findings related to growth on HIM. However. WC must stress rhc point that HM
and HM + C18:1 have a composition far simpler than Hlhl, which limits comparisons.
From the above results, WC can conclude that INIPOL EAP 12 promotes the n-
hexadecane biodegradation by a pure marine strain. An analogous conclusion was
reported by Basscrcs and Ladoussc (1992) for bactcriul communities. Hcxadccanc is
only degraded after mctabo!Ism of the fatty acids prcscnt in 111~ rcrtiliycr. Electron
microscopy rcvcaled that during growth on Hlhl. cells rend IO rnakc clumps but
their morphology remains like that obscrvcd when grown on l-151 (Guuthier ct al,
1992). Thus INIPOL EAP 22 dots not affect ccl1 morphology (Figure. 3).

639
Figure 3 ; Scanning electron micrograph of Marinobacter hydrocarbonoclasticus
growth at 32“C on HIM. Cells were fixed at the end of exponential phase
A : Gx4000; B : Gx20000. (prepared and photographed by C. Bezac)

Acknowledgements. This work was carried out in the framework of Cycles

biog4ochimiques : Bvolution de la mat&e organique en milieu littoral” and supported by the
Sock%?Nationale ELF Aquitaine, the Centre National de la Recherche Scientifique (CNRS) and
the Programme Interdiscipiinaire de la Recherche sur t’fnvironnement (PIREN).
Urea titration was performed by MS B. Tanguy from lnstitut P. Ricard (Les Embiez- FRANCE).

REFERENCES
-Al-Mallah, M. , Goutx, M., Mille, G. and Bertrand, J. C. (1990). Oil Chem. Poll., 6, 289-
305.
-Amino, A. and Kerouel, R. (1982). Can. I. Fish. Aqua. Science., 39, 174-177.
-Atlas, R. M. (1981). Microbial Rev., 45, 180-209.
-Atlas, R. M. and Bartha, R. (1973). Environ. Sci. Technol. 7, 538-541.
-Bartha, R. (1986). Microb. Ecol., 12, 155-172.
-Basseres, A. and Ladousse, A. (1992) Proceedings of the CONCAWE/DGMK Scientific
Seminar “Remediation of oil Spills” Hambourg.
-Bertrand, J. C., Almallah. M., Acquaviva, M. and Mille, G. (1990). Lett. Appl.
Microbial. 11, 260-263.
-Chakrabarty, A. M. (1985). Trends Biotechnol., 3, 28-32.
-Gauthier, M., Lafay, B., Christen, R., Fernandez, L. Acquaviva, M., Bonin, P. and
Bertrand. J. C. (1992). Int. I. Syst. Bacterial. 42, 568-576.
-Ladousse, A. and Tramier, B. (1991). Proceedings of the 1991 oil spill conference,
577-581.
-Leahy, J. G. and Colwell, R. R. (1990). Microbial. Rev., 54, 305-315.
-Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). .I Biol Chem , 95,
2102-2107.
-Mille, G., El Jammal, T., Doumenq, P. and Bertrand, J. C. (1992). The Scien. Total
Environ. 113,209-228.
-Olivieri, R., Robertiello, A. and Degen, L. (1978). Marine Pollut Bull., 9, 217-220.
-Pritchard, P. H. (1991). I. Hazard. Matter., 28, 115-130.
Safferman, S. I. (1991). Proceedings of the 1991 oil spill conference, 571-576.
-Tabak, H. H., Haines, J. R., Venosa, A. D., Glaser, J. A., Desai, S. and Nisamaneepong,
W. (1991). Procedings of the 1991 oil spill conference, 583-590.

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