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Enhanced bioremediation of oil spills in the sea
Eliora Z Ron1,2 and Eugene Rosenberg1
Hydrocarbon-degrading bacteria are ubiquitous in the sea,
including hydrocarbonoclastic bacteria that utilize
hydrocarbons almost exclusively as carbon and energy
sources. However, the rates at which they naturally degrade
petroleum following an oil spill appear to be too slow to prevent
oil from reaching the shore and causing environmental
damage, as has been documented in the Exxon Valdez and
Gulf of Mexico disasters. Unfortunately, there is, at present, no
experimentally demonstrated methodology for accelerating the
degradation of hydrocarbons in the sea. The rate-limiting factor
for petroleum degradation in the sea is availability of nitrogen
and phosphorus. Oleophilic fertilizers, such as Inipol EAP 22
and urea-formaldehyde polymers, have stimulated
hydrocarbon degradation on shorelines but are less effective in
open systems. We suggest uric acid as a potentially useful
fertilizer enhancing bioremediation at sea.
Addresses
1
Department of Molecular Microbiology and Biotechnology, Faculty of
Life Sciences, Tel Aviv University, Tel Aviv, Israel
2
MIGAL – Galil Research Center, Kiriat Shmona, Israel
Corresponding author: Ron, Eliora Z (eliora@post.tau.ac.il)
Current Opinion in Biotechnology 2014, 27:191–194
This review comes from a themed issue on Environmental biotech-
nology
Edited by Hauke Harms and Howard Junca
For a complete overview see the Issue and the Editorial
Available online 19th March 2014
0958-1669/$ – see front matter, # 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.copbio.2014.02.004
Introduction
Oil spills from the Exxon Valdez disaster and the blowout
of the Deepwater Horizon drilling rig accentuate the need
for effective bioremediation methods for treating oil spills
in the sea. Although technologies for enhancing bioreme-
diation of petroleum pollution in soil and reactors have
been known for several years [1], there is, at present, no
experimentally demonstrated methodology for accelerat-
ing the degradation of hydrocarbons in the sea.
The breakdown of hydrocarbons requires an appropriate
consortium of hydrocarbon-degrading microorganisms
and conditions that allow these microorganisms to grow
and multiply. Two essential characteristics of hydro-
carbon-oxidizing microorganisms are (a) membrane-
bound, compound class-specific dioxygenases [2] and
(b) mechanisms for optimizing contact between the
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microbes and the water-insoluble hydrocarbons [3].
Because the oxygenases are group specific, for example,
some degrade specific fractions of alkanes, others work on
aromatics or cyclic hydrocarbons, etc., it follows that only
a mixture of different microorganisms can efficiently
degrade crude oil and petroleum fractions. Hydrocarbon
oxidizers are located in virtually all natural environments,
although with large variations in cell concentration [4].
This is not surprising because hydrocarbons have been
present on land and in water for millions of years as a
result of natural oil seeps and plant synthesis [5].
Hydrocarbon-degrading bacteria in the
marine environment
Biodegradation mediated by indigenous microbial com-
munities is the ultimate fate of the majority of oil hydro-
carbon that enters the marine environment [6], where
hydrocarbon-degrading microorganisms are ubiquitous
[7]. Microbial biodegradation was shown to occur natu-
rally in remediating oil contamination associated with
several oil spills [8,9]. However, the rates of biodegrada-
tion are slow, and consequentially there are serious toxic
effects on marine life in the water and on the shoreline.
There is a growing body of data indicating which particu-
lar microorganisms are involved under in situ conditions at
oil spill sites. Of particular interest is the group of micro-
organisms, referred to as hydrocarbonoclastic bacteria
[7] that utilize hydrocarbons and hydrocarbon degra-
dation products almost exclusively as carbon and energy
sources [10]. They include strains of Alcanivorax
[11,12], Cycloclasticus [13], Oleiphilus [14], Oleispira
[15] Thalassolituus [16] and Planomicrobium [17]. Alca-
nivorax strains were most abundant following an oil spill
as determined by 16S rRNA gene analysis [18]. In our
opinion, research should be directed to establishing in situ
conditions for rapid growth of the oil-degrading bacteria.
Consideration should be given to ‘seeding’ oil spills near
shore with appropriate strains of Cycloclasticus because
these bacteria degrade the toxic polycyclic aromatic
hydrocarbons.
It is difficult to estimate the natural rates at which
hydrocarbons are degraded in seawater following
petroleum oil spills because much of the hydrocarbon
is removed by chemical and physical processes. Some of
the oil is photo-oxidized, the low molecular weight frac-
tions are volatilized, and currents move the oil to shore
and/or out to sea. The fact that the highly branched
hydrocarbons, such as hopane, which are difficult to
biodegrade, accumulate relative to straight chain hydro-
carbons of the same molecular weight, suggests that
Current Opinion in Biotechnology 2014, 27:191–194
192 Environmental biotechnology
microbial degradation plays a significant role in removing
the oil [19].
The need for physical interactions between
bacteria and hydrocarbons
The low solubility of hydrocarbons in water, coupled to
the fact that the first step in hydrocarbon degradation
involves membrane-bound oxygenases, makes it essential
for bacteria to come into direct contact with their hydro-
carbon substrates. Two general biological strategies have
been suggested for enhancing contact between bacteria
and water-insoluble hydrocarbons: adhesion mechanisms
and emulsification of the hydrocarbon [4].
To understand the special cell surface properties of
bacteria that allow them to grow on hydrocarbons, it is
necessary to consider the dynamics of petroleum degra-
dation in natural environments. Following an oil spill in
the sea, hydrocarbons rise to the surface and come into
contact with air. Some of the low molecular weight
hydrocarbons volatilize; the remainder is metabolized
relatively rapidly by microorganisms, which take up
soluble hydrocarbons. These bacteria do not adhere to
oil and do not have high cell-surface hydrophobicity. The
next stage of degradation involves microorganisms with
high-cell-surface hydrophobicity, which can adhere to the
residual high molecular-weight hydrocarbons. Adherence
is due to hydrophobic fimbriae, fibrils, outer membrane
lipids and proteins and certain small cell-surface mol-
ecules, such as gramicidin S and prodigiosin. Bacterial
capsules and other anionic exopolysaccharides appear to
inhibit adhesion to hydrocarbons.
Hydrocarbon-degrading bacteria often produce bioemul-
sifiers [20]. These can be divided into low-molecular-
weight molecules that lower surface and interfacial ten-
sions efficiently and high-molecular-weight polymers that
bind tightly to surfaces. Some bioemulsifiers enhance the
growth of bacteria on hydrophobic water-insoluble sub-
strates by increasing their bioavailability, presumably by
increasing their surface area, desorbing them from sur-
faces and increasing their apparent solubility. Bioemulsi-
fiers also play an important role in regulating the
attachment–detachment of microorganisms to and from
surfaces. Addition of artificial chemical emulsifiers, as was
performed in the Deepwater Horizon blowout, can have
negative effects. More than 700 000 gallons of the dis-
persant Corexit were applied directly at the wellhead.
Two years after the spill, studies showed Corexit
increased the toxicity of the oil by 52 times [21].
Nutritional requirements for the growth of
hydrocarbon-degrading bacteria
Hydrocarbons, as the name implies, contain only hydro-
gen and carbon. Thus, for bacteria to grow and multiply
on hydrocarbons in seawater they require utilizable ad-
equate sources of nitrogen, phosphorus, sulfur, iron and
Current Opinion in Biotechnology 2014, 27:191–194
oxygen (anaerobic degradation of hydrocarbons can also
occur at slow rates). Since seawater is a desert when it
comes to nitrogen and phosphorus, these two elements
must be supplied for efficient biodegradation of hydro-
carbons. In fact, the rate-limiting process in bioremedia-
tion of petroleum pollution in the sea is provision of
nitrogen and phosphorus [3]. In principle, approximately
150 g of nitrogen and 30 g of phosphorus are consumed in
the conversion of 1 kg of hydrocarbon to cell material.
The nitrogen and phosphorus requirement can be met
with fertilizers, such as ammonium phosphate, nitrates,
phosphates and urea. However, all of these compounds
have a high water solubility which reduces their effec-
tiveness in open systems, such as the sea, because of rapid
dilution. The first attempt to overcome this problem was
the use of paraffinized urea and octyl phosphate [22].
Although this mixture was effective, it is not a practical
solution to the problem because of cost and the high ratio
of C to N. Subsequently, there have been several other
attempts to provide oleophilic fertilizers for the treatment
of oil slicks.
An oleophilic fertilizer (Inipol EAP 22 = oleic acid, urea,
lauryl phosphate) was used in the bioremediation of
polluted shorelines following the Exxon Valdez spill
[23]. Initial reports of success [24] have been challenged
[25]. There are at least three problems with Inipol EAP
22. First, it contains large amounts of oleic acid, which
serve as an alternative carbon source, thereby increasing
the C:N ratio in the environment. Second, it contains
toxic compounds. Third, as soon as the fertilizer comes
into contact with water, the emulsion breaks, releasing
urea into the water phase where it is not available for the
microorganism.
Another approach has been the use of water-insoluble
polymers, based on urea-formaldehyde formulations that
adhere to oil [26]. This latter fertilizer was used success-
fully to bioremediate a heavily oil-contaminated sandy
beach, but the polymer would not be effective in the sea
because its high density causes it to sink. Other slow-
release or oleophilic fertilizers that have been considered
include chitosan [27], Customblen [28] and lecithin
[29]. Although these fertilizers stimulate petroleum bior-
emediation to a more or less extent on oil-contaminated
shorelines, none of them are effective in treating oil at sea.
The utilization of chitosan and urea-formaldehyde poly-
mers requires the production of extracellular hydrolyses.
Thus, when using these fertilizers it may be advantageous
to ‘seed’ oil licks with hydrocarbon-degrading bacteria that
contain the appropriate depolymerase. The interesting
possibility of using nitrogen-fixing, hydrocarbon-oxidizers
has been considered [30–32]. Since nitrogen is often the
limiting factor for growth of microorganisms following an
oil spill, one might expect a strong selection for nitrogen-
fixing, hydrocarbon-oxidizers following an oil spill. In 1967,
Cody (325) claimed to have isolated microorganisms, from
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 Enhanced bioremediation of oil spills in the sea Ron and Rosenberg 193
soil exposed to methane, that concomitantly convert
hydrocarbons, such as n-tetradecane, and atmospheric
nitrogen into cellular material. Since that time, there have
been few reports that conclusively demonstrate N2 fixation
coupled to growth on hydrocarbons larger than ethane.
However, there have been several publications that con-
sortia of bacteria can support hydrocarbon degradation
through N2 fixation [30]. To our knowledge, there is only
one report of a marine microorganism that couples N2
fixation and hydrocarbon oxidation [32].
Uric acid as a natural fertilizer for the
treatment of oil pollution in the sea
Uric acid is the major nitrogen waste product of birds,
bats, terrestrial reptiles, and many insects. It has a low
solubility in water and adheres to hydrocarbons [33].
These properties, in addition to the fact that uric acid is
the major component of inexpensive and commercially
available guano fertilizer, suggested to us that it might
be a useful nitrogen source for bioremediation of
petroleum pollutants in open systems. Being an abun-
dant natural product, many different species of bacteria
are known to use uric acid as a nitrogen source [34],
including hydrocarbonoclastic Alcanivorax strains
[33,35]. Using a simulated open system, it was demon-
strated that commercial uric acid was an effective source
of nitrogen for the growth of marine bacteria on crude
oil. Bacterial cultures reached over 108 cells/ml and 70%
of the crude oil was degraded [35]. In control exper-
iments using water soluble ammonium sulfate and phos-
phate in place of uric acid and phosphate in the
simulated open system, bacterial concentration reached
only 106 cells/ml and showed no detectable hydrocarbon
degradation. In our opinion, uric acid is a potentially
useful nitrogen and phosphorus fertilizer for the treat-
ment of oil spills in the sea.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
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