Desalination 211 (2007) 286295

Enhanced bioremediation of crude oil utilizing lipophilic

fertilizers
M. Nikolopouloua, N. Pasadakisb, N. Kalogerakisa*
a

Department of Environmental Engineering, bDepartment of Mineral Resources Engineering,

Technical University of Crete, Polytechneioupolis, 73100 Chania, Greece
Tel. +30 (282) 10-37794; Fax +30 (282) 10-37852; email: kalogera@enveng.tuc.gr

Received 1 December 2005; revised 27 January 2006; accepted 16 February 2006

Abstract
Bioremediation is being increasingly seen as an affective, environmentally friendly treatment for contaminated
shorelines from marine oil spills. Oil bioremediation is limited by the availability of nitrogen and phosphorous,
which are needed by the bacteria and are not present in sufficient amounts for the biodegradation of the spilled
hydrocarbons. The supply of these two essential elements as water-soluble salts presents several problems. These
include the rapid dilution of the salts in the large volumes of polluted sea water as well as their utilization by other
bacteria that do not degrade oil. In addition, by increasing the concentration of mobile nitrogen further environmental problems could potentially be created. As an alternative, hydrophobic sources of nitrogen and phosphorous
have been used that have a low solubility in water and hence, they can overcome these problems, however, many of
these bioremediation agents have proven to be somewhat toxic. The aim of this study was to examine the effects of
lipophilic fertilizers of natural origin (uric acid and lecithin) on the degradation of crude oil hydrocarbons in
comparison with the degradation that is achieved by commercial bioremediation agents. Biodegradation was quantified by GC/MS analysis of selected components. Petroleum degraders were measured by MPN analysis. From a
series of 18 days long experiments it was found that the saturated fraction of the residual oil was degraded more
readily and extensively than the aromatic fraction and the bacterial growth of the biostimulated solutions is much
greater compared to the control. The results showed that the treatment of oil spills with uric acid and lecithin is very
effective in a period of almost 7 days. Uric acid and lecithin can stimulate microbial growth from 3.65 to
1.9104 MPN/mL within 7 days and thus lead to extensive degradation of oil hydrocarbons in comparison to the
control and S200 solutions. Uric acid and lecithin proved to be excellent biostimulant agents in combating oil
spills.
Keywords: Oil spills; Biostimulation; Lipophlic nutrients; Bioremediation agents
*Corresponding author.
Presented at the 9th Environmental Science and Technology Symposium, September 13, 2005, Rhodes, Greece.
Organized by the Global NEST organization and prepared with the editorial help of the University of Aegean,
Mytilene, Greece and the University of Salerno, Fisciano (SA), Italy.
0011-9164/07/$ See front matter 2007 Published by Elsevier B.V.
doi:10.1016/j.desal.2006.02.095

M. Nikolopoulou et al. / Desalination 211 (2007) 286295

1. Introduction
Modern society continues to rely primarily on
the use of petroleum hydrocarbons for its energy
needs. Despite recent technological advances,
accidental spills of crude oil and its refined products occur on a frequent basis during routine operations of extraction, transportation, storage, refining and distribution. It is estimated that between
1.7 and 8.8 million metric tons of oil are released
into the worlds water every year, of which more
than 90% is directly related to human activities
including deliberate waste disposal [1].
Marine shorelines are important public and
ecological resources that serve as a home to a
variety of wildlife and provide public recreation.
Marine oil spills, particularly large scale spill accidents, have posed great threats and caused extensive damage to marine coastal environments.
For example, the oil spill from the Exxon Valdez
in 1989 and the Prestige in 2002 led to the mortality of thousands of seabirds and marine mammals, a significant reduction in population of many
intertidal and subtidal organisms, and led to many
and many other long term environmental impacts
[2,3].
Biodegradation as a natural process may proceed slowly, depending on the type of oil (i.e.,
light crude oils degrade faster than heavier oils).
Bioremediation strategies are based on the application of various methodologies to increase the
rate or extent of the biodegradation process. The
success of oil spill bioremediation depends on our
ability to optimize various physical, chemical, and
biological conditions in the contaminated environment. The most important requirement is the
presence of microorganisms with the appropriate
metabolic capabilities. If these microorganisms are
present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and
oxygen are present and that the pH is between 6
and 9 [4].
In marine environments, nutrient limitation is
generally correlated to the low background lev-

287

els of nitrogen and phosphorus in seawater [5].

Biostimulation involves the addition of rate-limiting nutrients to accelerate the biodegradation
process. A commonly used strategy has been to
add nutrients at concentrations that approach a
stoichiometric ratio of C:N:P of 100:5:1.
1.1. Nutrients application
The first approach to treat an oil spill includes
the application of commercial inorganic fertilizers and mineral nutrient salts. However these
water soluble nutrients were readily washed out
by the wave action.
Many attempts have been made to design nutrient delivery systems that overcome the washout problems characteristic of intertidal environments [6]. Use of slow release fertilizers is one of
the approaches used to provide continuous sources
of nutrients to oil contaminated areas. Slow release fertilizers are normally in solid form that
consists of inorganic nutrients coated with hydrophobic materials like paraffin or vegetable oils.
This approach may also cost less than adding
water-soluble nutrients due to the less frequent
application. Slow release fertilizers have shown
to be promising from several bioremediation studies. However, the major challenge for this technology is the control the release rates so that optimal nutrient concentrations can be maintained in
the water over long time periods [1].
Another approach to overcome the problem
of water-soluble nutrients being rapidly washed
out is to utilize oleophilic organic nutrients [7,8].
The rationale for this strategy is that oil biodegradation mainly occurs at the oil-water interface;
since oleophilic fertilizers are able to adhere to
oil and provide nutrients at the oil-water interface, enhanced biodegradation should result without the need to increase nutrient concentrations
in the bulk water. A well-known oleophilic fertilizer is Inipol EAP 22, a microemulsion containing urea as a nitrogen source, lauryl phosphate
(as phosphorus source), 2-butoxy-1-ethanol as a
surfactant, and oleic acid to give the material its

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hydrophobicity. This fertilizer has been subjected

to extensive studies under various shoreline conditions and was successfully used in oil bioremediation on the shorelines of Prince William Sound.
Other studies have focused on the 2-butoxy-ethanol which is present in Inipol and on its potential
toxicity to wildlife and cleanup workers [9]. Other
oleophilic organic fertilizers include polymerized
urea and formaldehyde, and some organic fertilizers derived from natural products such as fishmeal [1012].
In summary, the effectiveness of these various types of nutrients depends on the characteristics of the contaminated environment. Slow-release fertilizers may be ideal nutrient sources if
the nutrient release rates can be controlled sufficiently well. Water-soluble fertilizers are likely
to be more cost-effective in low-energy and finegrained shorelines where water transport is limited whereas, oleophilic fertilizers may be more
suitable in high-energy, coarse-grained beaches.
Nonetheless, successful application of bioremediation products will always require appropriate
testing and evaluation based on the specific conditions of each contaminated site [1].
The oil surface area is important because
growth of oil degraders occurs almost exclusively
at the oilwater interface [5]. In this work we focus on the development of new environmentally
friendly, inexpensive fertilizers that can bind at
this oilwater interface and thus facilitate microbial growth.
Due to the potential toxicity of the 2-butoxyethanol component of Inipol oleophilic fertilizer,
attempts have to be made to use less toxic, biodegradable and of natural origin fertilizers. Urea and
especially uric acid are of natural origin. Uric acid
is the major nitrogen waste product of birds, terrestrial reptiles, and many insects. It has a low
solubility in water and is the major component of
guano fertilizer, suggesting that it might be a useful nitrogen source for the bioremediation of petroleum compounds in open systems. Furthermore, uric acid binds to crude oil and is therefore

available to bacteria which grow at the hydrocarbon-water interface. Uric acid can (i) serve as a
nitrogen source for hydrocarbon degrading bacteria and (ii) bind to crude oil, thereby making it
a potentially useful nitrogen fertilizer for the
bioremediation of petroleum spills in open systems [13]. As source of phosphorous another lipophilic natural fertilizer such as Soya bean lecithin was used. Natural phospholipids such as lecithin are in fact oil soluble, easy to get at low cost
as by-product of oil seeds industry and have good
dispersant properties [14].

2. Materials and methods

2.1. Experimental design
The procedure consists of an experimental
shaker flask setup and the specific set of microbiological and chemical analyses that are performed on individual product samples. The test
flasks (labeled with unique identifiers) are prepared and set up on an orbital shaker at day 0 to
reflect the treatments indicated in Table 1.
Each 250-ml borosilicate glass Erlenmeyer
flask was charged with the following materials at
the onset of the experiment: 100 mL of seawater,
0.5 g of weathered crude oil, the biostimulation
agent at its appropriate concentration and inorganic mineral nutrients if required by the biostimulant vendor. The oil was Arabian Light crude
oil weathered artificially according to ASTM
method D 2892 [15]. The biostimulation additives
(one bioremediation agent and two fertilizers)
were introduced into the solutions in such amounts
so that optimized nutrient amendment is achieved,
namely, 2 g N/100 g of crude oil and 0.2 g P/100
g of crude oil. Uric acid and L-a-phosphatidylcholine (L-a-lecithin) derived from soybean, Type
II-S, with a purity of about 19% were supplied by
Sigma Chemical Co. The prepared flasks were
shaken at 200 rpm and 20C until the time that
they were removed for sampling. The treatments
were sampled three times over an 18-day period;

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289

Table 1
Experimental design

Treatment
Control
S-200
Uric acid + lecithin (UL)

No of samples at sampling times

Total number of analytical determinations

Day 0

Day 7

Day 18

Microbial counts

GC/MS

triplicate
duplicate
duplicate

duplicate
duplicate
duplicate

triplicate
triplicate
triplicate

nine triplicates
nine triplicates
nine triplicates

8
7
7

i.e., at day 0, day 7 and day 18. The entire flask

was used for analysis, a 1-mL aliquot was removed
from each flask for microbiological analysis and
the remainder of each flask was used for the
chemical analysis. The bioremediation agent S200 was applied according to manufacturer instructions. The bioremedia-tion agent S-200 was
used at the Prestige oil spill accident in Spain and
was kindly offered by IEP Europe, S.L. Co.
(Madrid-Spain).
2.2. Microbiological analyses
The microbial population in the flasks was
estimated by the most probable number (MPN)
method for hydrocarbon degraders [16]. The
growth medium was a Bushnell-Hass minimal
salts medium (BHS) supplemented with 3.5%
NaCl and crude oil as the hydrocarbon substrate.
The MPN plates were 96-well microtiter tissue
culture plates, with each well containing 180 l
BHS, 5 L crude oil and 20 L of the appropriate
dilution of sample. The samples were diluted in a
9 ml Bushnell-Hass solution (pH 7) and 3.5%
NaCl. Tenfold serial dilutions were performed, and
the plates were inoculated by adding 20 L of
each dilution to one of the 12 rows of eight wells.
The 1010 dilution was inoculated into row 11, the
109 dilution was inoculated into row 10, and so
on. The first row of each plate was inoculated with
20 L of undiluted sample, and row 12 remained
uninoculated to serve as a sterile control. The inoculated plates were incubated at 20C for
2 weeks. At the end of the incubation period,

20 L of p-iodonitrotetrazolium violet dye

(7.5 g/L) was added to each well of the tissue culture plates and allowed to stand at room temperature for 1 h. The dye turns from colorless to red
(when reduced) in the presence of actively respiring microorganisms. The MPNs were calculated
using a spreadsheet program [17].
2.3. Chemical analysis
Quantification of the hydrocarbon target
analytes was performed by gas chromatography/
mass spectrometry. The nonaqueous flasks contents were extracted by adding approximately
50 mL of dichloromethane spiked with a surrogate recovery standard (4 ng/L of each d10phenanthrene, 5-androstane in solvent of the final extract). After mixing for several minutes, the
flask was set aside to allow the dichloromethane
and water layers to partition. The dichloromethane
layer was drained by passing through a funnel
packed with anhydrous sodium sulfate. Subsequently, the dichloromethane was evaporated in
a Kuderna-Danish concentrator to approximately
5 mL and afterwards was blown down to dryness
with nitrogen to approximately 1.5 mL. 50 mg of
oil from this concentrated sample and a premeasured volume of 10 mL dichloromethane were
cleanup by passing through a funnel packed with
anhydrous alumina. A known volume of elluent
was blown down to dryness (nitrogen) and then
was dissolved in 1 mL of hexane for use on the
autosampler of the GC/MS instrument. To this
solution, 25 L of a 400 ng/L solution of the

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internal standards was added. The final concentration of the internal standards in each sample
was 10 ng/L. This solution contained 7 deuterated compounds: d8-naphthalene, d10-anthracene,
d12-chrysene, d12-perylene, d10-acenaphthene, d10phenanthrene and d4-1,4-dichlorobenzene in dichloromethane solution. For quantitative analyses, the GC/MS instrument was operated in the
Selected Ion Monitoring (SIM) mode at a scan
rate of greater than 1.5 scans per second, to maximize the linear quantitative range and precision
of the instrument [18]. The standard hydrocarbon
mix for the calibration curve was obtained from
Absolute Standards Inc. The surrogate and internal standards were obtained from Supelco Co.

3. Results and discussion

3.1. Visual observations on oil droplets (Fig. 1)
Immediately after the application of the
biostimulation additives the crude oil was dispersed to small droplets (micelles). When the
bioremediation agent S200 was added, the oil was
dispersed near the surface layer, whereas when
the combination of uric acid and lecithin was applied, the crude oil was dispersed in the water
column and the solution turned black. On the other
hand, in the control treatment where no additives
were applied, tar balls were formed.
By visual observation it was not possible to
determine the amount of crude oil in each treatment that was biodegraded over time; however,
one could readily see a decrease of the floating
oil over the 18 days of observation. The apparent
biodegradation was visually more for the uric acid
lecithin-UL treatment followed by the S200 treatment and with no apparent change for the control.
3.2. Microbial growth
The growth of the indigenous population in
the control solution was slowly increasing. Thus,

over the first 7 days an increase of 12.33% in the

microbial mass was only observed and after 18
days the total microbial mass increased only by
30%.
When the bioremediation agent S200 was
added, the microbial growth was high. In particular, during the first 7 days the microbial mass increased from 2.3 to 2.3103 MPN/mL; and after
18 days the total microbial mass remained practically constant.
Microbial growth in the presence of uric acid
and lecithin was also favored and the treatment
exhibited a continuous biomass increase throughout the experiment. In particular, during the first
7 days the microbial mass increased from 3.65 to
1.9104 MPN/mL and then to 8.3105 by day 18.
The above results confirm the well known fact
that in natural seawater we can have significant
microbial growth only if all essential nutrients are
present.
3.3. Monitoring of composition changes with GC
The effectiveness of fertilizer in stimulating
biodegradation in the tests is illustrated in a qualitative manner by inspecting the trends in gas chromatographs traces. Figs. 2, 3 and 4 present the
gas chromatographs of the initial oil and the oils
extracted after 7 and 18 days of incubation in the
laboratory at room temperature.
The traces clearly suggest that biodegradation
of both resolved and unresolved hydrocarbons in
the stimulated flasks was faster and more extensive than in the untreated control.
The samples maintained their overall profile
characteristics but had undergone some degradation, especially of the n-alkanes C16-C24, with
an increase in the proportion of the unresolved
complex mixture (UCM). In the S-200 solution
(shown in Fig. 3) biodegradation is evidenced by
diminution of n-alkane peaks in the range C16C24 and a slightly higher UCM profile. Biodegradation in the UL solution shown in Fig. 4, continued with a further loss of n-alkanes in the C16-

M. Nikolopoulou et al. / Desalination 211 (2007) 286295

CC0
Formation of tar balls.

CC18

S200-0

S20018

Oil dispersion in the water phase and

formation of small petroleum droplets
at the surface.

291

UL0

UL18

Black color, oil is dispersed in

the water column.

Fig. 1. Visual changes of the oil in the control, S200 and UL (uric acidlecithin) solutions after 18 days of incubation.

t = 0 days
t = 7 days
t =18 day

Fig. 2. Gas chromatographs of control solutions at day 0, 7 and 18.

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M. Nikolopoulou et al. / Desalination 211 (2007) 286295

t = 0 days
t = 7 days
t =18 days

Fig. 3. Gas chromatographs of S200 solutions at day 0, 7 and 18.

t = 0 days
t = 7 days
t =18 days

Fig. 4. Gas chromatographs of UL (uric acidlecithin) solutions at day 0, 7 and 18.

M. Nikolopoulou et al. / Desalination 211 (2007) 286295

100%

1000000

Control

S-200

UL

90%

% Biodegradation

100000

MPN (counts/mL)

293

10000

1000

100

Control

S200

UL

80%
70%
60%
50%
40%
30%
20%

10

10%

0%

10

15

20

Time (days)

Day 7

Day 18

Fig. 5. Microbial growth in control, S200 and UL (uric

acidlecithin) solutions.

Fig. 6. Biodegradation of C19-C34 n-alkanes after 7 and

18 days of treatment in control, S200 and UL (uric acid
lecithin) solutions.

C34 range and an associated increase in the proportion of UCM. The n-alkanes were completely
degraded within a week in the UL solution.
According to the trend of the chromatographs
for the control solution shown in Fig. 2, there is
no particular fluctuation in the form of the peaks
with respect to time. This suggests that in the absence of nutrients the indigenous microorganisms
can not cope with the high charge of organic carbon which creates rapidly limiting conditions in
terms of nitrogen and phosphorous. The comparison of the chromatographs shows that in the solutions where uric acid and lecithin (UL) were applied, an increase in the rate of degradation was
observed as the days of reaction passed by. It is
also observed that all the components of the weathered crude oil have nearly been removed as shown
in Fig. 4. The importance of dissolution of the oil
slick that is observed at the application of lecithin
in the solution should be emphasized and hence,
the beneficial effect of the combined application
of uric acid and lecithin on the biodegradation of
oil slicks should be noted. As shown in Fig. 3, for
the particular experimental conditions employed
in this study, the bioremediation agent S200 did
not enhance substantially the biodegradation of
crude oil although the oil was dispersed in small
droplets during its application in a period of 18
days.

4. Conclusions
This study aimed to evaluate the biodegradation of crude oil that is achieved when natural,
lipophilic nutrients are used. A set of chemical
and microbiological experiments were run and the
observed trend in the chromatographs from the
examined solutions showed that lipophilic nutrients enhance the biodegradation of crude oil. The
apparent reduction in petroleum hydrocarbons that
was achieved with the formulation UL (uric acid
lecithin) over a period of 18 days is very encouraging (with final removal of C19-C34 n-alkanes
of 83% as shown in Fig. 6).
In comparison, the commercial bioremediation
agent S200 eventually was not as effective in degrading petroleum hydrocarbons in crude oil for
this particular set of experiments. This contradicts
previous studies using S200 to stimulate indigenous hydrocarbon degraders. It should be noted
however, that S200 was found to be very effective in combined biostimulationbiaugmentation
experiments with isolated hydrocarbon degrading consortia [19].
The GC profiles after 7 and 18 days showed
that biodegradation in both resolved and unresolved hydrocarbons in the solutions were nutrients had been applied is more extensive than that
in the control solutions. However most of the

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M. Nikolopoulou et al. / Desalination 211 (2007) 286295

crude oil fractions were totally utilized after 18

days of treatment. The application of nutrients in
the solutions enhanced the growth of the hydrocarbon degraders as was estimated by the MPN
method in comparison with the control solution
where no nutrients were added.
As shown by the chromatographs (Fig. 5), the
saturated fraction of the residual oil is degraded
more extensively than the aromatic fraction. Lecithin supports the higher biodegradation rate, comparable to that obtained by using soluble phosphates. Both uric acid and lecithin are suitable
lipophlic nutrient sources and enhance microbial
growth during the first 7 days.
The fact that remains however is that in the
first week the overall growth is not very high when
in real life immediate hydrocarbon degradation
is needed. Considering this, besides the use of
biostimulation additives (like UL) we may have
to supplement with the addition of hydrocarbon
degrading consortia (bioaugmentation) particularly when the oil spill approaches near the shore
line.
Acknowledgements
Financial support was provided in part through
Project HPMD-CT-2001-00060 of the Marie Curie Development Host Fellowship Program.
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