Journal of Environmental Protection, 2011, 2, 47-55
doi:10.4236/jep.2011.21005 Published Online March 2011 (http://www.SciRP.org/journal/jep)
47
Potential Approaches to Improving Biodegradation
of Hydrocarbons for Bioremediation of Crude Oil
Pollution
Qingren Wang, Shouan Zhang, Yuncong Li, Waldemar Klassen
Tropical Research and Education Center, University of Florida, Homestead, FL, USA.
Received September 20th, 2010; revised November 3rd, 2010; accepted December 29th, 2010.
ABSTRACT
With increasing demands of fossil fuel energy, extensive exploration of natural sources has caused a number of large
scale accidental spills of crude oil and resulted in some significantly environmental disasters. The consequence of oil
pollution to environment and human health has brought a serious challenge to environmental scientists. Physical and
chemical approaches to cleanup oil spills are too expensive and create adverse effects. Bioremediation has shown a
great potential and competitive privilege because of environment friendly and cost effective. A number of efficient mi-
crobial strains have been identified and isolated, which can effectively degrade various components of petroleum oil.
However, the biodegradation efficiency is usually limited by abiotic factors, such as temperature and pH, which are
hardly to be contro lled in the in situ condition but adequate o xygen supply a nd nutrient balancing are of importance to
impact microbial function s. Therefore, this review especially addresses potential approaches to improving bioremedia-
tion of crude oil by supplying solid oxygen and adjusting C: N: P ratio to optimize microbial activities in order to im-
prove the effectiveness an d efficacy of bioremediation o f crude oil pollu tants. In addition, it also elucidates advantages
of bioremediation, isolation of selective microbial strains, and evaluation of the biodegradation rates.
Keywords: Biodegradation, Bioremediation, Crude Oil, Hydro c ar b on, Nutrient Balance, Oxygen
1. Critical Importance and Advantages of
Bioremediation
Oil contamination with petroleum and petroleum-based
hydrocarbons in accidental spills has caused critical con-
cerns in environment, ecological systems, human health,
tourism and recreation activities. Such pollution to water
and soil has become often in recent decades with the in-
creasing demand of fossil fuel energy requiring offshore
drilling due to the global population growth and persis-
tent persuasion for an increase of GDP. Indeed oil spill
has resulted in some environmental disasters throughout
the world. For example, the Lakeview Gusher spill in
California reached up to 1 200 000 tons of crude oil from
May 1910 to September 1911, which was the largest spill
in the world history [1]. Since the late of 1970s, there has
been a number of large spills [2,3] around the world (Ta -
ble 1) and the consequence of oil spill pollution can last
decades. For instance, the smallest spill among 13 major
spills occurred in 1989 by the Exxon Valdez in Prince
William Sound of Alaska caused about 100 tons of oil
still remained in the beaches of Prince William Sound as
of 2001 [4]. The incident of oil spill from BP in the Gulf
Coast of Mexico from April to July, 2010, has caused
almost 600 000 tons of crude oil spilled along the Gulf
Coast. The total amount of all major spills was as much
as 37 billion barrels of crude oil, which exceeds the total
amount of crude oil consumption for the entire world
annually (30 billion barrels in 2006) [5]. All these spills
have caused tremendous damage to ecological and envi-
ronmental systems, especially to many plant species, a
wide array of animals, human health, which resulted in
the alteration of the coast aesthetics for tourism and rec-
reations.
Oil spills can leave a legacy for decades, even centu-
ries into the future. The deaths of marine animals and
migratory birds and impacts of their losses on marine
ecosystems, and on human health effects by oil spills are
difficult to evaluate [6]. Cleanup efforts will require
decades of dedicated work, and conversion of a toxic
enironment to a healthy one will need long time and v

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution 48
Table 1. Major oil spills in the world by order of quantity [2].
Date Location Spill type Tons of crude oil Barrels
5/14/1910–9/10/1911 U.S. Kern County, California Lakeview Gusher 1 200 000 9 000 000
4/20–7/15, 2010 U.S. Gulf of Mexico Deepwater horizon 560 000 – 585 000 4 100 000 – 4 300 000
1/23/1991 Iraq, Persian Gulf and Kuwait Gulf War oil spill 270 000 – 820 000 2 000 000 – 6 000 000
6/3/1979–3/23/1980 Mexico, Gulf of Mexico Ixtoc I 454 000 – 480 000 3 329 000- 3 520 000
7/19/1979 Trinidad and Tobago Atlantic Empress/Aegean Captain287 000 2 105 000
3/2/1992 Uzbekistan Fergana Valley 285 000 2 090 000
2/4/1983 Iran, Persian Gulf Nowruz Field Platform 260 000 1 907 000
5/28/1991 Angola ABT Summer offshore 260 000 1 907 000
8/6/1983 South Africa, Saldanha Bay Castillo de Bellver 252 000 1 848 000
3/16/1978 France, Brittany Amoco Cadiz 223 000 1 635 000
4/11/1991 Italy, Mediterranean Sea Near GenoaMT Haven 144 000 1 056 000
11/10/1988 Canada Odyssey 132 000 968 000
3/24/1989 U.S. Prince William Sound, Alaska Exxon Valdez oil tanker 35 065 – 103 896 257 000 – 750 000 [3]
cost tremendously. Toxicity of crude oil often includes
necrosis and congestion of the liver, fat degeneration,
and dissociation of hepatocytes. Birds and animals in oil
contaminated area usually have black emulsion in the
digestive tract with a petroleum odor, which leads to de-
crease in the absorption of nutrients and eventually to
death due to a series of consequences, such as rupture of
capillaries and hemorrhage, hepatocellular dissociation,
hemosiderosis, renal tubular necrosis, and anemia [7].
Crude oil consists of a number of rather complicated
components, which are toxic and can excert side effects
on the environment and ecological systems. For instance,
the aromatics in crude oil produce particular adverse ef-
fects to the local microbial flora. It was found that
α-pinene, limonene, camphene, and isobornyl acetate
were inhibitory to microorganisms. Phenolic and quinonic
naphthalene derivatives inhibited the growth of the mi-
crobial cells [8]. Increase of naphthalene, 2-methyl-
naphthalene and pyrene can cause prolonged lag phase
and reduce the growth rates of two bacteria [9]. In terms
of the toxic effects of cyclohexane on the energy trans-
duction in saccharomyces cerevisiae, cyclohexane inhib-
ited oxygen uptake in intact cells and isolated mitochon-
dria [10]. Therefore, immediate actions should be taken
to remove or remediate the contaminant after an acci-
dental spill. Physical cleanup is expensive, and the dis-
ruption to the habitats may result in an even worse im-
pact than the oil pollution itself, and such cleanup on the
floating water or within plant communities is almost im-
possible. Chemical approach, such as application of dis-
persion would cause environmental side effects. For ex-
ample, coral reef can be affected by crude oil and dis-
persants. Early developmental forms (like coral larvae)
are particularly sensitive to such toxic effects, and oil
slicks can significantly reduce larvae development and
viability [11]. Natural oxidation by weathering takes
decades due to the lack of oxygen and nutrients in the
water for microbial organisms.
Bioremediation through hydrocarbon biodegradation
using selected microbial organisms has provided a fa-
vorable opportunity because it is environmentally friendly
and cost effective. Those microbial species or particular
strains can digest hydrocarbons and utilize the resulting
compound carbon as food and energy sources for growth
and reproduction. Simultaneously the hydrocarbons are
hydrolyzed from toxic and complicated organic com-
pounds into nontoxic and simple inorganic compounds,
such as CO2 and H2O along with microbial biomass ac-
cumulation, through oxidation under aerobic conditions.
Under certain circumstances, some anaerobic microbial
organisms can degrade hydrocarbons as well through
reduction. For instance, a benzene-tolerant strain, Fla-
vobacterium sp. DS-711 isolated from deep-sea sedi-
ments of 1 945 m had as much as more than 90% of
n-alkanes in kerosene degraded [12]. However, oxidation
by aerobic microbial organisms has been commonly con-
sidered to be a predominant and effective approach to the
hydrocarbon degradation. In addition, under anaerobic
conditions, some intermediate materials, such as fermen-
tation products, ethanol or methane (CH4) can be pro-
duced [13]. A large number of related studies, such as
specific microbial strain screen and isolation, bench- or
lab-scale trials under different environmental conditions,
i.e., pH and temperature, have been conducted with

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution49
promising results. However, the implementation of such
technology in a field scale is still very limited. A pilot in
situ trial by application of P. aeruginosa to degrade hy-
drocarbons for cleanup of the oil spilled from the Exxon
Valdez in Prince William Sound, Alaska [14] seemed
unsuccessful due to the low temperature and a lack of
essential nutrients for microbial growth and activities.
2. Isolation and Identification of Microbial
Strains to Selectively Degrade
Hydrocarbons
The petroleum crude oil usually consists of 83 - 87%
carbon and 10 - 14% hydrogen, 0.1 - 2% nitrogen, 0.1 -
1.5% oxygen, 0.5 - 6% sulfur, and < 0.1% metals [15].
The predominant hydrocarbons are theoretically degrad-
able but the components are rather complicated. It con-
tains aliphatic and polycyclic aromatic hydrocarbons
(PAH), for example, crude oil consists of paraffins 15 -
60%, naphthenes 30 - 60%, aromatics 3-30% and asphal-
tics 6% by weight [16]. Therefore, the specific microbial
strain screen and isolation need to be performed by sup-
plying petroleum crude oil or a particular component, if
the selective strain is going to be utilized to degrade that
component, as carbon for food and energy source with
mineral nutrients essentially to build up the microbial
biomass.
To isolate a microbial hydrocarbon degrader, a com-
mon approach is to use an enrichment culture system in
which the candidate strain from oil contaminated water
or soil samples is cultured in mineral salts medium
(MSM) consisting of essential macro- and micro-nutrients
(0.4% NH4NO3, 0.47% KH2PO4, 0.0119% Na2HPO4,
0.001% CaCl2·2H2O, 0.1% MgSO4·7H2O, 0.001%
MnSO4·4H2O, and 0.0015 FeSO4·4H2O at pH 7.0 with
phosphate buffer) [17]. Basically, microbial organisms
are transferred from samples collected to the above MSM
medium and cultured at 30˚C in a rotary shaker at 150
rpm with 0.1% yeast extract until turbid growth is ob-
served. The bacterial culture is diluted and spread on
MSM agar plates containing crude oil (1%) as carbon
source for selective isolation of petroleum degrader. The
plates are sealed and incubated at 30˚C until appearance
of several colonies. Individual colonies can be purified
by repeating the culture on MSM agar plates containing
1% crude oil. Identification of the candidate strains will
be performed based on physiological and biochemical
tests, or 16S rRNA sequencing.
Physiological and biochemical tests: using pheno-
typic and biochemical characterizations to identify spe-
cific strains can be performed as described by Yumoto et
al. [18].
DNA base composition and DNA-DNA hybridiza-
tion: The DNA content isolated from bacterial cells can
be determined fluorometrically using photo-biotin-labeled
DNA probes and microplates [19] and compared with
strains in the national collection of microorganisms. For
such an approach, Pseudomonas aeruginosa JCM596 2T
and Serratia marcescens JCM 1239T can serve as refer-
ence strains.
Phylog enetic analysis using 16S rRNA gene sequence
comparison: Almost the full length of 16S rRNA genes
of bacteria has been amplified by PCR with following
sets of primers 5’-GAGTTTGATCCTGGCTCAG-3’ and
5’-AAGGAGGTGATCCAGCC-3’ corresponding to the
positions 9 to 27 and 1525 to 1541, respectively [17].
3. Monitoring and Evaluation of
Biodegradation Rate of Hydrocarbons
To evaluate the biodegradation process of various hy-
drocarbons, cultured media can be extracted at certain
time intervals with dichloromethane. The extract can be
analyzed by a gas chromatography (GC), GC-MS (mass
spectrometer), gas-liquid chromatography (GLC), or high
performance liquid chromatography (HPLC).
With GC analysis, the carrier gas is helium, the injec-
tor and detector temperatures are set at 250˚C and 300˚C,
respectively, for analysis of total petroleum hydrocarbons
(TPH). However, for gasoline analysis, Wongsa et al. [17]
suggested that the column temperature is first maintained
at 35˚C for 5 min, then increased to 220˚C; for diesel oil
analysis, the column temperature is set at 50˚C and then
ramped to 270˚C with a regime of 5˚C·min -1. In the case
of lubricating oil, 320˚C is needed for both injector and
detector temperatures, and the column temperature can
be set at 100˚C initially and ramped to 320˚C at a rate of
10˚C·min -1.
For GC-MS analysis, resulting chromatograms can be
analyzed by various particular software packages, such
as Saturn Software GC/MS Workstation Version 5.52, to
identify petroleum components after the spectra have
been obtained at temperatures similar to those described
in the GC analysis. Detailed information has been pro-
vided elsewhere by other researchers [20].
4. Potential Approaches to Improving
Biodegradation of Hydrocarbons and
Bioremediation of Oil Pollutants
4.1. Performances of Various Bacterial Strains
Bioremediation approaches, i.e. using selected indige-
nous microbial organisms to degrade hydrocarbons, are
currently receiving favorable publicity because bioreme-
diation is environment friendly and cost effective.
Among those microorganisms, the genus Pseudomnas,
particularly P. putida F1 is one of the most well-studied
hydrocarbon degrading bacterial strains, this strain and

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution 50
several others are commercially available (ATCC, The
Essentials of Life Science Research), having approved
the capability to utilize organic compounds from the ge-
neric group aliphatic, cyclo-aliphatic, aromatic and/or
polynuclear aromatic hydrocarbons. Pseudomnas can
facilitate the degradation and utilization of carbon de-
rived from complicated compounds as its food and en-
ergy sources with metabolic plasmids. The strains iso-
lated from contaminated sites [14,21,22] have shown
promising potential in biodegradation of hydrocarbons
and bioremediation of contaminated sites mostly in
bench scale studies. For instance, the bioremediation rate
of naphthalene using P. putida can reach as high as 61
mg L-1 hr-1 [23]. Strains of P. aeruginosa WatG and Ser-
ratia marcescens HokM, isolated in Japan [17], showed
that about 90-95% of diesel oil and kerosene can be de-
graded within 2 and 3 weeks, and petroleum hydrocarbon
(TPH) can be degraded by 72% in 4 weeks (Figure 1).
Moriya and Horikoshi [12] reported that a strain of Fla-
vobacterium sp. named DS-711 isolated from deep sea
sediment degraded 90% of n-alkane in kerosene, which is
greater than toluene-tolerant P. putida. A strain of Bacil-
lus subtilis is a good degrader of both hydrocarbons with
degradability of 98% n-hexadecane and 75% naphthalene
[24].
4.2. Biosurfactant Production
To effectively degrade hydrocarbons of crude oil, emul-
sification with a surfactant is of importance due to their
low water solubility, especially polyaromatic compo-
nents in solid and liquid discharges of petroleum. Some
strains, such as P. putida, and B. subtilis, can produce
rhamnolipid biosurfactant, which can dramatically en-
hance aqueous dispersion via emulsification, and stimu-
late the biodegradation of organic compounds [25,26].
The emulsification plays an important role in the degra-
dation of organic compounds, especially for polyaro-
matic hydrocarbons, such as naphthalene and n-paraffin
fractions, and such emulsification usually can be ob-
served in 24-48 hrs after inoculation with some effective
microbial strains [27]. Other researchers indicated that
the dissolution rates for hydrophobic particles into the
culture media during the bioremediation process were up
to 4 times greater compared to mass transfer rates into
abiotic controls due to the production of biosurfactant by
P. putida [23].
4.3. Application of Bacterial Consortium
Recent research indicates that use of mixed bacterial
consortium is more efficient in biodegradation of crude
oil than individual bacterial strains. For example, ac-
cording to Sathish Kumar et al. [28], the mixed consor-
tium of four bacterial strains degraded a maximum of
77% crude oil, followed by 69% by Pseudomonas sp.
BPS1-8, 64% by Bacillus sp. ISS1-7, 45% by Pseudo-
monas sp. HPS2-5, and 41% by Gorynebacterium sp.
BPS2-6 at 1% crude oil concentration. Increasing crude
oil concentration from 1 to 12%, the degradation rate by
the same consortium was decreased but still reached up
to 45%.
Figure 1. Biodegradation of different hydrocarbon compounds of petroleum products by a selective strain, WatG of Pseudo-
monas aeruginosa (adapted from Wongsa et al.) [17].

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution51
4.4. Biodegradation Time and Efficiency
Regarding the degradation time, it varies from one re-
searcher to the others. For instance, Atlas and Bartha [29]
observed that the degradation started after a 2 to 4 day
lag period, and reached its maximum within 2 weeks
with their continuous monitoring on CO2 evolution. At
this time of degradation, up to 60% of the crude oil and
75% of the model hydrocarbon mixture were degraded
by Flavobacterium sp. and Brevibacterium sp. isolated
from coastal waters after each was added at the final
level of 1 ml per 100 ml artificial sea water. Berwick [30]
indicated that about 98% of the solvent (carbon tetra-
chloride) extractable oil was degraded over 83 days and
the degradation in percent was in the following order:
aromatics > saturates > heterocyclics > asphalts, but the
degradation rate for any of these fractions was above
94%.
4.5. Immobilizing Microbial Cells
Hydrocarbon biodegradation can be significantly im-
proved by immobilizing microbial cells. For example,
using the bacterial consortium MPD-M isolated from
sediments associated with Colombian mangrove roots
can effectively degrade hydrocarbons in water with sa-
linities varying from 0 to 180 g/L. However, the effec-
tiveness was 4 and 7 times greater with immobilized cells
on polypropylene fibers compared to free living cells
[31]. Moslemy et al. [32] demonstrated that an enriched
bacterial consortium encapsulated in gellan gum mi-
crobeads (16-53 µm dia.) at the rate of 2.6 mg·cells/g
bead degraded over 90% gasoline hydrocarbons within
5-10 days for the initial concentration of 50-600 mg/L.
However, degrading the same amount of gasoline hy-
drocarbons by free cells at equivalent levels required as
long as 30 days. The improved effectiveness of encapsu-
lation may result from potentially reducing biotic and
abiotic stresses, providing a number of advantages, in-
cluding protecting cells from the toxic effects of hazard-
ous compounds [33-35], and increasing their survival and
metabolic activities [36,37].
4.6. Potential Implementation and in Situ
Application
Successful implementations of crude oil biodegradation
in the field scale have been reported. Bacterial strains
such as P. aeruginosa have been applied to degrade hy-
drocarbons since 1989 for cleanup of the oil spilled from
the Exxon Valdez in Prince William Sound, Alaska [14].
Efficient enzymes extracted from the bacterium Bacillus
cereus DQ01 isolated from oil fields have been proven to
digest the hydrocarbon, n-hexadecane [38]. Alcanivorax
borkumensis is a recently discovered hydrocarbonoclastic
bacterium. According to Martin dos Santos et al. [39], it
might be the most important global oil degrader discov-
ered up to date.
4.7. Impacts of Environmental Conditions and/or
Abiotic Factors on Biodegradation
The mechanism whereby microorganisms degrade hy-
drocarbons has not been fully elucidated. It is known that
some selective strains possess a capability to tolerate a
certain concentration of hydrocarbons and can utilize
them as their carbon and energy sources for growth and
reproduction. Therefore, the end products of hydrocarbon
biodegradation under aerobic conditions are simply CO2,
H2O and the accumulation of microbial biomass. Since
the oxidation or hydrolysis of those hydrocarbons is ac-
complished by microbes, any factors influencing micro-
bial growth and activities can definitely impact the bio-
degradation rate and the effectiveness. Moreover certain
abiotic conditions such as temperature, pH, oxygen sup-
ply and nutrient balance, have been proved to play a cru-
cial role in the oxidation and hydrolysis involved. For
instance, temperature of 35˚C and pH 7 were found to be
optimum for maximum degradation of crude oil by a
consortium of Pseudomonas strains [28]. Mittal and
Singh [40] observed that using degrading consortia of
Pseudomonas sp. for bioaugmentation of polycyclic
aromatic hydrocarbon (PAH) of polluted soil with addi-
tion of nutrients and other environmental factors, i.e.,
tilling (aeration) resulted in 79% removal of PAH in 60
days, while only 30% removal was achieved by indige-
nous microflora alone. The result was obtained from the
nutrient ratio of C:N:P in 120:10:1 based on Gibb’s for-
mula (assuming crude oil contains 78% carbon) but the
optimal ratio is unknown. Oxygen and nutrient supply
are important to optimize the microbial activity because
inhibition of biodegradation by nutrient or oxygen limi-
tation or toxic effects exerted by volatile hydrocarbons
may occur due to high concentrations of undispersed
hydrocarbons in water [28].
The evidence in improvement of hydrocarbon or crude
oil biodegradation by aeration or oxygen supply has been
observed by a number of researchers [41-44]. The addi-
tion of nutrients adjusts the essential nutrient balance for
microbial growth and reproduction, and oxygen supply
can maintain the aerobic environmental condition to
stimulate the microbial oxidation and hydrolysis of hy-
drocarbon compounds. As matter of fact, in soil or water
with high levels of hydrocarbon contamination, the oxy-
gen demand often exceeds the supply, which is the rea-
son that the amount of oxygen supply was the most im-
portant single factor affecting biodegradation of petro-
leum [45].

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution 52
4.8. Oxygen Supply and Nutrient Balancing to
Improve Hydrocarbon Biodegradation for
Bioremediation of Oil Pollutants
Both biotic and abiotic conditions play a crucial role in
improvement of the bioremediation efficiency due to
optimization of function of indigenous microbial strains.
With application of efficient strains isolated, abiotic con-
dition improvement can definitely increase the biodegra-
dation efficiency and effectiveness. Among which, tem-
perature and pH are adjustable factors in a bench scale
trial but unrealistic to be implemented with an in situ
approach. Nutrient balancing, especially the supply of
essential nutrients such as N and P can improve the bio-
degradation efficiency by optimizing the bacterial C:N:P
ratio. However, appropriate rate and application method
are of great importance as insufficient rate cannot bal-
ance the nutrient ratio but excess amount will produce
toxic effects to microbial organisms. In addition, the im-
pact of nutrient supply on water quality may bring public
concerns on a possible eutrophication risk. The limited
quantity of supply is required only to provide an appro-
priate rate to microbial organisms for their growth and
reproduction. Therefore, it is urgently needed to study
the bacterial consumption rate and the optimum ratio of
such nutrients required by efficient microbial strains.
Nutrient fate and consequences of biodegradation of hy-
drocarbons associated with nutrient supply to influence
the environment and water quality should be monitored.
Also, to prevent toxic effects on microbial organisms by
abrupt supply of these nutrients, slow release controlled
by polymers can be an ideal approach as it provides pro-
long and constant supply of nutrients to microbes. Oxy-
gen supply has been evidently proved to be a stimulating
approach to assist microbial degradation of hydrocarbons
[46] because oxidation and hydrolysis are primary pro-
cedures to break down the complicated hydrocarbon
compounds to be utilized by microbial organisms for
food.
Regarding oxygen source, it requires a slow but a con-
sistent supply to create an aerobic environment for the
microbial strains. The introduction of solid peroxygen
materials provides a viable alternative for meeting the
oxygen demand by microbial organisms [46]. These ma-
terials are primarily peroxide salts of calcium and mag-
nesium: CaO2 and MgO2, which can release oxygen at
enhanced levels over extended time periods as described
below:
CaO2 + 2 H2O → Ca (OH)2 + H2O2
2 H2O2 → O2 + 2 H2O
MgO2 + H2O → 1/2 O2 + Mg (OH)2
Indeed, calcium or magnesium peroxide is a powerful
oxidizing agent and it breaks down into lime, water and
oxygen, which does not form any persistent, toxic resid-
ual compounds. The other ideal oxide material is calcium
superoxide, CaO4 or Ca(O2)2, which contains higher per-
centage of stored oxygen than CaO2, which has been
used in emergency breathing apparatus for miners and as
auxiliary oxygen sources for astronauts. Both of them
have similar physical and chemical characteristics but the
former can release a double amount of O2 with water
compared to the latter with the following reaction [47,
48]:
CaO4 + xH2O → CaO2·xH2O + O2
↓
Ca(OH)2 + xH2O2
↓
O2 + xH2O
From which, double amount of oxygen can be released
from CaO4 as compared to CaO2 to meet the microbial
requirement for oxygen but there is no toxic substance
produced. In addition, to avoid possible toxic effects by
CaO4, polymers can be applied to separate each compo-
nent to prevent direct contacts of microbial organisms
with calcium superoxide. The production of CaO4 can be
accomplished by the reaction of potassium superoxide
(KO2), which is commercially available, with calcium
chloride (CaCl2) in accordance with the following equa-
tion [49]:
2KO2 + CaCl2 → CaO4 + 2KCl
Therefore, to improve the hydrocarbon degradation ef-
ficiency and effectiveness, it is important to utilize in-
digenous microbial strains isolated specifically for crude
oil degradation that are commercially available, or iden-
tified and isolated from enrichment culture and screening.
Apply solid oxygen materials, such as calcium superox-
ide, and adjust nutrient balance, especially to optimize
C:N:P ratio for efficient degradation of hydrocarbons of
crude oil under optimum environmental conditions, such
as appropriate temperature and pH values in the media.
Regarding the nutrient supply, the controlled release
substrate with optimal ratio can provide the microbial
organisms a slow and constant nutrient supply under the
controlled conditions. Previous studies have displayed
that the addition of essential metabolic nutrients, N and P,
to oil contaminated beaches is an effective approach for
stimulating bioremediation of oil pollutants by indige-
nous microbial biomass [50-52]. However, the applica-
tion of excessive amount of nutrients over metabolic
needs by microbes can result in extra bioremediation
costs and potential marine eutrophication impacts [53].
The use of slow release nutrients with an appropriate rate
may provide a continuous nutrient supply by maintaining
a sufficient nutrient status for the perpetuation of micro-
bial metabolic activities without causing environmental

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution53
concerns and save the cost [54]. Xu et al. [55] found out
that an addition of 0.8% of slow-release fertilizer, Os-
moc o te TM consisting of 18, 4.8, and 8.3% N-P-K (w/w)
to oil polluted sediments was sufficient to maximize
metabolic activity of the microbial biomass and the bio-
degradation of straight-chain alkanes (C10-C33); and ap-
plication of 1.5% rate resulted in optimal biodegradation
of recalcitrant branched-chain alkanes, such as pristine
and phytane. The application of soluble nutrients to the
oil contaminated sites has shown an especially promising
potential of the indigenous microbial organisms for
stimulating biodegradation of petroleum hydrocarbons in
the tropical environment of Singapore with high tem-
perature and humidity [56]. Therefore, to improve the
biodegradation efficiency and implementation, integrat-
ing various components, such as microbial strains in
consortium, solid oxygen source, appropriate rate of nu-
trients with controlled release pattern, into a granule
formulation with an oleophilic matrix, may provide an
ideal approach to improving bioremediation of crude oil
pollutants.
REFERENCES
[1] S. Harvey, “California’s legendary oil spill,” Los Angeles
Times, 13 June 2010.
http://articles.latimes.com/2010/jun/13/local/la-me-then-2
0100613
[2] Anonymous, “Oil spill,”2010.
http://en.wikipedia.org/wiki/oil_spill
[3] E. Bluemink, “Size of Exxon Spill Remains Disputed,”
Anchorage Daily News, 2010.
http://www.adn.com/2010/06/05/1309722/size-of/esxon-s
pll-remains-disputed.html
[4] B. Marsh, “Even with a Cleanup, Spilled Oil Stays with
Us,” The New York Time s , 6 June 2010.
[5] I. Mundi, “World Crude Oil Consumption by Year”, 2010.
http://www.indexmundi.com/energy. aspx
[6] J. T. Tevvors and M. H. Jr. Saier, “The Legacy of Oil
Spills,” Water, Air and Soil Pollution, Vol. 211, No. 1,
2010, pp. 1-3. doi:10.1007/s11270-010-0527-5
[7] R. A. Khan and P. Ryan, “Long Term Effects of Crude
Oil on Common Murres (Uria Eagle) Following Reha-
bilitation,” Bulletin of Environmental Contamination and
Toxicology, Vol. 46, No. 2, 1991, pp. 216-222.
doi:10.1007/BF01691940
[8] J. Sikkema, A. M. deBont and B. Poolman, “Mechanisms
of Membrane Toxicity of Hydrocarbons,” Microbial Re-
view, Vol. 59, 1995, pp. 201-222.
[9] J. A. Calder and J. H. Lader, “Effect of Dissolved Aro-
matic Hydrocarbons on the Growth of Marine Bacteria in
Batch Culture,” Applied Environmental Microbiology,
Vol. 32, No. 1, 1976, pp. 95-101.
[10] S. Uribe, P. Rangel, G. Espinotal and G. Aguirre, “Effects
of Cyclohexane, an Industrial Solvent, on the Yeast Sac-
charomyces Cerevisiae and on Isolated Yeast Mitochon-
dria,” Applied Environmental Microbiology, Vol. 56,
1990, pp. 2114-2119.
[11] C. Hansen, “Coral Reefs and Oil Spills: What You Might
not Know,” The Sticky Tongue, Herpetology, Zoology
and Conservation, 2010.
http://thestickytongue.com/2010/08/02/coral-reefs-and-oil
-spills-what-you-might-not-know.
[12] K. Moriya and K. Horikoshi, “Isolation of Ben-
zene-Tolerant Bacterium and Its Hydrocarbon Degrada-
tion,” Journal of Fermentation and Bioengineering, Vol.
76, 1993, No. 3, pp. 168-173.
[13] E. A. Paul and F.E. Clark, “Soil Microbiology and Bio-
chemistry,” 2nd Ed., Academic Press, San Diego, 1996.
[14] S. Harvey, I. Elashvilli, J. J. Valdes, D. Kamely and A. M.
Chakrabarty, “Enhanced Removal of Exxon Valdez
Spilled Oil from Alaskan Gravel by a Microbial Surfac-
tant,” Nature Biotechnology, Vol. 8, No. 3, 1990, pp.
228-230. doi:10.1038/nbt0390-228
[15] J. G. Speight, “The Chemistry and Technology of Petro-
leum,” Marcel Dekker, New York, 1999.
[16] N. J. Hyne, “Nontechnical Guide to Petroleum Geology,
Exploration, Drilling, and Production,” PennWell Corpo-
ration, Tulsa, 2001.
[17] P. Wongsa, M. Tanaka, A. Ueno, M. Hasanuzzaman, I.
Yumoto and H. Okuyama, “Isolation and Characterization
of Novel Strains of Pseudomonas Aeruginosa and Serra-
tia Marcescens Possessing High Efficiency to Degrade
Gasoline, Kerosene, Diesel Oil and Lubricating Oil,”
Current Microbiology, Vol. 49, 2004, pp. 415-422.
doi:10.1007/s00284-004-4347-y
[18] I. Yumoto, T. Kusano, T. Shingyo, Y. Nodasaka, H. Ma-
tsuyama and H. Okuyama, “Assignment of Pseudomonas
sp. Strain E-3 to Pseudomonas Psychrophila Sp. Nov., a
New Facultatively Psychrophilic Bacterium,” Estremo-
philes, Vol. 5, No. 5, 2001, pp. 343-349.
[19] T. Ezaki, Y. Hashimoto and E. Yabuuchi, “Fluorometric
Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridi-
zation in Microdilution Wells as an Alternative to Mem-
brane Filter Hybridization in Which Radioisotopes are
Used to Determine Genetic Relatedness among Bacterial
Strains,” International Journal in Systematic Bacteriol-
ogy, Vol. 39, No. 3, 1989, pp. 224-229.
doi:10.1099/00207713-39-3-224
[20] S. H. Tzing, J. Y. Chang, A. Ghule, J. J. Chang, B. Lo
and Y. C. Ling, “A Simple and Rapid Method for Identi-
fying the Source of Spilled Oil Using an Electronic Nose:
Confirmation by Gas Chromatography with Mass Spec-
trometry,” Rapid Communication in Mass Spectrometry,
Vol.17, No. 16, 2003, pp. 1873-1880.
doi:10.1002/rcm.1127
[21] J. Olsen, “Pseudomonas Degradation of Hydrocarbons,”
United States Patent # 4508824, United States Patent Of-
fice, 1985.
[22] R. G. Binder, K. Numata, D. A. Lowe, T. Murakami and
J. L. Brown, “Isolation and Characterization of a Pseu-
domonas Strain Producing Glutaryl-7-Aminocephalospo-

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution 54
ranic Acid Acylase,” Applied and Environmental Micro-
biology, Vol. 59, No. 10, 1993, pp. 3321-3326.
[23] R. Yu, M. Nemati, G. Hill and J. Haddley, “Mass Trans-
fer and Bioremediation of Naphthalene and Methyl
Naphthalenes in Baffled and Bead Mill Bioreactors,” The
Canadian Journal of Chemical Engineering, Vol. 84, No.
3, 2006, pp. 349-355. doi:10.1002/cjce.5450840311
[24] N. Christova, B. Tuleva and B. Nikolova-Damyanova,
“Enhanced Hydrocarbon Biodegradation by a Newly Iso-
loated Bacillus Subtilis Strain,” Zeitschrift für Naturfor-
schung, Vol. 59c, 2003, pp. 205-208.
[25] A. Oberbremer, R. Muller-Hurtig and F. Wagner, “Effect
of the Addition of Microbial Surfactants on Hydrocarbon
Degradation in a Soil Population in a Stirred Reactor,”
Applied Microbiology and Biotechnology, Vol. 32, No. 4,
1990, pp. 485-489. doi:10.1007/BF00903788
[26] Y. Zhang and R. M. Miller, “Enhanced Octadecane Dis-
persion and Biodegradation by a Pseudomonas Rham-
nolipid Surfactant (Biosurfactant),” Applied Environ-
mental Microbiology, Vol. 58, No. 10, 1992, pp. 3276-
3282.
[27] J. E. Zajic and B. Supplisson, “Emulsificaiton and Deg-
radation of ‘Bunker C’ Fuel Oil by Microorganisms,”
Biotechlogy and Bioengineering, Vol. 14, 1972, pp. 331-
343. doi:10.1002/bit.260140306
[28] M. Sathishkumar, A. R. Binupriya, S. H. Baik and S. E.
Yun, “Biodegradation of Crude Oil by Individual Bacte-
rial Strains and a Mixed Bacterial Consortium Isolated
from Hydrocarbon Contaminated Areas,” Clean, Vol. 36,
No. 1, 2008, pp. 92-96. doi:10.1002/bit.260140303
[29] R. M. Atlas and R. Bartha, “Degradation and Mineraliza-
tion of Petroleum by Two Bacteria Isolated from Coastal
Waters,” Biotechnology and Bioengineering, Vol. 14, No.
3, 1971, pp. 297-308.
[30] P. G. Berwick, “Physical and Chemical Conditions for
Microbial Oil Degradation,” Biotechnology And Bioengi-
neering, Vol. 26, No. 11, 1984, pp. 1294-1305.
doi:10.1002/bit.260261106
[31] M. P. Diaz, K. G. Boyd, S. J. W. Grigson and J. G. Bur-
gess, “Biodegradation of Crude Oil Across a Wide Range
of Salinities by an Extremely Halotolerant Bacterial
Consortium Mpd-M, Immobilized onto Polypropylene
Fibers,” Biotechnology and Bioengineering, Vol. 79, 2002,
pp. 145-153. doi:10.1002/bit.10318
[32] P. Moslemy, R. J. Neufeld and S. R. Guiot, “Biodegrada-
tion of Gasoline by Gellan Gum-Encapsulated Bacterial
Cells,” Biotechnology and Bioengineering, Vol. 80, No. 2,
2002, pp. 175-184. doi:10.1002/bit.10358
[33] H. Bettmann and H. J. Rehm, “Degradation of Phenol by
Polymer Entrapped Microorganisms,” Applied Microbi-
ology and Biotechnology, Vol. 20, No. 5, 1984, pp. 285-
290. doi:10.1007/BF00270587
[34] S. Manohar and T. B. Karegoudar, “Degradation of Naph-
thalene by Cells of Pseudomonas Sp. Strain Ngk 1 Im-
mobilized in Alginate, Agar and Polyacrylamid,” Applied
Microbiology and Biotechnology, Vol. 49, No. 6, 1998,
pp. 785-792. doi:10.1007/s002530051247
[35] M. L. Paje, P. Marks and I. Couperwhite, “Degradation of
Benzene by a Rhodococcus Sp. Using Immobilized Cell
Systems,” World Journal of Microbiology and Biotech-
nology, Vol. 14, 1998, pp. 675-680.
doi:10.1023/A:1008898922908
[36] B. M. Hall, A. J. CcLoughlin, K. T. Leung, J. T. Trevors
and H. Lee, “Transport and Survival of Alginate-En-
capsu-Lated and Free Lux-Lac Marked Pseudononas
Areruginousa UG2Lr Cells in Soil,” FEMS Microbiology
and Ecology, Vol. 26, 1998, pp. 51-61.
doi:10.1111/j.1574-6941.1998.tb01561.x
[37] T. Suzuki, T. Yamaguchi and M. Ishida, “Immobilization
of Prototheca Zopfi in Calcium-Alginate Beads for the
Degradation of Hydrocarbons,” Process of Biochemistry,
Vol. 33, 1998, pp. 541-546.
doi:10.1016/S0032-9592(98)00022-3
[38] H. Wang and Y. Chen, “The Microbial Hydrocarbon
Diet,” Science and Technology News, 2005.
http://www.physorg.com/news163937198.html
[39] V. A. P. Martins dos Santos, M. M. Yakimov, K. N.
Timmis and P. N. Golyshin, “Genomic Insights into Oil
Biodegradation in Marine Systems: Chapter 9,” E. Diaz
Ed., Microbial Biodegradation: Genomics and Molecular
Biology, Caister Academic Press, U.K., 2008.
[40] A. Mittal and P. Singh, “Polycyclic Aromatic Hydrocar-
bon Degradation by Developed Consortium in Micro-
cosms Study,” The Internet Journal of Microbiology, Vol.
7, No. 1, 2009, pp. 1-12.
[41] G. Soli and E. M. Bens, “Bacteria Which Attack Petro-
leum Hydrocarbons in a Saline Medium,” Biotechnology
and Bioengineering, Vol. 14, No. 3, 1971, pp. 319-330.
doi:10.1002/bit.260140305
[42] R. Atlas, “Microbial Hydrocarbon Degradation-Bioreme-
Diation of Oil Spills,” Journal of Chemical Technology &
Biotechnology, Vol. 52, No. 2, 1991, pp. 149-156.
doi:10.1002/jctb.280520202
[43] D. Miethe, V. Riis and W. Babel, “The Relationship be-
tween the Microbial Activity of the Autochthonos Micro-
organisms of Pristine and Contaminated Soils and Their
Potential for The Degradation of Mineral Oil Hydrocar-
bons,” Acta Biotechnologica, Vol. 14, No. 2, 1993, pp.
131-140. doi:10.1002/abio.370140204
[44] S. R. Peressutti, H. M. Alvarez and O. H. Pucci, “Dy-
namics of Hydrocarbon-Degrading Bacteriocenosis of an
Experiment on Oil Pollution in Patagonian Soil,” Interna-
tional Biodeterioration and Biodegradation, Vol. 52, No.
1, 2003, pp. 21-30. doi:10.1016/S0964-8305(02)00102-6
[45] J. S. Devinny and R. L. Islander, “Oxygen Limitation in
Land Treatment of Concentrated Wastes,” Hazardous
Waste And Hazardous Material, Vol. 6, No. 4, 1989, pp.
421-433. doi:10.1089/hwm.1989.6.421
[46] J. W. Davis, R. J. West and G. M. Kecka, “Enhanced
Land Treatment of Petroleum-Contaminated Soils Using
Solid Peroxygen Materials,” Remediation Journal, Vol. 7,
No. 2, 1997, pp. 67-81. doi:10.1002/rem.3440070205
[47] R. S. Johnston, E. D. Osgood and R. R. Miller, “Analysis
of Mixed Oxides of Calcium,” Analytical Chemistry, Vol.

Potential Approaches to Improving Biodegradation of Hydrocarbons for Bioremediation of Crude Oil Pollution 55
30, No. 4, 1958, pp. 511-513.
doi:10.1021/ac60136a018
[48] E. V. Ballou, P. C. Wood and L. Spitze, “The Preparation
of Calcium Superoxide from Calcium Peroxide Diper-
oxyhydrate,” Industrial and Engineering Chemistry
Product Research and Development, Vol. 16, No. 2, 1993,
pp. 180-186. doi:10.1021/i360062a015
[49] J. S. Hashman, E. City, J. C. Renforth and V. J. W. Berk-
stresser, “Production of Calcium Superoxide,” United
States Patent #3119665, United States Patent Office,
1964.
[50] R. M. Atlas and R. Bartha, “Hydrocarbon Biodegradation
and Oil Spill Bioremediation,” Advances in Microbial
Ecology, Vol. 12, 1992, 287-338.
[51] A. D. Venosa, “Oil Spill Bioremediation on Coastal
Shorelines: a Critique,” S. K. Sikdar and R. L. Irvine,
Eds., Bioremediation: Principles and Practice, Lancaster,
PA, 1998, pp. 280-296.
[52] C. R. Johnson and K.M. Scow, “Effect of Nitrogen and
Phosphorus Addition on Phenanthrene Biodegradation in
Four Soils,” Biodegradation, Vol. 10, No. 1, 1999, pp.
43-50. doi:10.1023/A:1008359606545
[53] J. T. Dibble and R. Bartha, “Effect of Environmental
Parametes on the Biodegradation of Oil Sludge,” Applied
and Environmental Microbiology, Vol. 37, No. 4, 1979,
pp. 729-739.
[54] E. Riser-Roberts, “Bioremediation of Petroleum Con-
taminated Sites,” Chelsea, MI, 1992, pp. A173-179.
[55] R. Xu, J. P. Obbard and E. T. C. Tay, “Optimization of
Slow-Release Fertilizer Dosage for Bioremediation of
Oil-Contaminated Beach Sediment in a Tropical Envi-
ronment,” World Journal of Microbiology and Biochem-
istry, Vol. 19, 2003, pp. 719-725.
doi:10.1023/A:1025116421986
[56] M. Mathew, J. P. Obbard, T. P. Ting, Y. H. Gin and H. M.
Tan, “Bioremediation of Oil Contaminated Beach Sedi-
ments Using Indigenous Microorganisms in Singapore,”
Acta Biotechnologica, Vol. 19, No. 3, 1999, pp. 225-233.
doi:10.1002/abio.370190309