REMEDIATION OF PETROLEUM HYDROCARBON

POLLUTED SYSTEMS

ABSTRACT
The irrepressible quest for a cheap source of energy to meet the extensive global
industrialization demand has expanded the frontiers of petroleum hydrocarbon
exploration. These exploration activities amongst others often result in pollution of the
environment, thus creating serious imbalance in the biotic and abiotic regimes of the
ecosystem. Several remediation alternatives have been in use for the restoration of
petroleum hydrocarbon polluted systems. In this paper, we present an overview of
bioremediation alternative vis-à-vis other cleanup methods and its adaptations in various
polluted systems.
INTRODUCTION
Accidental and deliberate crude oil spills have been, and still continue to be, a
significant source of environmental pollution, and poses a serious environmental
problem, due to the possibility of air, water and soil contamination (Trindade et al.,
2005). For example, approx. 6 ´107 barrels of oil was spread over 2 ´107 m3 soil and 320
oil lakes were created across the desert during the first Gulf War in Kuwait (Al-Saleh and
Obuekwe, 2005). The processes leading to the eventual removal of hydrocarbon
pollutants from the environment has been extensively documented and involves the trio
of physical, chemical and biological alternatives. However, bioremediation which is
defined as any process that uses microorganisms or their enzymes to return the
environment altered by contaminants to its original condition, is an attractive process due
to its cost effectiveness and the benefit of pollutant mineralization to CO2 and H2O (da
Cunha, 1996). It also provides highly efficient and environmentally safe cleanup tools
(Margesin,2000). This technology accelerates the naturally occurring biodegradation
under optimized conditions such as oxygen supply, temperature, pH, the presence or
addition of suitable microbial population (bioaugmentation) and nutrients
(biostimulation), water content and mixing (Trindade et al., 2005). In this paper, we
present an overview of bioremediation alternative vis-à-vis other cleanup methods, and
its adaptations in various polluted systems.
BIOREMEDIATION TECHNOLOGY
Simply defined, bioremediation is the use of biological systems to destroy or
reduce the concentrations of hazardous wastes from contaminated sites. Such systems
have the potentially broad-spectrum site applications including ground water, soils,
lagoons, sludge and process waste-streams, and it has been used in very large scale
applications such as the shoreline cleanup efforts in Alaska, resulting from the oil tanker
“Exxon Valdez” oil spill in 1989 (Caplan, 1993). Bioremediation strategy can be as
simple as applying a garden fertilizer to an oil-contaminated beach, or as complex as an
engineered treatment “cell” where soils or other media are manipulated, aerated, heated,
or treated with various chemical compounds to promote degradation (Hildebrandt and
Wilson, 1991). The bioremediation strategy of choice ultimately will depend on the
peculiarity of the contaminated site. Many published articles have documented the
potentials of microorganisms to degrade oil both in the laboratory and in field trials. A
number of the scientific papers including several review articles covered aspects of the
biodegradation process as well as results from controlled field experiments designed to
evaluate degradation rates in various environments (Gunkel and Gassmann, 1980; Atlas,
1981; Halmos, 1985). Furthermore, some studies carried out following major oil spills
like the Amoco Cadiz have assessed oil degradation in the environment and confirmed
the reliability of bioremediation process. Crude oil is a complex but biodegradable
mixture of hydrocarbons, and the observation that hydrocarbon degraders can be enriched
in many, if not most, types of environments (Atlas, 1981) have contributed to the
development of oil bioremediation techniques (Margesin and Schinner, 1997). Although
the optimum temperature for biodegradation of petroleum products has generally been
found to be in the range of 20 – 30oC (Atlas and Bartha, 1992), local environmental
conditions may select for a population with a varying optimum temperature.
CONDITION FOR BIOREMEDIATION
 Bioremediation technology accelerates the naturally occurring biodegradation
under optimized conditions such as oxygen supply, temperature, pH, presence or addition
of suitable microbial population (bioaugmentation) and nutrients (biostimulation),water
content and mixing (Trindade et al., 2005). At specific sites where the contaminants are
petroleum products, the spectrum of necessary professional expertise is greatly expanded.
Moreover, the microbial composition, contaminant type, geology of polluted site and
chemical conditions at the contaminated site are of great importance in bioremediation.
(Aichberger et al., 2005).
The various classifications of microbes are shown in the diagrams below.

Microbes’ classification by temperatures.

Microbes’ classification by pH.

Microbe classification by Water Activity.

BIOAUGMENTATION AND BIOSTIMULATION

 Bioaugmentation can be defined as the introduction of a large number of
exogenous microorganisms into the environment of a biotreatment. Diverse
microorganisms, including many species of bacteria and fungi are known to degrade
hydrocarbons. The most prevalent bacterial hydrocarbon degraders belong to the genera
Pseudomonas, Achromobacter, Flavobacterium, Rhodococcus, and Acinetobacter.
Penicillium, Aspergillus, Fusarium, and Cladosporium are most frequently isolated
hydrocarbon degrading filamentous fungi. Among the yeasts Candida, Rhodotorula,
Aureobasidium, and Sporobolomyces are the hydrocarbon degraders most often reported
(Van Hamme et al. 2003). Environmental and nutritional factors influence the presence,
survival, or activity of microorganisms in contaminated soils. There are at least four
different routes that result in the development of microbes capable of degradation of
hydrocarbons at a certain site:
1. The indigenous microflora is exposed to the contaminant long enough for genetic
evolution to create a capacity to degrade the compound(s).
2. The indigenous microflora, adapted to the local conditions, is exposed to one or more
contaminating xenobiotic compounds. The bacteria acquire genes and degradation
pathways from bacterial cells immigrating from elsewhere.
3. The indigenous, well-adapted microflora is maintained ex-situ and then artificially
supplied with the required degradative capacity.
4. A bacterium that is thought to be competitive at the contaminated site is chosen. This
may be a strain that is known to degrade the contaminant or one that is specifically
constructed for this purpose.

Meanwhile, biostimulation in the other hand is the addition of nutrients to aid in

the growth of the indigenous microbe population. Major nutrients: carbon, nitrogen,
phosphorous, oxygen, and water (A major source of this is NPK fertilizer). Oxygen
supply and an appropriate temperature for the microbes. Nutrients must be available and
in contact with microbes to ensure and facilitate the microbial activities.
BIODEGRADATION TRANSFORMATION MECHANISMS
 Numerous microorganisms use HCs as a source of carbon and energy, however,
aerobic and anaerobic species have different enzymatic systems and metabolic pathways
for degradation of HCs. It is important to note that, for the majority of microorganisms,
the rate of biodegradation decreases in this order: n-alkanes, simple aromatic HCs
(benzene, toluene, etc.), branched alkanes, cycloalkanes, isoprenes and the condensed
polyaromatic HCs. (Heath et al. 1997). The recent review of Van Hamme et al (2003)
gives a comprehensive picture of the latest advances in petroleum microbiology. Below
we will point out only major mechanisms of biodegradation of various HCs.
In most cases, degradation of aliphatic HCs begins with an oxidation of
extraterminal methyl groups to primary alcohol groups, though intraterminal oxidation
has been also described (Gottshalk 1982). The primary alcohols are then oxidized to
aldehydes, which, in turn, under action of NAD dependent dehydrogenase are oxidized to
corresponding fatty acids. These are degraded by b-oxidation or are used by the cells as a
building material (Gottshalk 1982; Sharma and Pant 2000). Anaerobic microorganisms
can also degrade aliphatic HCs. The principal step of anaerobic alkane degradation, as
well as anaerobic degradation of aromatic HCs, is the carboxylation of substrate
molecules (Vasu et al. 1977; So and Young 1999). Various substances can act as carbon
donors, which is included in carboxylic group formed, namely: fumarate (when
degrading toluene, xylene and alkanes), bicarbonate (when degrading naphthalene and
alkanes) etc. (Young and Phelps 2005). Further decomposition proceeds via the known b-
oxidation pathway (Gottshalk 1982).

Mechanism of n-alkanes biotransformation by microorganisms is presented in the

figure below.
The mechanism of anaerobic degradation of alkanes (So and Young 1999; Young and Phelps 2005)

It is interesting also to have a brief look on mechanism of cycloalkanes

biodegradation because the strains capable of utilizing these substrates (Gordonia,
Xanthobacter) have specific enzymatic systems differing from those used by
microorganisms for acyclic alkane oxidation. The pathway of cycloalkane degradation
was studied and presented (Cerniglia and Yang 1984; Tadashi et al. 2004) as shown
below.

The mechanism of bacterial degradation of cycloalkanes in aerobic conditions (Cerniglia and Yang 1984)

There are two basic strategies utilized by microorganisms to degrade aromatic

compounds. The first strategy is used by aerobic microorganisms and involves the
oxidation of the aromatic ring into dihydroxyaromatic compounds (catechol or
hydroquinone intermediates) with subsequent oxidizing cleavage of the aromatic ring.
Oxidative ring cleavage of the catechol intermediate can occur in two ways: intradiol (or
ortho) cleavage to give a muconic acid or extradiol (or meta) cleavage to give a
hydroxymuconaldehydic acid derivative (Bugg and Winfield 1998). Long aliphatic
substitute of aromatic compounds are decomposed by b-oxidation to shorter ones and the
formed intermediates undergo degradation of aromatic ring by one of mechanisms
outlined previously. The second strategy is used by anaerobic microorganisms and
involves the reduction of the aromatic ring with the subsequent defragmentation of
formed cycloalkane derivatives (Neidle et al. 1989; Mason and Cammack 1992; Asturias
and Timmis, 1993; Nakatsu and Wyndham 1993; Massey 1994; Haak et al. 1995; van der
Meer 1997).
The principal pathways of aromatic structure fission by aerobic microorganism

The principal stage of aromatic HCs destruction in anaerobic conditions is also

formation of carboxylic derivative of substrate. Again various substances can act as
carbon donors, which is included in carboxylic group formed, namely: fumarate (when
degrading toluol, xylol), bicarbonate (when degrading ethyl- and propylbenzene,
polyaromatic CHs) etc. (Young and Phelps 2005). Then reduction of aromatic ring with
further fragmentation of cyclohexane derivatives occur. Anaerobic biodegradation of
aromatic structure is presented to demonstrates ethylbenzene and toluene degradation
using bicarbonate and fumarate as carbon donor for carboxylic group formation.
The pathway of Ethylbenzene degradation using bicarbonate as carbon

The pathway of anaerobic aromatic hydrocarbons degradation (Rabus et al. 2002)

Likewise, some denitrifying strains transform aromatic substrates to benzoic acid and
then to benzoyl-CoA. The latter is further reduced to cyclohexenyl-CoA derivatives
which then are hydrolytically decomposed, and the formed products undergo b-oxidation
(Rabus et al. 2002; Young and Phelps 2005). Polyaromatic HCs are most recalcitrant
compounds of all the oil constituents (Heath et al. 1997).
There is a fundamental distinction in mechanisms of polyaromatic molecules fission by
microorganisms. Bacteria and some green algae oxidize polyaromatic hydrocarbons
(PAHs) using both atoms of the oxygen molecule (reaction catalyzed by dioxygenases),
thus cis-dihydrodiols are obtained which then are transformed to catechols by
dehydrogenation.
Some fungi oxidize PAHs by means of cytochrome P-450 monooxygenase by
incorporating only one atom of the oxygen molecule into PAH or by the action of
peroxidase, which in presence of H2O2 transforms PAH to aryl radical undergoing further
oxidation to form quinone. However, these fungi are most likely not involved in the
degradation of oil or fuels (Steffen et al. 2002).
There are essential differences in mechanisms of HC degradation by aerobic and
anaerobic microorganisms. Aerobic degradation begins with the oxidation of varying
groups of atoms of a substrate molecule by different enzyme systems. Unlike aerobic
processes, the mechanism of anaerobic HC degradation starts with attachment of some
groups of atoms to a substrate molecule.

The major pathway in the microbial metabolism of PAHs

 IN SITU AND EX SITU BIOREMEDIATION
Ex situ bioremediation is a different approach that utilizes specially
constructed treatment facility. It is more expensive than in situ bioremediation. In
Situ Bioremediation entails the creation of subsurface environmental conditions
conducive to the degradation of chemicals (i.e., the target chemical) via microbial
catalyzed biochemical reactions. This is a technical way of saying that certain microbes
can degrade specific chemicals in the subsurface by optimizing their environmental
conditions (which causes them to grow and reproduce) (Cookson, 1995). In turn, the
microbes produce enzymes that are utilized to derive energy and that are instrumental in
the degradation of target chemicals. In order to accomplish this chain of events, several
crucial aspects must converge, according to the National Research Council (NRC, 1993):
• The type of microorganisms,
• The type of contaminant, and,
• The geological conditions at the site.
Once converged, such conditions accelerate microbial activity that, in turn, cause target
chemical “biological” destruction. This bioremediation solution yields an elegant and
cost-effective way to attack chemicals in the environment using naturally occurring
microbes.
MICROORGANISMS (OR MICROBES)
The basic premise of bioremediation is to accelerate microbial activity using nutrients (i.e.,
phosphorus, nitrogen) and substrate (i.e., food) to create conditions conducive to biodegradation
of a target chemical or contaminant. Microorganisms (or microbes) are microscopic organisms
that have a natural capability to degrade or destroy a wide range of organic and inorganic
chemicals. Such microbes and the processes by which such degradation occurs are important to
understand. Microbes can use a variety of organic chemicals for their own growth and
propagation. These organic chemicals may serve various functions but primarily may be used as
either a carbon source for growth or as a source of electrons for energy. Microbes extract energy
via catalyzing energy–yielding biochemical reactions, thus enzymes produced by the microbe
can cleave chemical bonds and assist in a transfer of electrons from a chemical compound. These
types of reactions are termed oxidation-reduction reactions, where the organic chemical
(contaminant) is oxidized (i.e., electrons are lost) and another chemical (or acceptor) gains
electrons (or is reduced).
CONTAMINANT OF CONCERN (COC)
The second important aspect to consider is whether, and to what extent, a contaminant of
concern (COC) is amenable to biodegradation. Some chemicals are easier to biodegrade than
others by the variety of microorganisms found in the subsurface.
In general, petroleum hydrocarbon compounds are relatively easy to biodegrade and have well-
developed bioremediation processes. Other COCs, such as chlorinated compounds, were once
thought to be recalcitrant but have received much more attention in the past five to 10 years.
These chemicals are now viewed as amenable to bioremediation under appropriate engineered
(or natural) conditions. Chlorinated compounds are degraded under a wide variety of conditions,
which must be rigorously monitored to define effectiveness and efficiency.
GEOLOGICAL ENVIRONMENT
In the subsurface, numerous factors affect contaminant distribution. Here, the effects of the
saturated zone are considered. Once a contaminant reaches the saturated zone and dissolves,
advection and dispersion play major roles in the subsequent distribution in the subsurface.
Advection is the movement of contaminants carried by groundwater in the direction of flow and
is controlled by the linear velocity of the groundwater. That is, dissolved contaminants generally
move in proportion to the groundwater velocity. Thus, an increase in groundwater velocity will
result in farther travel of the contaminant. Dispersion is the “spreading out” of a contaminant
plume and is composed of both molecular diffusion and mechanical mixing. Many factors affect
dispersion, including pore size, path length, friction in the pores due to soil particles, and organic
carbon content, which will affect the retardation of the COC, thus limiting dispersion of the
subsurface matrix. Some of the important parameters for ascertaining subsurface effects include:
• Groundwater flow directions and velocities,
• Transport parameters (dispersion coefficients),
• Contaminant distribution and concentrations,
• Degradation rates (kinetics),
• Contaminant retardation, and biological process involved.
Typical examples of an in situ and ex situ bioremediation scenario are shown below.

In situ groundwater remediation.

Ex situ remediation (Bioreactor).

The In situ remediation could involve: Volatilization, Soil leaching, Prepared bed,
Biopile, Phytoremediation, Bioventing, Biosparing and Composting. Meanwhile, Ex situ
remediation includes: Bioreactors and Biofilters.
 PERFORMANCE BEFORE & AFTER TREATMENT
A comparism of the performance of the Total Petroleum Hydrocarbon (TPH) and
Polycyclic Aromatic Hydrocarbon (PAH) before and after the treatment is presented
below (Stanley Abraham et, al.).
 CONCLUSION AND RECOMMENDATION

The quest for a cheap source of energy coupled with the extensive rate of
industrialization has expanded the frontiers of petroleum hydrocarbon exploration with its
attendant negative consequence being the pollution of the environment. Several
remediation alternatives have been in use for the restoration of polluted systems.
Bioremediation, which exploits the biodegradative abilities of, live organisms and/or their
products have proven to be the preferred alternative in the long-term restoration of
petroleum hydrocarbon polluted systems, with the added advantage of cost efficiency and
environmental friendliness. The biodegradative abilities of organisms and/or their
products have proven to be the preferred alternative in the long-term restoration of
petroleum hydrocarbon polluted systems, with the added advantage of cost efficiency and
environmental friendliness.
Reduced transportation and disposal fees is also a vital advantage of bioremediation
especially the in situ bioremediation, therefore it should be explored and optimized to
ensure economic breakthrough while environmental sustainability is also ensured.
However, there is the need for further studies towards optimizing the process conditions
for the application of bioremediation strategies in diverse climatic zones especially in
extreme environments. There is also the need for the evolvement of a consortium of
microbes which can effect degradation on a wide range of hydrocarbon.
 QURAN 30 VERSE 41.
Mischief has appeared on land and sea because of (the meed) that the
hands of men have earned, that ((Allah)) may give them a taste of
some of their deeds: in order that they may turn back (from Evil). (Yusuf
Ali Translation).

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www.ejfoundation.org/reports.html
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