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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