Phytoremediation of Crude Oil Contaminated Marine Water with Halophytes

endowed with the Capacity to Catabolize Naphthalene

Kalyani Rajalingham

Introduction

Petroleum Spills in Marine Systems

Bioremediation is the process by which contaminants are eliminated using

microorganisms. Phytoremediation, on the other hand, is the use of plants to

achieve the same goal. Contaminants are classified into three general

categories: biodegradable, persistent, or recalcitrant (hard to degrade) (Dua et

al. 2002). Petroleum spills in the ocean in particular results in (i) a barrier

between air, and water, (ii) and toxicity to aquatic life. Long term exposure to

petroleum has been linked to liver, and kidney diseases, to damage to the bone

marrow, and elevated risk of cancer (Jain et al., 2011). Though oil is a

degradable substance, often, without the assistance of bioremediation, much

damage can ensue between the time of spill, and the end of natural degradation.

The rate of biodegradation depends on the number of hydrocarbon degrading

microorganisms, the degradation capacity of the said population, abiotic

factors that affect the growth rates of hydrocarbon degrading microorganisms,

and abiotic conditions (such as temperature) (Atlas, 1991, Jain et al., 2011).

Persistence of petroleum in the environment was found to depend on

hydrocarbon mixture itself (Atlas, 1991). Further, the various types of

hydrocarbons are degraded by distinct microorganisms (Atlas, 1991).

Current methods used to remediate oil spills involve sorbents, vacuuming, low-

pressure flushing, removal of vegetation, or allowing nature to clean up the

spill. However, the three most marked methods involve the addition of either

dispersants, fertilizers, or microorganisms. The first method consists of using

dispersants to increase the surface area of the spill to permit rapid

remediation by microorganisms (Atlas, 1991). The second method consists of

adding fertilizer to promote microbial growth (Atlas, 1991). It has been found

that the use of an oleophilic fertilizer could remove oil spills in 10 days

(Jain et al., 2011, Atlas, 1991). The third method consists of adding

microorganisms or genetically modified microorganisms to the system. However,

the latter (GMO) has not undergone any field trials (Atlas, 1991).

Petroleum hydrocarbons is composed of alkanes, cycloalkanes, and aromatics

(Bartha, 1986). Petroleum, like any natural compound, is biodegradable by a

number of microorganisms; a number of microorganisms can utilize petroleum as

the sole carbon source (Jain et al., 2011). In an old spill, hydrocarbon

degrading microorganisms represent about 1-10% of the population while in an

uncontaminated area, this number reduces to about 1% (Atlas, 1991). Old

petroleum spills have been noted to harbour 5 distinct types of microorganisms

- α-proteobacteria, β-proteobacteria, δ-proteobacteria, γ-proteobacteria, and

the CFB group (Cappello et al., 2007). Currently, petroleum spills in flowing

media are not remediated using genetically modified organisms due to safety

concerns, containment issues, and unknown ecological effects (Atlas, 1991).

Chakrabarty engineered a hydrocarbon-degrading pseudomonas that could

degrade low weight aromatic hydrocarbons (Atlas, 1991). But it is believed that

genetically modified microorganisms is not a solution to this problem.

Phytoremediation of Petroleum Spills

A potential solution to this problem comes in the form of plants (Table 1). It

has been shown that hydrocarbon contaminated soil can be remediated using Zea

mays, and Pennisetum pupureum for instance. Hydrocarbons levels were found to

decrease by 77.5% (Zea mays), and 83% (Pennisetum pupureum) in two weeks using

phytoremediation (Ighovie and Ikechukwu, 2014). Soil hydrocarbon levels were

shown to decrease by 66% by Axonopus sp. (Ighovie and Ikechukwu, 2014). El-

Bakatoushi, (2011) stated that in choosing a plant for phytoremediation, one

must first consider the potential of the plant to tolerate the contaminant;

typically, the plant selected can be found growing on contaminated sites (El-

Bakatoushi, 2011). Further, petroleum at low levels can stimulate growth of

plant due to the presence of naphthenic acids; however, moderate to high levels

of petroleum is noxious to plants (El-Bakatoushi, 2011). Organic compounds in

soil, unlike the ocean, are harder to absorb due to their association to other

compounds. However, plants can tackle contaminants by absorption, or by

tackling those on the surface of leaves (Kathi and Khan, 2011).

Table 1: Phytoremediation of oil. (Source: Ighovie and Ikechukwu, 2014)

Extreme Halophytes

Marine systems necessitate extreme halophiles for remediation. Limoniastrum

monopetalum, for instance, is a salt tolerance plant that can be found on oil

contaminated soil. However, the performance of Limoniastrum monopetalum in

marine waters is not known. As such, an alternative is the marine algae. They

thrive only in marine water, and usually require an attachment surface (which

can be provided). In this study, the model organism chosen to achieve the

purpose is Macrocystis pyrifera, or the Giant kelp because it has a height of

45m. Macrocystis pyrifera typically however does not tolerate high oil

concentrations.

Naphtalene degrading pathway from Pseudomonas putida

Naphthalene, an aromatic hydrocarbon, is commonly found in crude oil.

Naphthalene degradation has been shown possible with the use of 6 enzymes in

Pseudomonas putida: A - naphthalene dioxygenase, B - cis-dihydrodiol

naphthalene dehydrogenase, C - 1,2-dihydroxynaphthalene dioxygenase, D - 2-

hydroxychromene-2-carboxylate isomerase, E - 2-hydroxybenzalpyruvate

aldolase, and F - salicylate dehydrogenase (Grund and Gunsalus, 1983, Figure 1).

Objective

Most studies focus on bioremediation of non-flowing systems (such as sand, or

soil), and as such there is a lack of experiments focusing on the use of

phytoremediation on flowing systems, especially marine systems. This paper is

not intended to solve the problem as a whole but rather attempts to actualize

an unexplored solution. In particular, the purpose of this study is to create a

transgenic halophilic Macrocystis pyrifera with hydrocarbon remediation

capabilities for naphthalene via insertion of a construct containing genes

necessary for naphthalene catabolism.

Figure 1: Naphthalene oxidation pathway. (Source: Grund and Gunsalus, 1983; Enzymes A, B, C, D, E, F, G, H, I, J, K, L, and M catalyze the reaction.)

Methodology

Hydrocarbon Degrading Constructs. Genes required for naphthalene

catabolism – NaphA – NaphL – will be excised from the pIG7 plasmid, and

tailored to a eukaryotic genome (Grund and Gunsalus, 1983, Figure 2). PCR will

be used to add necessary segments.

Figure 2: Hydrocarbon degrading construct. (SM=Selection Marker; P=Promoter; NaphA-L total length = 25.2kb)

Plant Material. Macrocystis pyrifera will be acquired from a source.

Seedlings of Macrocystis pyrifera will be obtained, and permitted to grow until

a particular height is reached (larger than the height of the tank).

Bacterial Strain. Pseudomonas putida will also be acquired, and grown in

broth.

Multiple Transformations. Construct size has not yet been computed,

however, the current construct is based on the work done by Grund and Gunsalus,

(1983). Plants will be transformed with a single construct (ex: NaphA-L) via

agrobacterium-mediated transformation. Tissue culture will be used to

regenerate the transgenic plant. Selection markers will be used to isolate

plants with construct. In the event that more than one construct is necessary,

multiple plants will be transformed, each with a particular construct.

Assuming that there are 2 constructs (NaphA-F, NaphG-K), 2 plants will be

transformed each with a different construct. Thereafter, crossing, and PCR

will be used to isolate plants possessing both constructs which will be subject

to experimentation.

Molecular Analyses. Copy number, and transcript levels will be

determined using qRT-PCR, and protein levels will be determined using a

Western blot. Morphological analysis will be performed to detect any

phenotypic changes should there be any.

Degradation Capacity. Multiple tanks will be filled with marine water,

and petroleum (simulating a real oil spill). Test plants (approximately 4 per

tank) will be placed in the tank, and TOC (total organic carbon) levels before

and after the treatment will be measured. TOC is typically measured by

collecting, burning a sample, and measuring the CO2 concentration emitted; the

[CO2] emitted is proportional to the amount of hydrocarbons present. The test

period will be 2 weeks. Four experiments will be conducted (1 control, 3

experimental). The experimental set-ups will consist of either a transgenic

plant alone, a transgenic plant supplemented with leaf surface Pseudomonas

putida, or a non-transgenic plant with leaf surface Pseudomonas putida (Figure

3).

Figure 3: Experimental set-ups. (AM = Agrobacterium mediated)

References

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