This document discusses using transgenic halophytic algae Macrocystis pyrifera for phytoremediation of naphthalene in marine oil spills. The objective is to insert genes for naphthalene degradation from Pseudomonas putida into M. pyrifera to create plants that can break down naphthalene. Experiments will transform M. pyrifera with these genes using agrobacterium, then test the transgenic plants' ability to degrade naphthalene from simulated oil spills in seawater tanks over 2 weeks, with or without additional P. putida on the leaves.

1. 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).

2. 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

3. 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)

4. 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.

5. 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

6. 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

7. 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
Atlas, R. (1991). Microbial hydrocarbon degradation-bioremediation of oil
spills. Journal Of Chemical Technology & Biotechnology 52, 149-156.
Bartha, R. (1986). Biotechnology of Petroleum Pollutant Biodegradation. Microb
Eco, 12, 155-172.

8. Cappello, S., Denaro, R., Genovese, M., Giuliano, L. and Yakimov, M. (2007).
Predominant growth of Alcanivorax during experiments on “oil spill
bioremediation” in mesocosms. Microbiological Research, 162(2), pp.185-190.
Dua, M., Singh, A., Sethunathan, N., & Johri, A. (2002). Biotechnology and
bioremediation: Successes and limitations. Applied Microbiology and
Biotechnology, 143-152.
El-Bakatoushi, R. (2011). Identification and characterization of up-regulated
genes in the halophyte Limoniastrum monopetalum (L.) Boiss grown under crude
oil pollution. Journal Of Genetic Engineering And Biotechnology 9, 137-148.
Grund, A., and Gunsalus, I. (1983). Cloning of genes for naphthalene metabolism
in Pseudomonas putida. Journal Of Bacteriology 156, 89-94.
Ighovie, E., and Ikechukwu, E. (2014). Phytoremediation of Crude Oil
Contaminated Soil with <i>Axonopus compressus</i> in the Niger
Delta Region of Nigeria. NR 05, 59-67.
Jain, P., Gupta, V., Gaur, R., Lowry, M., Jaroli, D., and Chauhan, U. (2011).
Bioremediation of Petroleum oil Contaminated Soil and Water. Research J. Of
Environmental Toxicology 5, 1-26.
Kathi, S., and Khan, A. (2011). Phytoremediation approaches to PAH contaminated
soil. Indian Journal Of Science And Technology 4, 56-63.