Science of the Total Environment 628–629 (2018) 1582–1599

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Review

Microbe and plant assisted-remediation of organic xenobiotics and its

enhancement by genetically modified organisms and recombinant
technology: A review
Imran Hussain a,b,1, Gajender Aleti a,1, Ravi Naidu c, Markus Puschenreiter d, Qaisar Mahmood f,
Mohammad Mahmudur Rahman c, Fang Wang e, Shahida Shaheen f,
Jabir Hussain Syed g,h,⁎, Thomas G. Reichenauer a,⁎⁎
a
AIT Austrian Institute of Technology, Centre for Energy, Environmental Resources and Technologies, Tulln, Austria
b
Department of Molecular Systems Biology, Faculty of Life Sciences, University of Vienna, Austria
c
Global Centre for Environmental Remediation (GCER), Faculty of Science, The University of Newcastle, Callaghan, New South Wales, Australia
d
Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria
e
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
f
Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan
g
Department of Meteorology, COMSATS Institute of Information Technology, Park Road Tarlai Kalan 45550, Islamabad, Pakistan
h
Department of Civil and Environmental Engineering, Hong Kong Polytechnic University, Hong Kong

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• This review offers details on enhanced

remediation by transgenic plants and
microbes.
• Use of transgenic plants for remediation
of organic compounds has enormous
potential.
• Synergistic interaction between recom-
binant bacteria and genetically modified
plants may enhance the remediation.
• Potential risks associated with altered
microbes and plants can be tackled by
abiding regulatory guidelines.
• Full scale remediation system may be
obtained by using biotechnological tools.

a r t i c l e i n f o a b s t r a c t

Article history: Environmental problems such as the deterioration of groundwater quality, soil degradation and various threats
Received 22 September 2017 to human, animal and ecosystem health are closely related to the presence of high concentrations of organic xe-
Received in revised form 31 January 2018 nobiotics in the environment. Employing appropriate technologies to remediate contaminated soils is crucial due
Accepted 3 February 2018
to the site-specificity of most remediation methods. The limitations of conventional remediation technologies in-
Available online 22 February 2018
clude poor environmental compatibility, high cost of implementation and poor public acceptability. This raises
Editor: P Holden the call to employ biological methods for remediation. Bioremediation and microbe-assisted bioremediation
(phytoremediation) offer many ecological and cost-associated benefits. The overall efficiency and performance
of bio- and phytoremediation approaches can be enhanced by genetically modified microbes and plants.

⁎ Correspondence to: J.H. Syed, Department of Meteorology, COMSATS Institute of Information Technology, Park Road Tarlai Kalan 45550, Islamabad, Pakistan.
⁎⁎ Correspondence to: T.G. Reichenauer, AIT Austrian Institute of Technology, Centre for Energy, Environmental Resources and Technologies, Tulln, Austria.
E-mail addresses: jabir.syed@comsats.edu.pk (J.H. Syed), thomas.reichenauer@ait.ac.at (T.G. Reichenauer).
1
Authors making an equal contribution.

https://doi.org/10.1016/j.scitotenv.2018.02.037
0048-9697/© 2018 Elsevier B.V. All rights reserved.
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599 1583

Keywords: Moreover, phytoremediation can also be stimulated by suitable plant-microbe partnerships, i.e. plant-endophytic
Organics or plant-rhizospheric associations. Synergistic interactions between recombinant bacteria and genetically mod-
Plant-rhizospheric partnership ified plants can further enhance the restoration of environments impacted by organic pollutants. Nevertheless,
Genetically modified plants
releasing genetically modified microbes and plants into the environment does pose potential risks. These can
Microbes
Recombinant technology
be minimized by adopting environmental biotechnological techniques and guidelines provided by environmen-
Health risks tal protection agencies and other regulatory frameworks. The current contribution provides a comprehensive
overview on enhanced bioremediation and phytoremediation approaches using transgenic plants and microbes.
It also sheds light on the mitigation of associated environmental risks.
© 2018 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1583
2. Microorganisms for the clean-up of contaminated environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584
2.1. Enhancement of bioremediation with engineered microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584
2.2. Enhancement of bioremediation with recombinant technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1584
3. Plant-assisted bioremediation of organic contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586
3.1. Phytoremediation and its processes/techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1586
3.2. Degradation mechanisms in plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1587
3.3. Phytoremediation technology for remediation of organics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1587
4. Plant-bacterial partnership for improved remediation of organic xenobiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1588
4.1. Endophytic-plant partnerships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1589
4.2. Rhizosphere-plant partnership . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1590
4.3. Prediction and annotation of genome sequences of xenobiotic-degrading bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 1590
5. Transgenic plants for remediation of organic contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1591
5.1. Transgenic plants for stimulated phytodegradation/rhizodegradation of organic pollutants . . . . . . . . . . . . . . . . . . . . . . . 1591
5.1.1. Herbicides and pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1591
5.1.2. Halogenated organic compounds and explosives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1592
6. Risks and mitigation through biotechnological advances associated with using genetically modified plants and microbes . . . . . . . . . . . . . 1593
7. Conclusions and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1593
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1594
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1594

1. Introduction
The post-World War II era witnessed a substantial increase in the
production and release of organic contaminants into the environment
around the globe. This increase corresponded with rapid industrializa-
tion, population growth, mechanized agricultural practices and military
activities (Arslan et al., 2015). Although certain organic compounds are
beneficial for industry and agricultural productivity, their excessive
usage creates many environmental problems. Kang (2014) noted the
detrimental implications of such organic contaminants in the environ-
ment and on human health. Many other researchers highlighted the ad-
verse consequences of organic contaminants on plants (Copaciu et al.,
2016; Noguera-Oviedo and Aga, 2016), soils (Pinedo et al., 2013; Teng
et al., 2014) and humans (Cachada et al., 2012; Malchi et al., 2014).
Most organic contaminants are hydrophobic in character and can re-
duce the quality of soils/sediments, pollute groundwater and cause se-
vere health risks for humans and animals (Liu et al., 2016; Ramadass
et al., 2015). Additionally, some organic contaminants (chlorinated sol-
vents, persistent organic pollutants (POPs) and explosives) have proved
to be carcinogenic, mutagenic and neurotoxic (Gilden et al., 2010;
Tabrez and Ahmad, 2009; Wasi et al., 2013).
The various classes and groups of organic contaminants differ in
their bioavailability and persistence in the environment. Chlorinated
solvents are the most common and widely distributed organic contam-
inants in the environment (Matteucci et al., 2015). They include trichlo-
roethylene (TCE), perchloroethylene (PCE), carbon tetrachloride and
chloroform. Most chlorinated hydrocarbons are of anthropogenic origin
and can spread throughout the environment via plastics, paints, adhe-
sive materials and gasoline (Rosner and Markowitz, 2013). Another no-
torious group of organic contaminants consists of persistent organic
pollutants (POPs) (Fernández-Cruz et al., 2017), which are lipophilic or-
ganic compounds that can resist photochemical degradation. They typ-
ically show a tendency to accumulate and persist in the environment for
longer periods (Cetin et al., 2017; Sharma et al., 2014a, 2014b). Most
POPs fall into three categories: organochlorine pesticides, industrial
chemicals and their by-products (Arslan et al., 2015; Beyer et al.,
2000; Muir and Howard, 2006). Within the domain of POPs, a “dirty
dozen” showing pronounced persistence, toxicity, as well as bioaccu-
mulation and biomagnification in human and animal fatty tissues was
defined (Xu et al., 2013; Martin et al., 2016). The pesticides and insecti-
cides on the list exhibit persistence and toxicity (Haffner and Schecter,
2014; Tavakoly Sany et al., 2015).
Explosives comprise another class of organic contaminants that
heavily pollute the environment due to their industrial-scale production
and exploitation (Kiiskila et al., 2015). Explosives are found at former
war zones, military firing ranges and their production plants. The most
prevalent contaminants associated with such areas are 2,4,6-trinitrotolu-
ene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine, hexogen (RDX) and
octahydro-1,3,5,7 tetranitro-1,3,5,7-tetrazocine, octogen (HMX) (Ali et
al., 2014; Douglas et al., 2012). These four explosives along with their
transformation products can cause toxic, lethal and detrimental effects
in many organisms, including humans (Wang et al., 2017). Direct contact
may cause severe health problems such as liver, skin and immune system
disorders. Lastly, pollution associated with petroleum hydrocarbons and
their derivatives is a well-established concern worldwide (Gerhardt et
al., 2017). Crude oil, gasoline, creosote and diesel are major constituents
of this group commonly designated as total petroleum hydrocarbons
(TPHs) (Yousaf et al., 2011; Afzal et al., 2014). All these organic xenobi-
otics are linked to cancer, mutations, immune system disorders, as well
as reproduction and birth defects (Gilden et al., 2010; Kang, 2014).
1584 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
The development of remediation technologies to reclaim contami-
nated sites is of paramount importance to protect human lives. A set
of physical, chemical, biological and integrated approaches are available
to remediate the pollution caused by organic xenobiotics (Gomes et al.,
2013; Abhilash et al., 2009; Khan et al., 2004). The conventional mea-
sures usually involve high application costs and energy inputs
(Kuppusamy et al., 2017). This has triggered intensified efforts to de-
velop more efficient, ecologically sustainable, environmentally friendly
and cost-effective remediation technologies utilizing advanced biotech-
nological tools and rhizosphere engineering (Kuppusamy et al., 2017;
Ibañez et al., 2016; Dixit et al., 2015; Truu et al., 2015). Bioremediation
involves using specific microbes to degrade organic contaminants – a
reasonable and effective method based on the unique catabolic poten-
tial of microbes (Cheng et al., 2017). Within the scope of bioremedia-
tion, phytoremediation offers promising advantages with the
synergistic use of plants and microbes (Vergani et al., 2017; Thijs et
al., 2016). Moreover, plants exhibit an array of additional decontamina-
tion mechanisms in comparison to microbes alone (Gerhardt et al.,
2016, 2009; Singh et al., 2016).
This review discusses the enhanced bioremediation and
phytoremediation of organic contaminants. It focuses on recent ad-
vances in augmented bioremediation involving recombinant technolo-
gies and phytoremediation with plant-endophytic/rhizospheric
partnerships, along with its further improvements using genetically
modified plants. Finally, also discussed here are the risks associated
with engineered plants and microbes along with their mitigation
using biotechnological advances.
2. Microorganisms for the clean-up of contaminated environments
The term bioremediation implies the conversion of organic contam-
inants into carbon dioxide, water and a transformed product created by
the actions of specific microbes (Hlihor et al., 2017a, 2017b; Chaudhry et
al., 2005; Sarkar, 2018). Principally, it involves the transformation of or-
ganic contaminants by the concerted action of microorganisms as part
of their metabolic processes (Ghosal et al., 2016). Both aerobic and
non-aerobic degradation pathways have been reported in the minerali-
zation and stabilization of organic contaminants. Many bioremediation
systems operate under aerobic conditions; anaerobic conditions, how-
ever, may be advantageous in degrading recalcitrant molecules such
as PCBs and PAHs (Passatore et al., 2014). Various bacterial genera
such as Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus and My-
cobacterium degrade pesticides and hydrocarbons under aerobic condi-
tions (Vergani et al., 2017). These heterotrophic bacterial genera utilize
contaminants as their sole source of carbon and energy. Anaerobic mi-
crobes have been frequently used to degrade PCBs as well as dechlori-
nate TCE and chloroform (Hlihor et al., 2017c).
The bioremediation efficiency may be limited especially when soils
are polluted with recalcitrant organic molecules. In such cases, the com-
bined use of microbes and plants (rhizoremediation) can enhance the
efficiency (Thijs et al., 2016). Plant roots provide microaerophilic condi-
tions, and root exudates additionally enhance the degradation of or-
ganic contaminants. Root exudates accelerate the degradability and
bioavailability of organic xenobiotics to microbes by: firstly, providing
substrate for microbial metabolism; and secondly, altering soil pH or
contaminant solubility. Four possible pathways have been reported for
enhancing the biodegradation of organic contaminants by root exu-
dates: 1) direct degradation by plant enzymes; 2) enhanced pollutant
bioavailability via secreting surfactants; 3) exploitation of structural
analogy and co-metabolic processes; and 4) stimulation by providing
a nutrient and energy flow (Martin et al., 2014).
Bioremediation is an attractive option for remediating organic con-
taminants because it offers many benefits, for instance cost-effective-
ness, efficiency, and environmental “friendliness” (Kästner and
Miltner, 2016; Chen et al., 2015; Kang et al., 2012). It has also been in-
cluded in the list of the “top ten technologies for improving human
and environmental health” (Viana and Sant'ana, 2017; Daar et al.,
2002). The discovery of many microbes and their unique catabolic char-
acteristics has boosted the application of bioremediation. Microbes can
degrade a large variety of organic xenobiotics by specific biochemical
processes under anaerobic or aerobic conditions (Kumar and Philip,
2017; Collins, 2001). Distinctive features of microbes (e.g. catabolic po-
tential, adaptation and surfactant production) render them favorable
candidates for contaminant transformation (Le et al., 2017; Schenk et
al., 2012). Various factors – soil pH, nutritional status, moisture, temper-
ature, redox potential and chemical composition of pollutants – can in-
fluence and potentially limit bioremediation efficiency (Shukla et al.,
2013). This calls for the need to improve bioremediation processes. Ap-
plication of microbes alone has shown limited efficiency due to multiple
factors including poor competitiveness and excessive contaminant con-
centrations in soil. Efficiency can be enhanced by applying various
amendments such as inorganic nutrients, surfactants, bulking agents,
compost as well as biochar (Wiszniewska et al., 2016). Such amend-
ments have been comprehensively reviewed in recent studies
(Kästner and Miltner, 2016, Lukić et al., 2016; Truu et al., 2015). The
present review focuses on improved bioremediation using transgenic
microbes. The development and use of transgenic microbes through re-
combinant technology is a straightforward approach.
2.1. Enhancement of bioremediation with engineered microbes
Engineered microbes for the transformation of organic xenobiotics
are constantly being developed to overcome the limitations of bioreme-
diation. During the early 1990s, genetic engineering techniques were
applied to exploit microbial metabolism for this purpose (Zwillich,
2000). This led to a rapid growth in biodegradation enterprises and ex-
panded the research in this emerging field (Jagtap and Bapat, 2017). The
regulatory challenges and enormous expenses involved in transgenic
research affected many companies. Over time, experiments and re-
search on genetically modified microorganisms gradually became con-
fined to academic and research institutes. Some studies such as Abril
et al. (1989) and Brinkmann and Reineke (1992) reported increased
degradation of 3- and 4-chlorotoluene and 3-chlorotoluene over the
control by inoculation of a genetically modified Pseudomonas sp. B13
strain. Some genetically engineered microorganisms outside the field
of bioremediation have also been reported (Coleman et al., 1997;
Pieper and Reineke, 2000; Sandermann, 1994; Yang et al., 2010). The ra-
diation-tolerant microbe Deinococcus radiodurans has been genetically
engineered for toluene degradation; it has not, however, been commer-
cialized to date for bioremediation purposes due to anticipated risks and
regulatory challenges (Ezezika and Singer, 2010; Lang and Wullbrandt,
1999). Failure to advance from research to commercialization is attrib-
uted to several factors such as challenges in gaining approval from reg-
ulatory bodies prior to release in the environment, public acceptability
and safety risks associated with engineered bacteria.
2.2. Enhancement of bioremediation with recombinant technology
The microbial metabolic potential can be exploited for environmen-
tal remediation and is a very economical and safe alternative compared
to other physicochemical techniques (Liu et al., 2017). Despite the pres-
ence of highly diverse and specialized microbial communities in the en-
vironment that can break down many pollutants, the natural
remediation process may be quite slow. This may lead to the accumula-
tion of pollutants in the environment, which can be hazardous (Ijoma
and Tekere, 2017; Cheng et al., 2017; Perpetuo et al., 2011). With the ad-
vent of the latest biotechnological techniques such as natural gene
transfer or recombinant DNA methods, bacterial strains (rhizospheric
and/or endophytic) can be engineered to produce specific enzymes
that promote the degradation of toxic organic substances (Pandotra et
al., 2018; Chakraborty and Das, 2016). Genetic engineering has the po-
tential to improve soil bioremediation technologies, and applying this
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
approach to plant-associated endophytic and rhizospheric bacteria can
help enhance the degradation of toxic compounds in contaminated
sites by phytoremediation (Fasani et al., 2017).
Previous studies (Gao et al., 2017; Chebbi et al., 2017) clearly re-
vealed oil biodegradation capabilities of four Pseudomonas strains and,
interestingly, genes encoding degradation enzymes were found on
extra-chromosomal plasmids. Four plasmids were isolated and intro-
duced in Pseudomonas spp., which enhanced the oil degradation poten-
tial by 10–100 times compared to natural isolates (Time Magazine,
1975). Studies related to the degradation of toxic organic contaminants
using genetically engineered microbes have been recently summarized
by Kumar et al. (Kumar et al., 2013). Agent Orange is one of the toxic
herbicides and defoliants used by the U.S. military during the Vietnam
War, and it was closely linked to increased incidences of cancer
(Ezezika and Singer, 2010). Agent Orange is a mixture of equal parts
of phenyl herbicides including 2,4-dichlorophenoxyacetic acid (2,4-D)
and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). A recombinant strain
of Burkholderia cepacia was tested for the degradation of Agent Orange
at the storage site of the U.S. Air Force in Pensacola, Florida (Chauhan
et al., 2008).
Cytochrome P450 monooxygenases (CYPs) are associated with the
degradation of the most toxic pollutants, for example naphthalene, fluo-
rine, acenaphthalene, acenaphthylene, and 9-methylanthracene. Previ-
ous studies (Chica et al., 2005; Pleiss, 2012) suggested that enzyme
activity could be increased by altering the active site through rational
design. Alterations at the F87 and Y96 sites within the CYP101 enzyme
tripled the oxidation potential of the enzyme in Pseudomonas putida
(Harford-Cross et al., 2000). Mutations at R47L and Y51F sites enhanced
the oxidation potential of the CYP102 enzyme for phenanthrene and
fluoranthene 40-fold and 10-fold, respectively, in Bacillus megaterium
(Carmichael and Wong, 2001). Moreover, a 30-fold increase in the
degradation potential of polyaromatic hydrocarbons was achieved
when CYP was mutated at three active sites (A74G/F87V/L188Q). A
simultaneous increase in the nicotinamide adenine dinucleotide
phosphate (NADPH) consumption rate was observed as well (Liu et
al., 2006).
PCBs are a class of aromatic chemicals encompassing 209 different
chemical structures (congeners). PCBs are used in a wide range of appli-
cations such as the manufacture of insecticides and electrical insulating
fluids in capacitors and transformers (Das and Adholeya, 2012). PCBs
also adversely affect human and environmental health. The biphenyl
dioxygenase enzyme degrades PCBs under aerobic conditions (Ang et
al., 2005). During this process, the enzyme incorporates two hydroxyl
groups into the aromatic ring of a PCB's congener, yielding a tricarbox-
ylic acid cycle intermediate (chlorobenzoate). The biphenyl
dioxygenase consists of a terminal dioxygenase, ferredoxin, and ferre-
doxin reductase encoded by the bph operon (Erickson and Mondello,
1992). Variants of bphA with increased capacities for PCB degradation
were produced from Burkholderia cepacia strain LB400, C. testosterone
B-365, and Rhodococcus globerulus P6 via the family shuffling approach
(Barriault et al., 2002). The hybrid bphA, in particular II-9 genes, were re-
ported to oxygenate persistent 2,6-dichlorobiphenyl up to 58%, while
the control oxygenated only up to 10% (Suenaga et al., 2002). Geneti-
cally modified strains of the plant-associated rhizospheric bacteria Pseu-
domonas fluorescens F113rifbph and Pseudomonas fluorescens
F113:1180 colonized plant roots similar to the wild-type rhizospheric
bacteria (Brazil et al., 1995; Villacieros et al., 2005). Both genetically
modified strains, however, showed a significantly increased degrada-
tion capacity for PCBs compared with wildtype rhizospheric bacteria.
This makes the manipulation of the rhizospheric bacteria a substantial
future prospect for bioremediation. Furthermore, the Pseudomonas
fluorescens F113: 1180 strain was tested negatively for antibiotic resis-
tance and the strain was considered suitable for in situ applications
(Villacieros et al., 2005). Gene bph of Pseudomonas fluorescens f113:
1180 is stable and therefore it is not clear if a horizontal gene transfer
among the rhizospheric bacteria will take place (Ramos et al., 1995).
1585
A genetically engineered poplar endophytic Pseudomonas putida
VM1441 (pNAH7) protected pea plants against the toxic effects of naph-
thalene. Furthermore, plants inoculated with that strain indicated an in-
crease in naphthalene degradation rates (up to 40%) compared to un-
inoculated plants in artificially contaminated soil (Germaine et al.,
2009). In one greenhouse study, a genetically modified endophyte
Burkholderia cepacia G4 showed increased toluene tolerance in the
yellow lupine plant and decreased phytovolatilization of toluene by
70%. Natural endophyte was conjugated by a plasmid (pTOM)
encoding enzymes that made toluene degradation possible (Barac
et al., 2004). Nonetheless, subsequent studies on poplar trees
suggested that the plasmid was prone to horizontal gene transfer
(Taghavi et al., 2005).
A specific bacterial glutathione-S-transferase (GST) isolated from
Burkholderia xenovorans LB400, BphKLB400 removed toxic chlorines
from organic pesticides and provided resistance to chloromequat chlo-
ride in pea plants (McGuinness et al., 2007). Further mutations in the
conserved amino acid site of GST involving substitution of Ala by Pro re-
sulted in a 2-fold increase in GST activity towards a number of com-
monly used chlorinated pesticides (McGuinness et al., 2006, 2007).
That study suggested that genetically modified endophytic or
rhizospheric bacteria are promising bioremediation agents for
dehalogenation of organic pollutants. Atrazine (2-chloro-4-
ethylamino-6-isopropylamino-1, 3, 5-s-triazine), with a half-life of
170 days is used to control weeds. Pseudomonas sp. ADP was reported
to enzymatically stabilize atrazine to the final, less toxic product called
cyanuric acid. Pseudomonas sp. ADP produced two forms of atrazine-
degrading enzymes – Atz (A, B, and C) and TriA – which may have dif-
ferent action mechanisms and produced cyanuric acid as the final deg-
radation product. Atz and TriA enzymes may have evolved recently to
degrade atrazine (De Souza et al., 1998).
Comamonas testosterone strain VP44 was transformed by two plas-
mids: pE43 comprising oxygenolytic ortho-dechlorination ohb gene;
and pPC3 comprising the hydrolytic para-dechlorination fcb gene. The
resulting two recombinants C. testosterone VP44 (pE43) and VP44
(pPC3) displayed affinity to cometabolite 95% concentrations of ortho-
and para-chlorobiphenyls as sole carbon sources (Hrywna et al.,
1999). Sphingomonas sp. CDS-1 was genetically modified to degrade or-
ganophosphate- and carbamate. In Pseudomonas putida DLL-1, a methyl
parathion hydrolase gene (mpd) with cognate regulator of a methyl
parathion (MP) was prepared by shotgun cloning. The DNA fragment
was further cloned into a broad host vector pBBR1MCS-2 to produce
pBBR-mpd (Liu et al., 2006). CDS-pBBR-mpd recombinant strain was
produced by transforming the recombinant plasmid into the
carbofuran-degrading strain Sphingomonas sp. CDS-1. The resulting ge-
netically engineered organism mediated methyl parathion degradation
nearly 7 times faster than the wild type strain (Liu et al., 2006).
Organophosphates such as paraoxon, parathion, chlorpyrifos disul-
foton, ruelene, carbophenothion, and dimeton are potent neurotoxins
that interfere with the breakdown of acetylcholinesterase at the synap-
tic junction and are involved in chromosomal aberrations and bladder
cancer (Das and Adholeya, 2012; Webster et al., 2002). Bacterial en-
zymes including phosphotriesterase (PTE) and organophosphorus hy-
drolase (OPH) catalyze the hydrolysis of these toxic organophosphates
by cleaving P\\O, P\\F, or P\\S bonds. The analysis of the three-dimen-
sional structure of PTE from Pseudomonas diminuta with a structural an-
alogue diethyl 4-methylbenzylphosphonate revealed three
hydrophobic binding pockets that were necessary for the orientation
of the substrate in the active site (Chen-Goodspeed et al., 2001a,
2001b). Moreover, triple mutations at key sites Ile106, F132G, and
His257 enhanced the catalytic properties. For example, substitution of
Ile106 by Ala eliminated organophosphates 90-fold; Sp-enantiomer
mutations at I106G/F132G showed 270-fold enhancements in Kcat
and Rp-enantiomers with a stable Kcat for Sp-enantiomers. Further-
more its substitution by His257 resulted in the reduced kinetic parame-
ters of PTE (Chen-Goodspeed et al., 2001a). Selected examples of
1586 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599

genetically engineered bacteria that degrade specific organic com-
pounds are summarized in Table 1.
3. Plant-assisted bioremediation of organic contaminants
3.1. Phytoremediation and its processes/techniques
Phytoremediation is a set of technologies using the natural potential
of specific plants and their associated microbes to detoxify, transform,
extract, sequester, or assimilate various toxic inorganic and organic pol-
lutants in the environment, especially soil, surface waters, groundwater
and air (Gerhardt et al., 2016). Plants have always been a basic source of
food, fuel (energy) and fibre. Over the past three decades, the utilization
of plants and catabolic potential of their associated microbes have be-
come accepted as a ‘green solution’ for reclaiming contaminated soils
and water (Truu et al., 2015). It offers many ecological and technical
benefits over conventional methods for remediating organic contami-
nants. Phytoremediation is considered to be environmentally friendly,
sustainable, solar-driven, cost effective and socially acceptable. It does,
however, have certain disadvantages that can limit its applicability, in-
cluding a longer time frame for achieving the remediation target, ineffi-
ciency in the presence of high pollutant levels, limited zone of
effectiveness and limitations in the case of low bioavailability
(Gerhardt et al., 2016). For a detailed description of the advantages
and disadvantages of phytoremediation, see earlier reviews (Afzal,
2010; Ali et al., 2013; Arslan et al., 2015; Asim Shahzad, 2015;
Chaudhry et al., 2005; Dhankher et al., 2012; Gerhardt et al., 2009;
Gkorezis et al., 2016; Gerhardt et al., 2017).
In 1980 the term phytoremediation was coined, followed by a rapid
expansion for treating a variety of pollutants in the last three decades
(Schwitzguébel, 2015). To date, it has been applied to remediate soils
or water bodies contaminated by heavy metals, petroleum hydrocar-
bons, chlorinated solvents, pesticides, explosives and radionucleotides
(Afzal et al., 2014; Aken and Doty, 2009; Álvarez et al., 2015; Kiiskila
et al., 2015; Zhang et al., 2016). Despite the proven effectiveness of
phytoremediation in different experimental contexts, i.e. laboratory,
greenhouse and in the field, this technology remains underutilized by
governments and the private sector. Recently, Gerhardt et al. (2017)
proposed different steps for commercial utilization of this green and
sustainable technology. Phytoremediation covers many strategies that
seek to remediate a wide range of organic and inorganic pollutants
using different natural processes. These are explained in more detail
below.
Phytoextraction refers to the uptake of contaminants through plant
roots and their translocation to harvestable plant parts such as shoots
(Mahar et al., 2016; Abhilash et al., 2012). It is also typically known as
phytoaccumulation or phytosequestration, mostly reported for heavy
metals (Ali et al., 2013). Rhizofiltration involves the removal of pollut-
ants from surface water or liquid waste streams by plant roots
(Dimitroula et al., 2015; Ali et al., 2013). It can be further classified
into blastofiltration (use of seedlings) and caulofiltration (use of plant
shoots). Phytostabilization or phytoimmobilization is the use of certain
plants to stabilize contaminants in soils in order to reduce their bioavail-
ability (Burges et al., 2016; Singh et al., 2012). Reduced bioavailability
may help prevent pollutants enter groundwater and the food chain
(Erakhrumen, 2011). Plant processes such as sorption (by roots), pre-
cipitation, complexation and metal valence reduction (in rhizosphere)
are mainly responsible for immobilizing metals in contaminated soils
(Wuana and Okieimen, 2011; Ghosh and Singh, 2005; Yoon et al.,
2006). Plants can secrete specific sets of redox enzymes that convert
more toxic metals species to less toxic species. Thus, it may help avoid
metal stress and damage in plants and other biota. Phytostabilization

Table 1
Recombinant technologies used for transgenic microbes with their significant findings.

Recombinant strategy Results References

Escherichia coli engineered to overproduce atrazine After 8 weeks, Atrazine concentration decreased by 52% in plots containing (Strong et al., 2000)
chlorohydrolase AtzA killed recombinant E. coli cells, and 77% in combination plots containing
phosphate and the enzyme
Plasmid with a transposon-based bioluminescence producing Higher Naphthalene gene expression lead to enhanced degradation rates of (Sayler and Ripp, 2000)
lux gene fused with a promoter for naphthalene catabolic the pollutant
genes
Engineered endophytic bacteria with toluene degrading The recombinant strain induced up to 50%–70% degradation of toluene. (Barac et al., 2004)
pTOD/pTOM plasmid
pNF142 plasmid, gfp Degradation of naphthalene in soil (Wang et al., 2008)
pTOM-Bu61 plasmid encoding constitutively expressed Inoculation of poplar had increased plant growth in the presence of toluene (Taghavi et al., 2005)
toluene degradation and substantially reduced concentrations of toluene released via
evapotranspiration
Arthrobacter globiformis encoding enzymes responsible for Degraded mixture of PCBs in soil polluted with Aroclor 1242 (Rodrigues et al., 2006)
ortodehalogenation of mono-,di-and trichlorobenzoates
pWW0 plasmid Genetically modified rhizospheric bacteria degraded petroleum (Jussila et al., 2007)
hydrocarbons
Transformed with the degradative plasmid pNB2 Removed 3-chloroaniline pollutant to large extent (Bathe et al., 2009)
Insertion of Azoreductase gene Direct Blue 71 concentration was decreased to 12.6 mg/L in 12 h (Jin et al., 2009)
Recombinant strain was developed by cloning fcb genes Was able to degrade 4-CBA in soil (Massa et al., 2009)
encoding dehalogenase into artificial plasmid pDH5
pWR1 plasmid 3-chlorobenzoate-degradation (Hartmann et al., 1979,
Reineke and Knackmuss,
1980)
TOL plasmid restructured to produce a pathway for processing a new substrate (Ramos et al., 1995)
4-ethylbenzoate
TOL operon of plasmid pWW0 with cognate regulatory gene Conversion of toluene into catechol intermediate was mediated by the TOL (Panke et al., 1998)
xylR enzymes
pPC3 from Arthrobacter globiformis and pE43 from Degradation of ortho and para monochlorobiphenyls (Hrywna et al., 1999)
Pseudomonas aeruginosa encompassing genes for
dehalogenation of chlorobenzoates
Biphenyl dioxygenase, bphA Site-directed mutagenesis of bphA gene resulted in broad congener (Erickson and Mondello, 1992)
specificity with increased activity
bphA1 gene (encoding a large subunit of biphenyl These hybrid strains gained resistance to grow on different aromatic (Suyama et al., 1996)
dioxygenase) is replaced with the todC1 gene (encoding hydrocarbons, and degraded chloroethenes, trichloroethylene and cis-1,
large subunit of toluene dioxygenase) 2-dichloroethylene more efficiently.
Genes coding for toluene 4-monooxygenase were cloned Recombinant organisms showed degradation of TCE (Winter et al., 1989)
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
is mostly not applicable as a permanent solution because contaminants
(heavy metals) remain in the soil, albeit with reduced mobility. None-
theless, it can be part of a management strategy for potential toxic con-
taminants (Ali et al., 2013; Vangronsveld et al., 2009).
Phytovolatilization refers to the most controversial process of
phytoremediation in which plants can be used to take up pollutants
from contaminated soils and transform them into a volatile form
followed by transfer to the atmosphere (Limmer and Burken, 2016).
This technique has limited applications because it can only be utilized
for organic pollutants and certain heavy metals or metalloids (Hg and
Se). Critics argued that phytovolatilization is merely the transfer of pol-
lutants from one compartment of the environment (soil) to another (at-
mosphere) (Ali et al., 2013; Padmavathiamma and Li, 2007). Some low
molecular weight volatile hydrocarbons can experience
phytovolatilization along the transpiration stream (Yavari et al., 2015).
Phytodesalination is the use of halophytic plants to remove salts
from salt-affected soils (Zorrig et al., 2012). Now that water scarcity
and desertification are two threats to human and ecosystem heath,
phytodesalination is emerging as a sustainable option for remediating
salt-affected land (Rabhi et al., 2010). Additionally, halophytic plants
can cope better with heavy metals than glycophytic plants
(Manousaki and Kalogerakis, 2011). Thus, halophytes can be used to si-
multaneously remediate salts and heavy metals in co-contaminated
soils. Phytotransformation (sometimes also termed phytodegradation)
refers to the transformation of organic pollutants by plant enzymes
(dehalogenase, nitroreductase, laccase and oxygenase) in any aerial
part of the plants themselves (Siciliano et al., 2002; Germida et al.,
2002). It mainly depends on plant metabolic activities and is not influ-
enced by rhizospheric microorganisms (Newman and Reynolds,
2004). Phytotransformation is applicable only in remediating organic
pollutants. It has been described in the literature as able to remediate
a variety of pollutants including chlorinated solvents, petroleum hydro-
carbons and synthetic herbicides or pesticides (Dhankher et al., 2012;
Chen et al., 2008; Doty, 2008; Wang et al., 2008). Al-Baldawi et al.
(2015) reported the phytotransformation of diesel-contaminated
water using Scirpus grossus; the results showed promising degradation
rates. In that study, the maximum total petroleum hydrocarbons
(TPHs) were recorded in stems and leaf samples (93.72%, C20-C34).
Finally, Rhizodegradation is the breakdown of organic contaminants
in soils by bacterial and fungal activity enhanced by the rhizospheric ef-
fect of certain vascular plants (Thijs et al., 2016; Truu et al., 2015).
Phytotransformation and rhizodegradation are described in more detail
in the following sections.
3.2. Degradation mechanisms in plants
In green plants the main mechanisms responsible for metabolic pro-
cessing of xenobiotic pollutants were termed the “green liver” by
Sandermann (1994) as an analogy for the processing of xenobiotic pol-
lutants in humans and animals. Recent biotechnological approaches for
bioremediation include biosorption, phytostabilization,
hyperaccumulation, dendroremediation, biostimulation,
mycoremediation, cyanoremediation and genoremediation, which
mainly depend on enhancing or preventing specified gene activities
(Mosa et al., 2016; Malik et al., 2016). This successful technique, how-
ever, faces challenges (Mani and Kumar, 2014). In this concept xenobi-
otic metabolism consists of three biochemical processes:
transformation (phase 1), conjugation (phase 2) and compartmentali-
zation (phase 3) (Reichenauer and Germida, 2008). Fig. 1 illustrates
the fate and degradation processes of a variety of organic xenobiotics
in plants. The only difference between human, animal and plant path-
ways is that plants lack an excretion system.
During phase 1, hydrophobic xenobiotics are chemically modified to
less hydrophobic metabolites by oxidation, reduction or hydrolysis, ox-
idative desulfuration and reduction by cytochrome P450 (Abhilash et
al., 2009). In transformation, the most common and vital reactions –
1587
catalyzed by prominent enzymatic families known as cytochrome
P450 (CYP) and carboxylesterases (CXE) – produce less toxic metabo-
lites (Reichenauer and Germida, 2008). During phase 2 (conjugation),
metabolites of the previous phase are directly converted into hydro-
philic compounds by conjugating with sugars or amino acids and gluta-
thione (Dietz and Schnoor, 2001). Finally, the conjugated molecules are
stored in vacuoles (apoplast) or assimilated into the cell wall in lignin,
pectin, and cellulose fractions in phase 3 (compartmentalization)
(Dietz and Schnoor, 2001). Previous studies (Theodoulou, 2000;
Schröder, 2007) categorized the final part of the xenobiotic degradation
mechanism into two independent processes. One was responsible for
transport and storage in vacuoles, whereas the second confined mole-
cules to the cell wall for binding or excretion. After being translocated
via plant roots, organic xenobiotics undergo different biotransformation
(green-liver model) for detoxification, i.e. sequestration in roots, trans-
port to aerial plant parts, and finally stored in vacuoles or transformed
by volatilization.
3.3. Phytoremediation technology for remediation of organics
After being taken up by plants, organic pollutants may be
translocated and accumulated in roots or shoots. The extent of plant up-
take depends on different physicochemical properties (e.g. octanol-
water partitioning coefficient and acidity constant) of the pollutants
(Gerhardt et al., 2017). The log value of the octanol-water partition co-
efficient (log Kow) provides a direct measure of the hydrophobicity or li-
pophilicity of organic compounds and their tendency to move through
lipid bilayers (Yang et al., 2017). Generally, a higher value of log Kow
(˃3 to 8) represents low to nil solubility in water – the scenario that pre-
vents their entry in plant roots but shows a tight sorption on soil parti-
cles and root surfaces. Organic compounds with a medium log Kow value
(0.5–3) indicate less hydrophobicity and can be taken up by plants.
Lastly, the lower values of log Kow (˂0.5) reveal higher solubility of or-
ganic compounds in aqueous media (Reichenauer and Germida,
2008). Most of the organic pollutants (TPH, polycyclic aromatic hydro-
carbons (PAHs), pesticides, chlorinated solvents and POPs) have log
Kow values in the 3.0–8.5 range and are subsequently resistant to plant
uptake. In addition to log Kow values, pollutant concentration and mor-
phological features of plant species are important factors affecting the
translocation of organic xenobiotics (Arslan et al., 2015).
Organic pollutants trigger drastic and deleterious effects on soils and
plants (Tao et al., 2017). These include deterioration of soil quality, nu-
trient imbalance and extreme hydrophobic conditions. Reduced seed
germination, lower chlorophyll pigment production, reduced root turn-
over, disturbed root/shoot growth and disruption of root architecture
are other potential negative outcomes. In this regard, plant selection is
crucial to phytoremediation strategies. The desirable plant characteris-
tics for successful phytoremediation of organic contaminants are: high
pollutant tolerance, robust plant growth and an effective enzymatic ac-
tion for pollutant degradation. It is well established that specific plants
can degrade a variety of organic xenobiotics such as petroleum hydro-
carbons, PCBs, chlorinated solvents, pesticides, POPs and the aforemen-
tioned ‘dirty dozen’. A vast body of literature confirms the suitability of
grasses, legumes, agronomic crops, ornamentals, herbs and trees for re-
mediation of organics in the environment. Table 2 summarizes different
plants used in decontaminating organic xenobiotics. In general, each
type of plant has a specific set of advantages over the others. Fibrous
roots, higher surface area, deeper penetration and more plant-pollutant
contact are the typical advantages offered by grasses. Symbiotic nitro-
gen fixation is typically associated with legumes, which can restore
the nitrogen pool of polluted soils.
Ornamentals can be used with no risk of introducing contaminants
or their metabolites into the food chain, and they help beautify such dis-
turbed lands. Herbs and trees have additional benefits such as easy
propagation, robust and perennial growth, and phreatophytic roots
with direct contact to the water table (thus facilitating remediation of
1588 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599

Sequestraon,

Phytovolalizaon,

Compartmentalizaon

Aer transport in
shoot Rhizobacteria
and Fungi

Organic pollutants e.g.

Xenobiocs, TNT,
POPs, PCB, VOCs, Rhizodegradaon
Phenols, Dyes,
Hydrocarbons, RDX

Endophyc
Genecally
microbes
Modified Bacteria

Fig. 1. Biochemical/biotechnological treatment of organic pollutants in rhizosphere.

contaminated groundwater as well). Phytoremediation is a site-specific
technology utilizing plant species selected specifically for the site condi-
tions. As well, various attempts have been made to increase the effi-
ciency of phytoremediation. These include soil amendments by adding
inorganic fertilizers, nutrients, bulking agents, aeration, compost and
biochar. The use of plants with associated suitable microbes (contami-
nant degraders and plant growth promoters) is a viable and promising
technology offering a number of benefits.
4. Plant-bacterial partnership for improved remediation of organic
xenobiotics
Many reports mentioned the use of plants alone to remediate or-
ganic pollutants. Nonetheless, the synergistic use of plants with specific
microbes is recommended for greater efficiency. Bacterial associations
with plants naturally encompass a remarkable catabolic diversity to de-
grade, transform or accumulate a broad spectrum of organic compounds

Table 2
Selected case studies (2005–2017) of plants used in different phytoremediation studies for organic pollutants.

Pentachlorophenol Salix Spp. (willow and Plant showed tolerance towards pollutant and degradation in rhizosphere (Mills et al., 2006)
poplar)
Insecticide Poplar and Willow Uptake of dieldrin by plant roots with no biodegradation reported (Skaates et al., 2005)
Chlorinated solvent Poplar Poplar trees reduced TCE evapotranspiration by 90% with specific actions (Weyens et al., 2009)
Explosives(TNT, RDX) Poplar Detoxification of 2, 4, 6-trinitrotoluene (TNT) and 1, 3, 5-Trinitro-1, 3, (Flokstra et al., 2008)
5-triazacyclohexane (RDX). But the plant uptake was very slow
Explosive (TNT) Marigold After 3 months of plant growth more than 90% degradation was reported (Hooda et al., 2016)
Explosives (TNT,RDX) Vetiver grass, canaray Identification of suitable plants for explosive remediation plus specific genes for (Kiiskila et al., 2015)
grass, fox sedge, rice degradation under field conditions
Pesticide (DDT) Willow Uptake and translocation of DDT by plants. Increase availability of DDT by combined (Mitton et al., 2012)
action of Tween 80 and carboxylic acid
Total petroleum Scirpus grossus Phytodegradation variability by mentioned plants under multiple pollutant levels. (Al-Baldawi et al., 2015)
hydrocarbons (TPH)
Polychlorinated biphenyl Zucchini (Cucurbita pepo) The dissipation rates of Aroclor 1242 and soil bacteria abundance were much higher (Qin et al., 2014)
(PCB) with only microbes and control treatments
Organic compounds Multiple plants Phytovolatilization for multiple organic pollutants are reported in review form (Limmer and Burken, 2016)
Explosives Multiple plants A comprehensive review on phytoremediation of explosive contaminated soils are (Kiiskila et al., 2015)
presented
TPH Italian ryegrass Enhancement of phytoremediation efficiency by addition wheat-straw biochar (Cao et al., 2016)
Polycyclic aromatic Medicago sativa Dissipation of selected polycyclic aromatic hydrocarbons (Fu et al., 2012)
hydrocarbons (PAHs)
PAHs Medicago sativa Promotion of rhizodegradtion of pyrene with combined action of plants and microbes (Fan et al., 2008)
TPH Ornamental plants Rhizodegradtion of TPH with ornamental plants; remediation and beatify contaminated (Liu et al., 2008, 2012; Suleiman
soils with specific plants and Bhat, 2003)
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
such as petroleum hydrocarbons, halogenated hydrocarbons, pesticides,
PCBs and PAHs (Xun et al., 2015; Glick, 2010; Meagher, 2000). In
rhizodegradation, microbes mostly target hazardous organic pollutants
that cannot enter the plants due to their hydrophobic nature (Chaudhry
et al., 2005; Gerhardt et al., 2009; Huang et al., 2004, 2005). Bacterial as-
sociations with plants can enhance the phytoremediation of organic
pollutants in the rhizosphere and stabilize them into less harmful me-
tabolites or even mineralize the complex organic compounds into car-
bon dioxide and water (Alexander, 2000; Dzantor, 2007; Macek et al.,
2000).
In such plant-bacterial interactions, bacteria may proliferate around
plant roots (as rhizospheric bacteria) and in the internal tissues (as en-
dophytes) without being pathogenic to their host plant (Compant et al.,
2005; Ryan et al., 2008; Sessitsch et al., 2005). Plants support bacterial
growth by providing residence and nutrients; in turn, bacteria metabo-
lize pollutants in the rhizosphere and endosphere. This synergistic rela-
tionship enhances the role of each partner (Feng et al., 2017; Macci et
al., 2013; Compant et al., 2010). Bacteria help their host plant to over-
come stress caused by pollutants and improve plant growth, thus en-
hancing shoot and root biomass, which in turn supports microbial
populations and the degradation of organic pollutants (Weyens et al.,
2013; Arshad et al., 2007). Moreover, rhizoremediation results in
broader microbial consortia with a faster degradation rate of contami-
nants compared to rates achieved by individual strains (Khan et al.,
2013).
Recently, several studies successfully exploited plant and rhizo-
sphere and endophytic bacterial partnerships to efficiently
phytoremediate organic contaminants from soil and water (Arslan et
al., 2015; Fatima et al., 2015; Khan et al., 2014). Various bacterial strains
with the metabolic potential to degrade specific toxic organic com-
pounds have been successfully isolated and characterized from a wide
range of contaminated environments. Organic compounds can be
biodegraded aerobically or anaerobically (Ghattas et al., 2017). Anaero-
bic bacteria reduced highly chlorinated PCBs into less chlorinated bi-
phenyls (Matturro et al., 2016). Various aerobic bacterial genera
including Burkholderia, Xenovorans, Pseudomonas, Mycobacterium and
Rhodococcus were extensively studied for the bioremediation of organic
pollutants (Song et al., 2015). Rhizospheric, endospermic and
phylospheric microbes can beneficially interact with the host plants, in-
cluding especially mutualism (Berendsen et al., 2012). Special charac-
teristics rendering them suitable for remediation purposes are the
presence of catabolic genes and enzymes (Gkorezis et al., 2016; Chen
et al., 2015; Glick, 2010; Arshad et al., 2007). These specific genes and
enzymes make them candidates for utilizing organic xenobiotics as car-
bon and energy sources. The following section discusses the selected
paradigms of potential applications of plant and associated endophytic
and rhizospheric bacteria, in order to shed light on the improvement
of rhizoremediation.
4.1. Endophytic-plant partnerships
Endophytic bacteria are known to colonize the internal tissues of
plants without serious consequences for their host (Feng et al., 2017;
Mitter et al., 2016). In fact, all plant species harbor endophytic bacteria
by establishing symbiotic or commensalistic relationships (Lòpez-
Fernàndez et al., 2016). Endophytic bacteria inhabit several plant tissues
including roots, stems, leaves, flowers and seeds (Hardoim et al., 2015).
Such bacteria thrive in the root cortex and xylary elements and, most
importantly, they have a recognized ability to degrade and mineralize
organic pollutants, potentially enhancing phytoremediation (Eevers et
al., 2016; Fatima et al., 2015; Weyens et al., 2009, 2010a, 2013). Endo-
phytic bacteria display beneficial effects on their host plants (Andreolli
et al., 2013; Compant et al., 2016; Ma et al., 2016). They assimilate nutri-
ents by phosphorous solubilization and synthesize siderophores (Afzal
et al., 2014). They also promote plant growth by synthesizing phyto-
1589
hormones such as ethylene-1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase and indole-3-acetic acid (IAA) (Taghavi et al., 2009).
Diazotrophic bacteria comprise a group of bacteria known for root
nodulation which fixes atmospheric nitrogen. Research (Afzal et al.,
2014; Compant et al., 2005, 2010; Ma et al., 2011; Ryan et al., 2008;
Weyens et al., 2009) confirms that cultivable endophytes can been
screened and isolated from grasses (tall fescue, ryegrass), herbs (penny-
cress, oak, and arabidopsis), woody trees (poplar, pine, forest trees) and
agronomic crops (wheat, maize and sugarcane). Conventional bioreme-
diation techniques for organic xenobiotics in soils and groundwater
such as soil washing, extraction, pump and treat, application of heat/ox-
ygen, and the addition of different chemicals or surfactants (Khan et al.,
2004) may cause secondary pollution. Endophytic bacteria encompass
diverse secondary metabolite potentials and have been shown to sup-
press plant pathogens by antibiosis, competition for ecological space
or nutrients, and by activating plant-induced systemic resistance
(Álvarez et al., 2015; Doty, 2008; Germaine et al., 2006; Ryan et al.,
2008; Van Aken et al., 2004). Endophytic bacteria have major advan-
tages over free-living rhizobacteria because they live inside the host tis-
sue and thus have less competition for space and nutrients than the
rhizobacteria population, which generally competes with a large num-
ber of soil microorganisms (Compant et al., 2016; Doty, 2008).
The endophytic niche is a hot spot for horizontal gene transfer, and
this naturally occurring process plays a vital role in the adaptation of mi-
crobial communities to environmental stress (Huang et al., 2015;
Taghavi et al., 2005; Wang et al., 2010). For example, the plasmid
pTOM-Bu61 encoding degradation enzymes can transfer naturally to
many plant endophytes and has contributed to efficient degradation
of toluene in poplar plants. Pseudomonas endophytes with plasmids
pWWO and pNAH7 also showed high rates of horizontal transfer into
a number of endophytes (Ryan et al., 2007). In another study, Barac et
al. (2004) reported the conjugative transformation of natural endo-
phytes for enhanced toluene degradation. A toluene-degradation plas-
mid (pTOM) of Burkholderia cepacia was transferred by conjugation
into a natural endophyte inhabiting yellow lupine. Intriguingly,
engineered endophytic bacteria reduced the plant transpiration to 50%
from 70% (as compared to un-inoculated control), and thus reduced
the toxicity in the host plant by degrading toluene. A number of recent
studies (Badri et al., 2009; Oliveira et al., 2014; Phillips et al., 2008;
Syranidou et al., 2016; Weyens et al., 2009) confirmed that endophytic
bacteria perform better in the rhizodegradation of organic pollutants
than do rhizosphere microbes. They can also facilitate the
phytoremediation performance by reducing the biotic and abiotic stress
of the host plant. The whole stress-reducing process is controlled by the
production of ACC-deaminase activity (e.g. Arshad et al., 2007; Saleem
et al., 2007).
The potential advantages of utilizing entophytic bacteria for im-
proved phytoremediation were summarized by Ryan et al. (2008).
They concluded that: a) genetic manipulation of a catabolic pathway in-
volved in xenobiotic degradation was easier in bacteria than in plants;
b) quantitative gene expression analysis of catabolic genes of endo-
phytic populations related to the degradation of pollutants could help
to assess the efficiency of the bioremediation process; and c) toxic pol-
lutants imbibed by the plant could be degraded by endophytes inter-
nally and might reduce phytotoxic effects on herbivorous fauna near
contaminated sites as well. Other studies address disadvantages
(Compant et al., 2016; Ma et al., 2016; Ryan et al., 2008). For instance,
phytoremediation technology: a) is limited to shallow depths of con-
taminated soil; b) may be slower than chemical treatments; (c) is
greatly influenced by choice of the plant species and may show seasonal
variations; (d) is limited by phytotoxic effects of toxic pollutants; and
(e) contaminant residues in plants may enter the food chain.
Another study (Vacheron et al., 2013) provides useful insights into
molecular mechanisms of endophytes' promotion of plant growth.
Given that endophytic bacteria harbor essential catabolic genes which
enhance xenobiotic degradation because they are imbibed and
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Table 3
Applications of plant-endophytic bacteria for remediation of organic contaminants in the soils.

Endophytic bacteria Plant association Pollutant type Case studies

Herbaspirillum sp. (K1) Wheat (Triticum aestivum) PCBs, TCP (Männistö et al., 2001)
Pseudomonas aeruginosa (R75) Wild rye (Elymus dauricus) Chlorobenzoic acids (Siciliano and Germida, 1998)
Pseudomonas putida (VM1450) Pea (Pisum sativum) Pesticide-2,4-D (Germaine et al., 2006)
Burkholderia cepacia (G4) Yellow lupine (Lupinus luteus) VOCs and toluene (Barac et al., 2004)
Burkholderia sp. (HU001) Willow (Salix spp.) Toluene (Weyens et al., 2013)
Burkholderia cepacia strain (FX2) Maize (Zea mays) Toluene (Wang et al., 2010)
Wheat (Triticum aestivum)
Pseudomonas sp. Poplar (Populus trichocarpa) MTBE, BTEX and TCE Germaine et al., 2004; (Moore et al., 2006)
Enterobacter sp. (PDN3) Poplar (Populus trichocarpa) BTEX (Kang et al., 2012)
Pseudomonas putida Pea (Pisum sativum) Naphthalene (Germaine et al., 2009)
Pseudomonas putida (W619-TCE) Poplar (Populus trichocarpa) TCE (Weyens et al., 2010a)
Bacillus cepacia (VM1468) Poplar (Populus trichocarpa) TCE (Weyens et al., 2010b)
Pseudomonas sp. (ITRI53 and MixRI75) Italian ryegrass (Lolium multiflorum) TPH (Afzal et al., 2012; Andria et al., 2009)
Enterobacter ludwigii strains Italian ryegrass (Lolium multiflorum) TPH (Yousaf et al., 2011)
Birdsfoot trefoil (Lotus carniculatus)
Alfalfa (Medicago sativa)
Pantoea sp. strain (ITSI10) Italian ryegrass (Lolium multiflorum) TPH (Yousaf et al., 2010)
Methylobacterium populi (BJ001) Poplar tissues (Populus deltoidesnigra) Explosives-TNT, RDX, HMX (Van Aken et al., 2004)
Achromobacter xylosoxidans Phragmites australis, Catechol and phenol (Ho et al., 2009)
Ipomoea aquatica
Vetiveria zizanioides
Sphingomonas sp. (D4) Cytisusstriatus Hexachlorocyclohexane (Becerra-Castro et al., 2013)
Consortium (CAP9) Agrostis 2,4,6-Trinitrotoluene (Thijs et al., 2014)
Enterobacter sp. (12J1) Wheat (Triticum aestivum) Pyrene (Sheng et al., 2008)
Maize (Zea mays)
Stenotrophomonas Sp. Zuchini (Cucurbita pepo) DDE and DDT (Eevers et al., 2016)
Sphingomonas sp.
Mesorhizobium sp. (HN3yfp) Ryegrass (Lolium multiflorum) Chlorpyrifos (Jabeen et al., 2016)

accumulated by the host plant, it is envisaged that the combined use of
endophytic bacteria and plants would considerably improve remedia-
tion and increase the rate of degradation of a broad spectrum of contam-
inants. Table 3 highlights the combined use of plants and endophytic
bacteria and their potential applications in degrading organic
contaminants.
4.2. Rhizosphere-plant partnership
The rhizosphere serves as a microhabitat around roots and typically
extends 1–2 mm around them (Liu et al., 2014). Generally, vegetated
soils have higher (10–100 times) microbial numbers and diversity com-
pared to bulk soils (Hinsinger et al., 2005; Wenzel, 2009). The larger mi-
crobial biomass in the rhizosphere is a function of rhizodeposits – a set
of organic compounds released by plant roots. These organic com-
pounds are divided into exudates, secretions, mucilages and lysates,
all of which microbes can use as a carbon and energy source (Martin
et al., 2014). Plant roots provide a favorable microenvironment to at-
tract chemotactic bacteria by secreting these organic compounds.
These organic compounds probably also change the physicochemical
properties of soils and potentially increase the bioavailability of pollut-
ants. Moreover, plant roots can also secrete specific organic compounds
(terpenes, flavonoids, phenols) that are structurally analogous to cer-
tain organic contaminants (petroleum hydrocarbons). This phenome-
non can induce the expression of degrading genes and increase the
abundance of the associated rhizospheric microbes (Xun et al., 2015).
Such enhanced catabolic gene expression has been successfully uti-
lized for the mineralization, degradation and sequestration of many or-
ganic pollutants (Andria et al., 2009; Yousaf et al., 2010; Meier et al.,
2016). Initially, rhizobacteria were extensively studied in agriculture
to improve plant-microbe interactions and help reduce stress and en-
hance growth and yield (Bhattacharyya and Jha, 2012; Saleem et al.,
2007). This concept garnered special attention in remediation trials de-
signed to improve the phytoremediation efficiency of organic xenobi-
otics (Yang et al., 2009). Recent studies demonstrated the potential
application of plant and rhizobacteria partnerships to clean up or-
ganic-contaminated soils (Abhilash et al., 2013; Brink, 2016; Qin et al.,
2014; Singh et al., 2016). Several studies have reported the improved
degradation of organic contaminants by combined usage of plant and
rhizobacteria (Table 4). For example, alfalfa in association with Pseudo-
monas fluorescens sp. F113 and Arabidopsis with Pseudomonas putida
Flav1-1 enhanced the degradation of several PCBs (Narasimhan et al.,
2003; Villacieros et al., 2005). Mehmannavaz et al. (2002) also reported
that the inoculating alfalfa with Sinorhizobium meliloti strain A-025, a
symbiotic N2-fixing rhizobacterium, enhanced the biotransformation
of many polychlorinated biphenyls. Especially root exudates tended to
induce catabolic pathways within rhizospheric bacteria, promoting the
degradation of several organic pollutants. The composition of root exu-
dates differs among plants, and many plant species exude phenols that
enrich organic-pollutant-degrading bacteria in the rhizosphere (Salt et
al., 1998).
In degrading PCBs, the presence of salicylate in root exudates report-
edly induces the expression of the bphA gene encoding biphenyl
dioxygenase in Pseudomonas sp. Cam-1. Conversely, terpenes inhibit
the same gene (Master and Mohn, 2001). The above-mentioned exam-
ples underline that plant-microbe interactions often have mutual bene-
fits for plants and rhizobacteria. Rhizobacteria capable of synthesizing
bio-surfactants have been shown to enhance the bioavailability of or-
ganic pollutants by releasing these pollutants from soil particles and ac-
celerating their degradation (Whitfield Åslund et al., 2010). These bio-
surfactants can help desorb many organic xenobiotics from soil parti-
cles. To date, rhizosphere engineering has attracted increasing interest
by modifying this microenvironment with nutrient adjustment, exu-
dates regulation and insertion of transgenic strains.
4.3. Prediction and annotation of genome sequences of xenobiotic-
degrading bacteria
Integration of various bioinformatics tools and molecular biology as
well as biochemistry databases has recently been discussed by Arora
and Bae (2014). The field of bioremediation and biodegradation has en-
tered the (meta) omics era (from genomics to metabolomics) with re-
cent advances being made in next generation sequencing (NGS).
Many contaminant-degrading bacterial genome sequences have been
subjected to NGS technology, and subsequent genes related to contam-
inant degradation have been identified by in silico prediction and
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599 1591

Table 4
Case studies of successful application of plant-rhizosphere bacteria for remediation of organic contaminants in the soils.

Rhizospheric bacteria Plant association Soil contaminant Case studies

Pseudomonas putida (Flav-1-1, PML2) Wall Cress (Arabidopsis thaliana) PCBs (Narasimhan et al., 2003)
Pseudomonas mendocina Black nightshade (Solanum PCBs (Ionescu et al., 2009)
nigrum)
Pseudomonas fluorescens Horseradish (Armoracia rusticana)
Bacillus pumilus Goat willow (Salix caprea)
Sphingobacterium mizutae Tobacco (Nicotiana tabaccum)
Burkholderia cepacia Alfalfa (Medicago sativa)
Burkholderia xenovorans (LB400) Austrian pine (Pinus nigra) PCBs (Leigh et al., 2006)
Goat willow (Salix caprea)
Sphingobium chlorophenolicum (ATCC 39723) Wheat (Triticum aestivum) Pentachlorophenol (Dams et al., 2007)
Streptomyces strains (M7, A5, A2, and A11) Maize (Zea mays) Lindane Pesticides (Álvarez et al., 2015)
Endosulfan degrading rhizospheric strains Khus (Vetiveria zizanioides) Endosulfan Pesticides (Singh et al., 2016)
(EAG-EC-12,13,14,15)
Pesticide tolerant rhizobacterial strains Okra (Abelmoschus esculentus) Bifenthrin Pesticide (Ahmad et al., 2015)
Sphingomonas herbicidovorans (AB042233) Maize (Zea mays) Hexachlorocyclo-hexane (Abhilash et al., 2013)
insecticide
Pseudomonas Sp. (ADP rifR) – Atrazine pesticides (Shaw et al., 2006; Silva et al.,
2004)
Pseudomonas putida (W619-TCE) Poplar (Populus deltoids) TCE (Weyens et al., 2015)
Indigenous degraders Switchgrass (Panicum virogatum) PCBs (Chekol et al., 2004)
Pseudomonas fluorescens (F113 Derivatives) Alfalfa (Medicago sativa) PCBs (Villacieros et al., 2005)
Rhizospheric bacterial strain (TP13) Tomato (Solanum lycopersicon) Phenol (Wang et al., 2014)
Indigenous microbes Alfalfa (Medicago sativa) Benzo[a]pyrene (Fu et al., 2012)
Bacterial consortia Maize (Zea mays) Petroleum hydrocarbons (Shahzad et al., 2016)
Fungal rhizospheric strain (Rhizophagus irregularis) Wheat (Triticum estivum) PAHs (Ingrid et al., 2016)
Indigenous degraders Italian ryegrass (Lolium perenne) Pentachlorophenol (He et al., 2005)
Azospirillum lipoferum Wheat (Triticum estivum) Petroleum hydrocarbons (Muratova et al., 2012)
Azospirillum brasilense Tall fescue (Festuca arundinacea) PAHs (Huang et al., 2004)
Enterobacter cloacae (CAL 2)
Pseudomonas putida (UW3, PCL1444)
Pseudomonas putida PCL1444 Bermuda grass (Lolium Naphthalene (Kuiper et al., 2004)
multiflorum)

annotation of metabolic pathways. This has improved our holistic un-
derstanding of the metabolic network of an interesting bacterium (Lee
et al., 2012; Schneiker et al., 2006; Vikram et al., 2013 ; Kumar et al.,
2012; Vilchez-Vargas et al., 2013). For instance, analysis of the complete
genome sequence revealed that Cupriavidus necator JMP134, which uti-
lizes aromatic substituted compounds as the sole carbon and energy
source, encompasses 300 genes related to degradation pathways for
various aromatic contaminants (Perez-Pantoja et al., 2008; Lykidis et
al., 2010). Another in silico study identified aromatic degradation path-
ways such as an ortho-pathway for protocatechuate and catechol,
phenylacetate, homogentisate and N-heterocyclic aromatic pathways
in the genome of Pseudomonas putida KT2440 (Jiménez et al., 2002).
The complete genome sequences have not only helped to predict
genes and their functions but also to identify novel biocatalysts for bio-
degradation (Vilchez-Vargas et al., 2013). Kim et al. (2008) integrated
genomic, proteomic and metabolomic approaches and identified 27 en-
zymes to construct the complete metabolic pathway for pyrene degra-
dation in Mycobacterium vanbaalenii PYR-1.
5. Transgenic plants for remediation of organic contaminants
Despite many studies having reported the successful transfer of
phytoremediation from the greenhouse to the field, this approach still
lacks widespread application. One possibility to overcome this might
be to develop transgenic plants to metabolize/degrade organic xenobi-
otics as well as to improve the ability of plants to complete the whole
process more rapidly. Identifying suitable genes for enhancing the deg-
radation/mineralization of xenobiotic pollutants, followed by their iso-
lation and transformation to a candidate plant, are the key steps
during the development of transgenic plants. Initially, transgenic plants
were produced to reduce insect damage as well as increase crop yields
in agriculture (Abhilash et al., 2009). They later gained acceptance in en-
vironmental sciences and were initially used in remediating heavy
metal-contaminated soils (Misra and Gedamu, 1989; Vatamaniuk et
al., 1999). For example, Misra and Gedamu (1989) reported Nicotiana
tabaccum as a transgenic plant expressing a metallotheionein gene for
higher tolerance to and uptake of cadmium. Tobacco (Solanum nigrum)
was the first to be used as a genetically modified plant for organic xeno-
biotics such as explosives and halogenated organics (French et al., 1999
and Doty et al., 2000). This species was modified to contain transgenes
for metabolizing xenobiotics and increasing resistance against pollut-
ants. The literature extensively documents the efficiency of transgenic
plants in removing chlorinated biphenyl, explosives and phenolic com-
pounds (Abhilash et al., 2009; Aken and Doty, 2009; Eapen et al., 2007).
5.1. Transgenic plants for stimulated phytodegradation/rhizodegradation of
organic pollutants
5.1.1. Herbicides and pesticides
In the agricultural sector, herbicides have become paramount in
preventing yield losses of valuable crops by weed infestation
(Kawahigashi et al., 2008; Keller et al., 2014). These herbicides, how-
ever, can damage the environment as pollutants (Gill and Garg, 2014;
Seth et al., 2012). Moreover, repeated application and extensive use
can lead to the development of herbicide-resistant weeds. This phe-
nomenon requires applying ever greater quantities, polluting soils and
nearby water bodies (Kawahigashi et al., 2006, 2007). Consequently,
there is an urgent need for the detoxification of herbicides from polluted
environments. Transgenic plants tolerant to herbicides have been suc-
cessfully developed and used in agriculture for many years. Among di-
verse enzymatic groups, two major enzymes known as cytochrome
P450 and glutathione S-transferase are key players for enhanced herbi-
cidal degradation (Duhoux et al., 2015). For example, the chemical con-
tamination by agrochemicals can be reduced by introducing transgenic
plants (Macek et al., 2008). Similar findings were reported by
Kawahigashi et al. (2006), who studied rice plants showing overexpres-
sion of transgenes such as CYP1A1, CYP2B6 and CYP2C19. The results
1592 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599

demonstrated that transgenic rice plants were more tolerant to herbi-
cides than non-transgenic plants due to an adapted metabolism.
Transgenic rice plants carrying CYP1A1 showed herbicide tolerance
towards atrazine, diuron, quizalofop-ethyl, and other herbicides and
they reduced the levels of atrazine and simazine in hydroponic solutions
(Kawahigashi et al., 2007). Small-scale batch and meso-scale green-
house experiments confirmed the ability of transgenic rice plants carry-
ing the CYP2B6 gene to reduce residual quantities of a group of
herbicides in soil and water (Kawahigashi et al., 2005a,b). Sixteen cyto-
chrome P450 species responsible for herbicide detoxification in Lolium
rigidum were reported by Fischer et al. (2001). Transgenic rice (having
a human gene CYP1A1) showed increased tolerance and degradation
activities on 10 of 13 herbicides belonging to different chemical groups
(Kawahigashi et al., 2008). Transgenic plants used for selected herbi-
cides and pesticides are summarized in Table 5.
5.1.2. Halogenated organic compounds and explosives
Halogenated organic compounds comprise a group of organic com-
pounds containing halogen atoms. They include common environmen-
tal pollutants such as chloroform, benzene, tetrachloride, vinyl chloride
and trichloroethylene (TCE). All can cause serious health problems and
are reported as being carcinogenic, neurotoxic and hepatotoxic (Chiu et
al., 2013). TCE and its derivative vinyl chloride (produced by microbial
respiration) are heavily used in many industries and can persist in the
environment for decades (Doty et al., 2017). Another very important
halogenated organic compound, ethylene dibromide (EDB), is used as
a fumigant and gasoline additive. Chloroform, a famous disinfection
agent to treat drinking water, is also considered a harmful pollutant
(Huang et al., 2014). A very common pollutant associated with petro-
leum industries, benzene, is a proven human carcinogen (Bahadar et
al., 2014).
To remediate such pollutants and many others, CYP2E1 is an exten-
sively studied cytochrome that can be used in transgenic plants (Zhang
et al., 2013; Abhilash et al., 2009). Transgenic tobacco plants (having
human cytochrome) can metabolize TCE, vinyl chloride, chloroform,
toluene and benzene (Doty et al., 2000; Doty, 2008). The same studies
confirmed the suitability of the bacterial gene (dhlAB) from
Xanthobacter for removing 1, 2-dichlorethene in plants. After successful
metabolism of TCE by transgenic tobacco plants (640-fold increase
versus non-transgenic plants), introducing P450 2E1 in deep-rooted,
fast-growing trees was suggested by Doty et al. (2000). It may stimulate
the uptake and dissipation of these organic contaminants. Use of
bacteria genes (XplA and XplB) from Rhodococcus conferred enhanced
removal of RDX in plants (Jackson et al., 2007). Similarly, a transgenic
hybrid poplar having overexpression of a rabbit gene (CYP450 2E1)
served to boost the removal of TCE, vinyl chloride, carbon tetrachloride,
benzene and chloroform from hydroponic solution (Doty et al., 2007).
Explosive pollutants are toxic and persistent in the environment at
military sites worldwide, where they can cause serious problems in
soil and groundwater (Seth et al., 2012). Among explosives, nitro-
substituted compounds such as 2, 4, 6-trinitrotoluene (TNT),
hexahydro-1, 3,5-trinitro-1,3,5-triazine (RDX) and glycerol trinitrate
(GTN) are listed as ‘priority pollutants’ and ‘possible human carcino-
gens’ by the U.S. EPA (Singh, 2013). Based on the literature, Yew et al.
(2016) documented that nitro-substituted explosives are toxic for all
classes of organisms (bacteria, algae, plants and mammals). Traditional
remediation techniques of explosives-contaminated sites are costly,
damaging to the environment and practically not feasible (Kiiskila et
al., 2015). As one of the top ten technologies for improving human
health, phytoremediation is an attractive alternative due to its
ecological and environmental benefits (Kiiskila et al., 2015; Daar et al.,
2002).
For enhanced utilization of phytoremediation in the remediation of
organic contaminants, different approaches may be combined in an in-
tegrated manner rather than focusing solely on endophytic/
rhizospheric plant partnerships and transgenic plants. These
approaches include the use of agronomic practices, omics technolo-
gies, transgenic, somaclonal variants and mutant lines. Recently,
Gerhardt et al. (2017) reviewed all the above-mentioned approaches
and their usage in phytoremediation. Thijs et al. (2016) recently pro-
posed the concept of engineering the metaorganisms to improve
phytoremediation at the field scale.

Table 5
Summary of transgenic plants used for selected herbicides and pesticides.

Target plant Gene (s) Enzymes involved Source Transgene effects References

Nicotiana tabaccum CYP105A1 Cytochrome Streptomyces Resistance to sulfonylurea (O'Keefe et al.,

P450 monoxygenase 1994)
Oryza sativa CYP2B6 Cytochrome Human Tolerance to group of herbicides (Hirose et al.,
P450 monoxygenase 2005)
Oryza sativa CYP2B22,CYP2C49 Cytochrome Pig Tolerance to several herbicides (Kawahigashi et
P450 monoxygenase al., 2006)
Nicotiana tabaccum CYP71A10 Cytochrome Glycine max Tolerance to phenyl urea herbicide (Siminszky et al.,
Arabidopsis thaliana P450 monoxygenase 1999)
Nicotiana tabaccum GstI-6His Glutathione S-transferases Maize Higher tolerance to variety of Aroclor (Karavangeli et al.,
(GST I) 2005)
Medicago sativa atzA Atrazine chlorohydrolase Bacteria Enhanced metabolic activity against atrazine (Wang et al.,
Nicotiana tabaccum 2005)
Arabidopsis thaliana Mn peroxidase Peroxidases Coriolus versicolor Enhanced removal of pentachlorophenol (PCP) (Iimura and Ka,
Nicotiana tabaccum gene 2002)
Oryza sativa Protox Protoporphyrinogen IX Bacillus subtilis Tolerance to diphenyl ether herbicide (Jung et al., 2008)
oxidase
Nicotiana tabaccum CYP1A1 Cytochrome Human Enhanced metabolic activity again metamitron Bode et al.,2004a
CYP1A2 P450 monoxygenase
Nicotiana tabaccum CYP1A1 Cytochrome Human Enhanced metabolic activity again atrazine (Ma et al., 2013)
CYP1A2 P450 monoxygenase
Nicotiana tabaccum CYP76B1 Cytochrome Helianthus tuberosus Tolerance to herbicide (Didierjean, 2002)
Arabidopsis thaliana P450 monoxygenase
Solanum tuberosum CYP1A1 Cytochrome Human Enhanced tolerance and metabolism of (Inui et al., 1999)
P450 monooxygenases herbicides
Solanum tuberosum CYP1A1, CYP2B6, Cytochrome Human Enhanced tolerance and metabolism of (Inui et al., 2000)
CYP2C19 P450 monooxygenases herbicides
Oryza sativa CYP1A1 Cytochrome P450 Human Enhanced herbicide metabolism (Kawahigashi,
monooxygenases 2009)
Nicotiana tabaccum ophc2 Organophosphorus Pseudomonas Enhanced degradation of organophosphorus (Wang et al.,
hydrolase (OPH) pseudoalcaligenes (methyl parathion). 2008)
 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
6. Risks and mitigation through biotechnological advances associ-
ated with using genetically modified plants and microbes
Although the use of advanced recombinant DNA technology to pro-
duce genetically modified organisms and the application of these mod-
ified microorganisms to remove environmental pollutants is very cost-
effective and environmentally friendly approach, field releases of trans-
genic microorganisms have been quite limited due to risks in the open
environment (Singh, 2013). Since the 1980s, advances in producing re-
combinant microbes has generated possible risks associated with the
release of such genetically engineered microorganisms (GEMs) in the
open environment (Singh et al., 2011). The regulatory hurdles in the re-
lease of GEMs are mostly due to safety concerns, legislative issues and
potential risks perceived by the public (Kritikos, 2018). For instance,
Chakrabarty et al. (1975) genetically engineered Burkholderia cepacia
to remove Agent Orange at the U.S. Air Force storage facility but failed
to receive approval for a field application because it raised concerns re-
garding its potential impact on the environment. Two international or-
ganizations, the United Nations Environmental Program (UNEP) and
the Organization for Economic Co-operation and Development
(OECD), are involved in the regulatory framework of genetically modi-
fied organisms (GMOs) or living modified organisms (LMOs). In prac-
tice, experimental studies related to the risk assessment often do not
incorporate all the norms suggested by regulatory agencies. It was
strongly suggested that the fate of the recombinant genes of the candi-
date strain has to be assessed in comparison to the corresponding wild-
type strain in the containment system (characterized by all relevant
field conditions) prior to its release. More importantly, the heterologous
gene in the host genome should not display horizontal transfer to any
large extent (Smith et al., 1992). The other challenges include assessing
the possible risks on ecosystem diversity and function.
Many microbial organisms commonly used for bioremediation may
have harmful impacts. For example, Burkholderia cepacia has bioremedi-
ation potential for toxic nitro-compounds; however, it has also been
identified as a causative agent of cystic fibrosis in humans and also pre-
sented multiple antibiotic resistances. For this reason the U.S. EPA raised
concerns over its use as an environmental protection agent. Another ex-
ample is the biodegradation of toluene, which is mediated by the unreg-
ulated activity of efflux pumps in Pseudomonas. Since these pumps
pump out various antibiotics and biocides (Davison, 2005), selecting
such microbes may increase the risk of introducing multidrug-resistant
bacteria into the environment and may eventually spread and produce
more virulent progeny (Davison, 2005). Another major concern in the
development of recombinant microbial systems is the horizontal trans-
fer of heterologous genes among the host organism and other closely re-
lated species living nearby. This may involve transduction and
conjugation. DNA transfer may occur even among interspecies under
different ecological conditions.
One of the major challenges related to both the public and scientific
communities is the containment of recombinant microbes after comple-
tion of the bioremediation process to minimize undesirable environ-
mental outcomes (Khan et al., 2016; Prakash et al., 2011). These
concerns reflect the capability of such GEMs to disrupt ecosystems by
further spreading after the degradation of the pollutant. To tackle
these risks, attempts are being made to control and track GEMs, and a
set of guidelines has been proposed by the International Technical
Guidelines for Safety in Biotechnology for microorganisms.
Designing GEMs by applying suitable genetic approaches to contain
the bacterial system may help minimize the anticipated risks. For in-
stance, transposition vectors constructed without antibiotic-resistant
genes are considered to be safe in the environment. Another contain-
ment approach mainly involves designing “suicidal GEMs”, but the tech-
nology has yet to be put into practice (Chamekh, 2015). This concept
involves programming the death of GEMs after toxin degradation in
the target site (Paul et al., 2005). Based on this concept, a genetic design
model was recently developed to control the fate of the GEMs after they
1593
have degraded the contaminants. The proposed design integrates the
killer–anti-killer genes, whose expression is triggered by the pollutant.
Upon pollutant degradation, killer genes are activated, eradicating the
GEMs. These killer genes, located on plasmids, can also induce death
in the microbial recipient upon a gene transfer, therefore mitigating
horizontal gene transfer. Such bacterial containment systems are
among the most promising approaches to mitigate the undesirable af-
fects associated with the release of GEMs in the open environment
(Paul et al., 2005). For example, the lethal gef gene is located on the
mini-Tn5 transposon regulated by the LacI repressor. Moreover, lacI
gene activation depends on the XylS positive activator, which is active
only in the presence of 3-methylbenzoate. Therefore, the cell remains
viable as long as 3-methylbenzoate is present. Depletion of the chemical
signal may reflect degradation or escape into the environment; the cell
is killed by the action of the lethal gene gef (Davison et al., 2002; Torres
et al., 2003).
A major drawback of the containment systems is that cells in some
cases may cease to respond to the toxic effect of the lethal gene if muta-
tion occurs. To circumvent this problem, double-contained strains have
been designed. For example the asd gene encodes diaminopimelic acid
synthesis, which is essential for cell wall synthesis. Pseudomonas putida
with a deletion of the asd gene was complemented with an external asd
gene under an XylS positive activator, the same activator that sup-
presses gef expression as described above. Thus, a strain deprived of 3-
methylbenzoate would die not only as a result of the lethal activity of
the gef protein but also due to the lack of diaminopimelic acid
(Ronchel and Ramos, 2001). These advanced technologies serve as a
good platform for future studies and may be introduced in the environ-
ment to control the proliferation of genetically modified microorgan-
isms after they fulfill the purpose of biodegrading pollutants.
7. Conclusions and recommendations
Organic xenobiotics are ever-increasing in the environment due to
rapid industrialization, population increase, mechanized agricultural
practices and military activities. Rapid environmental clean-up and sta-
bilization of organic xenobiotics is highly desirable to support the sus-
tainability of natural ecosystems. The presence of plants and
associated microbes in contaminated areas is due to the xenobiotic-re-
sistant genes they naturally contain; these genes help chelate, traffic
and sequestrate xenobiotics. The biological approaches involving use
of efficient plants and rhizobacteria/genetically engineered microbes
are always safer and preferable over costly physicochemical methods
of environmental remediation.
The bottleneck in using such biological techniques is the slow
growth kinetics of microbial metabolism, which may be caused by envi-
ronmental factors or even inhibited by pollutants. The result is an accu-
mulation of these pollutants of concern. To overcome these challenges,
future research must be directed towards developing efficiently grow-
ing rhizobacterial species capable of promoting growth of associated
plant species. Such combinations would greatly improve environmental
remediation. Bioremediation efficiency can be optimized by inserting
special features into the respective microbes. The required traits in mi-
crobes can be inserted via natural gene transfer or recombinant DNA/
RNA technology. Phytoremediation efficiency can be augmented by im-
proving plant-microbe interactions via rhizosphere engineering. Syner-
gistic use of suitable plants and efficient microbes can greatly enhance
remediation efficiency. Furthermore, this partnership can also be in-
creased by inoculating seeds/plants with specific endophytic and
rhizospheric microbes. Microbial strains having both features – plant-
growth-promoting as well as contaminant-degrading abilities – offer
an efficient, cost-effective and environmentally safer option for biore-
mediation and phytoremediation. Lastly, the use of genetically modified
plants can further exploit this plant microbe partnership and offer time-
efficient remediation options.
1594 I. Hussain et al. / Science of the Total Environment 628–629 (2018) 1582–1599
Environmental biotechnology can promote the study of the specific
genes responsible for xenobiotic resistance and for the nature of the up-
take, accumulation and detoxification pathways of xenobiotics. There-
fore, xenobiotic-resistant and tolerant accumulator plants and
associated microbial species can be isolated and further modified for
the decontamination of polluted sites by using DNA and RNA recombi-
nant technologies, which tackle xenobiotics by activating the responsive
genes of the respective organisms. Another helpful approach for the ex-
ploration and activation of stress-related genes is to investigate extra-
cellular and intracellular signal transduction processes of such plants
and microbes. This can be done by using the Ca calmodulin system, hor-
mones, ROS signaling, and the mitogen-activated protein kinase
(MAPK) phosphorylation flow. Such strategies can open a gateway for
a more thorough understanding and improvement of phytoremediation
techniques.
Another challenge of future research will be to counteract the pro-
duction of undesirable proteins (enzymes) that potentially disturb the
functioning of the rhizosphere community or the metabolism in plants.
Using genetically modified microbes and plants poses risks in the open
environment. The regulatory hurdles regarding their release are mostly
due to safety concerns, legislative issues and public perceptions. The po-
tential risks associated with altered microbes and plants can be tackled
by adhering to regulatory guidelines and applying biotechnological ad-
vances. Certain risks may still come to light and would then require fur-
ther investigation to generate appropriate technical regulatory
guidelines.
Acknowledgments
This work was partly funded by AIT Austrian Institute of Technology
(project number 1.G2.00126.0.0) Vienna, Austria, Department of Health
and Environment, under the umbrella of business unit Environmental
Resources and Technologies. Imran Hussain gratefully acknowledges
the Higher Education Commission (HEC Overseas Scholarship Phase I,
Batch 4) of Pakistan for financial support during his four-year stay for
undertaking a doctoral degree in Vienna.
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