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Chemosphere 90 (2013) 1317–1332

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Review

Plant–bacteria partnerships for the remediation of hydrocarbon contaminated soils

Sumia Khan, Muhammad Afzal ⇑, Samina Iqbal, Qaiser M. Khan
National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan

h i g h l i g h t s

" Plant–bacteria partnerships is an emerging pollutant remediation approach.

" Plant associated rhizo- and endophytic bacteria can enhance pollutant degradation.
" Plant stimulates the bacteria to degrade organic contaminants in the soil.
" Plant associated-bacteria can reduce phytotoxicity and evapotranspiration of hydrocarbons.

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

Article history: Plant–bacteria partnerships have been extensively studied and applied to improve crop yield. In addition
Received 5 June 2012 to their application in agriculture, a promising field to exploit plant–bacteria partnerships is the remedi-
Received in revised form 8 September 2012 ation of soil and water polluted with hydrocarbons. Application of effective plant–bacteria partnerships
Accepted 10 September 2012
for the remediation of hydrocarbons depend mainly on the presence and metabolic activities of plant
Available online 8 October 2012
associated rhizo- and endophytic bacteria possessing specific genes required for the degradation of
hydrocarbon pollutants. Plants and their associated bacteria interact with each other whereby plant sup-
Keywords:
plies the bacteria with a special carbon source that stimulates the bacteria to degrade organic contami-
Hydrocarbons
Phytoremediation
nants in the soil. In return, plant associated-bacteria can support their host plant to overcome
Plant growth promotion contaminated-induced stress responses, and improve plant growth and development. In addition, plants
Biodegradation further get benefits from their associated-bacteria possessing hydrocarbon-degradation potential, lead-
Rhizobacteria ing to enhanced hydrocarbon mineralization and lowering of both phytotoxicity and evapotranspiration
Endophytic bacteria of volatile hydrocarbons. A better understanding of plant–bacteria partnerships could be exploited to
enhance the remediation of hydrocarbon contaminated soils in conjunction with sustainable production
of non-food crops for biomass and biofuel production.
Ó 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1318
2. HC remediation by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1318
3. HC remediation by plant–bacteria partnerships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1319
4. RB with the capacity to enhance HC phytoremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1322
5. EB with the capacity to enhance HC phytoremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1324
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326
7. Future prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1326

⇑ Corresponding author. Tel.: +92 41 2651475; fax: +92 41 2651472.

E-mail address: manibge@yahoo.com (M. Afzal).

0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2012.09.045
 Author's personal copy
1318 S. Khan et al. / Chemosphere 90 (2013) 1317–1332
1. Introduction
Although petroleum is one of the main components of our mod-
ern industrial society, during the processes of extraction, refining,
transport and use, an increasing number of sites is seriously con-
taminated by hydrocarbons (HCs) world-wide (Banks et al., 2003;
Truu et al., 2007; Nam et al., 2008; Rojo, 2009). Presence of HC pol-
lutants in the environment not only adversely affect human health
but also plant growth and development (Joner et al., 2004; Meudec
et al., 2007; Euliss et al., 2008; Peng et al., 2009; Nie et al., 2011;
Rojo-Nieto and Perales-Vargas-Machuca, 2012). Furthermore,
these strongly influence soil chemical properties, and soil microor-
ganisms’ population and activities (MacNaughton et al., 1999;
Lapinskienė et al., 2006; Kisic et al., 2009; Barrutia et al., 2011;
Guo et al., 2012; Leme et al., 2012). The toxicity of low molecular
weight HCs is considered to inhibit plant growth and development.
Furthermore, hydrophobic properties of HCs reduce the ability of
plants and microorganisms to absorb water and nutrients from
the soil (Kirk et al., 2005; Kechavarzi et al., 2007; Nie et al., 2011).
Therefore, the remediation of HC contaminated soils is one of the
hot topics in the field of environmental sciences and engineering
(Ciric et al., 2009; Gan et al., 2009; Gurska et al., 2009; Kathi and
Khan, 2011; Nie et al., 2011; Basumatary et al., 2012).
At present, remediation treatments available for HC polluted
soils are usually expensive and environmentally invasive (Pandey
et al., 2009; Weyens et al., 2009a, 2010b). As a complement to tra-
ditional remediation treatments, currently emerging phytoremedi-
ation technology might provide a more cost-effective and
environmentally friendly HC remediation approach. Phytoremedia-
tion involves the use of plants to extract, sequester, and/or detoxify
hazardous organic and inorganic contaminants from soil, water and
air (Salt et al., 1995; Meagher, 2000; Pilon-Smits, 2005; Sandhu
et al., 2007; Ali et al., 2012; Kabra et al., 2012). As plants use sun-
light, water and carbon dioxide for their growth and development,
the in situ application of this technology requires low inputs and
maintenance. Furthermore, it causes minimum site disturbance,
therefore it is highly acceptable in community (Weyens et al.,
2010b, 2010c; Nesterenko-Malkovskaya et al., 2012). However,
there are some disadvantages/limitations of phytoremediation,
i.e. it is a long time process, not as efficient in the presence of high
concentration of pollutants. The zone of effectiveness of phyto-
remediation is generally dictated by the depth of root growth, only
the plant with extensive root system can be applied. Moreover, con-
taminants which are highly sorbed to soil particles may not be
treatable due to limited bioaccessibility or bioavailability. More
importantly, the effectiveness of phytoremediation varies with
environmental conditions, including soil physicochemical proper-
ties and seasonal temperature fluctuations (Segura et al., 2009).
Although there have been some reports whereby plants alone
were used successfully for bioremediation of HC from contami-
nated soils (Liste and Alexander, 2000; Newman and Reynolds,
2004; Euliss et al., 2008; Gerhardt et al., 2009; Peng et al., 2009;
Zhang et al., 2012), the use of plants in conjunction with HC-
degrading and/or plant growth-promoting bacteria (PGPB) offers
much more potential for the remediation of HC contaminated soils
(McGuinness and Dowling, 2009; Afzal et al., 2011; Teng et al.,
2011; Yousaf et al., 2011). Bacteria possessing HC-degradation
pathways and metabolic activities improve plant tolerance to HC
pollutants by degrading these organic compounds (Escalante-
Espinosa et al., 2005; Alarcón et al., 2008; Robert et al., 2008). Addi-
tionally, PGPB mitigate plant stress responses, and enhance plant
growth and development (Weyens et al., 2009c, 2009d; Golubev
et al., 2011; Afzal et al., 2012). Beside the fact that bacterial appli-
cation to plants vegetated in contaminated soil enhances biomass
production, for the purposes of its use in different industries, the
exploration of plant–bacteria associations for the reclamation of
polluted environments is an emerging field (Weyens et al.,
2010a, 2010b, 2010c). Due to high potential of plant–bacteria part-
nerships to remediate contaminated soil and water, the use of this
technology to remediate different environmental pollutants has
been increasingly investigated (Germaine et al., 2006, 2009; Wey-
ens et al., 2009c, 2010b, 2010c; Glick, 2010; Yousaf et al., 2010b;
Afzal et al., 2011, 2012).
Plant-associated bacteria, such as rhizobacteria (RB) and endo-
phytic bacteria (EB), have been shown to contribute to biodegrada-
tion of toxic organic compounds in polluted soil and could have
potential for improving phytoremediation (Siciliano et al., 2001;
Germaine et al., 2006, 2009; McGuinness and Dowling, 2009;
Weyens et al., 2009c, 2009d; Yousaf et al., 2010a; Afzal et al.,
2012). RB colonize the root environment, where they can involve
in the degradation of organic contaminants including HCs (Alkorta
and Garbisu, 2001; Glick, 2003; Abhilash et al., 2011; Afzal et al.,
2011). RB which exert a beneficial effect on the plant being colo-
nized are termed ‘plant growth promoting rhizobacteria’ (PGPR).
PGPR may benefit the host by causing plant growth promotion or
biological disease control. EB colonize in the internal tissues of
the plant and generally do not confer pathogenic effects to their
host (Compant et al., 2010). HC-degrading EB can produce various
enzymes to degrade HCs and reduce both the phytotoxicity and
evapotranspiration of volatile HCs (Germaine et al., 2009; Weyens
et al., 2009d; Yousaf et al., 2011; Li et al., 2012). Recently,
HC-degrading bacteria (up to 9  107 cells g1) were isolated from
the surfaces of leaves of two legumes, peas and beans (Ali et al.,
2012). It was recommended that phytoremediation through phyl-
osphere technology could be useful in remediating atmospheric
HC pollutants.
Although plant–bacteria partnerships have been extensively
investigated; most of the studies aimed to explore either plant–
pathogen interactions or nitrogen fixation. From the last decade,
the ecology of bacteria in the rhizosphere and endosphere of differ-
ent plants was focused to improve the phytoremediation of soil
contaminated with different organic pollutants including HCs
(Siciliano et al., 2001; Yousaf et al., 2010a). More recently, many re-
ports pointed out that successful colonization of inoculated RB
and/or EB in different compartments of plant is an important factor
to enhance the efficiency of phytoremediation (Nie et al., 2011;
Yousaf et al., 2011; Afzal et al., 2012; Wang et al., 2012a). In this
paper, we review the current knowledge about the plant–bacteria
partnerships that can be applied for the remediation of HCs from
the polluted environment. Furthermore, colonization and meta-
bolic activities of plant growth-promoting and HC-degrading RB
and EB regarding phytoremediation of HCs will be addressed.
2. HC remediation by plants
It is a general concept that plants are basic source of fiber, fuel
and food. However, their possible roles to remove contaminants
from the soil and water have been recognized in the last two dec-
ades. The particular tendency to tolerate, immobilize, accumulate,
degrade and remove large amount of organic pollutants from the
soil and water has opened up the possibility to use plants for the
remediation of HCs polluted environment (van der Lelie et al.,
2001; Garbisu et al., 2002; Peña-Castro et al., 2006; Ying et al.,
2011). Plants can absorb minute quantity of HCs from the soil
and translocate them into their different parts (McCrady et al.,
1987; Sheng et al., 2008a). HCs uptake and accumulation in the
root and shoot of plants vegetated in contaminated soil are associ-
ated with contaminant concentration in the soil (Brooks et al.,
1998; Wild et al., 2005; Parrish et al., 2006; Lu et al., 2010b). Once
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S. Khan et al. / Chemosphere 90 (2013) 1317–1332
HCs move into the plant, these may have multiple fates; some HC
compounds can be sequestered in root tissue, some can be trans-
ported into shoots and then leaves, where they can be stored in
the vacuole or volatilized into the atmosphere (McCrady et al.,
1987; McFarlane, 1995; Reichenauer and Germida, 2008; Sheng
et al., 2008a). The term ‘‘green liver’’ was coined by Sandermann
(1992) to describe the metabolic processing of foreign chemicals
(xenobiotics) by plants. Sandermann compared the metabolism
of xenobiotics in plants with that in the mammalian liver, which
can be divided into three phases: transformation (Phase I), conju-
gation (Phase II), and excretion (Phase III). In plants, the metabo-
lism of xenobiotics also works in three phases, except that there
is no excretion system analogous to the mammalian one. Thus, in
plants Phase III is the step where the modified xenobiotics are
sequestered by the cells, that is, the chemicals are ‘‘stored’’ in the
vacuole or on the cell walls. Plants may therefore be considered
as a ‘‘green liver’’, acting as an important global sink for environ-
mental chemicals. It is also well documented that plants can de-
grade/transform organic pollutants into less toxic form, through
oxidation and/or reduction, by the virtue of their own enzymes
(Salt et al., 1998; Dietz and Schnoor, 2001; Pilon-Smits, 2005;
Couto et al., 2011; Basumatary et al., 2012). Most likely, plants
produce organic contaminant-degrading enzymes to overcome dif-
ferent allelochemicals released by competing organisms, including
microbes, insects and other plants (Singer, 2006).
Majority of the HCs cannot move considerably into plants from
the soil due to a log Kow > 4, i.e. equilibrium constant that provide
an indication of constituent sorption onto soil (Alkorta and
Garbisu, 2001; Newman and Reynolds, 2004; Meng et al., 2011).
Main mechanism involved in the phytoremediation of HCs is be-
lieved to be biodegradation in the rhizosphere (Kechavarzi et al.,
2007; Yi and Crowley, 2007; Vangronsveld et al., 2009; Tang
et al., 2010b). In the rhizosphere, plants support HC-degrading mi-
crobes that are involved in the mineralization of HCs (Donnelly
et al., 1994; Muratova et al., 2003; Juhanson et al., 2007; Nie
et al., 2009; Shibata and Robert, 2009). A number of plant species
were tested for their abilities to tolerate and remove the HCs from
the soil. Among others, alfalfa (Medicago sativa var. Harpe), rye-
grass (Lolium multiflorum L.), birdsfoot trefoil (Lotus corniculatus
var. Leo), sorghum (Sorghum bicolor L.), maize (Zea mays L.), ber-
muda grass (Cynodon dactylon L.), legumes and beggar ticks (Bidens
cernua L.) (Kaimi et al., 2006, 2007; Shirdam et al., 2008; Tang et al.,
2010b; Yousaf et al., 2010b; Hall et al., 2011) have been shown to
be suitable for the removal of HCs from the soil. For example, root
activities of alfalfa (M. sativa var. Harpe) and perennial ryegrass
(Lolium perenne var. Affinity) enhanced the population of RB pos-
sessing HC degradation capability (Kirk et al., 2005; Schwab
et al., 2006; Rezek et al., 2008; Sorkhoh et al., 2010). Similarly in
the rhizosphere of goosegrass (Eleusine indica), vegetated in HC
contaminated soil, 72 times greater bacterial numbers were ob-
served than in the unvegetated soil (Lu et al., 2010a).
It is generally believed that for the cleanup of soil contaminated
with HCs, plant should have the ability to tolerate high concentra-
tions of these pollutants and possess extensive root system. Many
studies have been focused on the use of grasses for the remediation
of HCs due to their ability to tolerate HCs, extensive root system,
large root surface area and deeper root penetration into the soil
(Aprill and Sims, 1990; Tesar et al., 2002; Cofield et al., 2008; Lee
et al., 2008; Rezek et al., 2008; Gaskin and Bentham, 2010; Yousaf
et al., 2011; Afzal et al., 2012). An additional advantage is that
grasses can efficiently remove the HCs from contaminated soil,
without any nutrient supplementation, and show extensive appli-
cation to the problems associated with HC contamination (Gaskin
and Bentham, 2010).
Several processes and mechanisms taking place in the areas
surrounding the roots provide an ideal environment for HCs
1319
degradation. Root growth and development in deeper soil increase
exchange of gases and provision of water, which improve soil
physicochemical and biological properties. Roots increase the bio-
availability of HCs by decreasing the surface and volume of soil
micropores (Mueller and Shann, 2006; Wang et al., 2011; Muratova
et al., 2012). These also enhance bacterial population, diversity and
activity, and consequently HC removal (Siciliano et al., 2002;
Newman and Reynolds, 2004; Gerhardt et al., 2009). Apparently
plants enhance soil microbial population and activity through the
release of organic compounds, e.g. amino acids, sugars, enzymes,
organic acids and carbohydrates, from the root system (Van Hecke
et al., 2005). Several compounds released by roots act as inducer
for microbial genes involved in HC degradation, or act as a come-
tabolite to assist microbial HC mineralization (Fletcher and Hegde,
1995; Leigh et al., 2002; Olson et al., 2003). Recently, it was re-
ported that root exudates supported the development of high
diversity of bacteria containing known HC-degrading genes
(Cébron et al., 2011; Toyama et al., 2011). It was also observed that
plant roots enhance the tolerance of soil microorganisms to petro-
leum HCs (Lin et al., 2005; Chen et al., 2007; Liu et al., 2011; Cao
et al., 2012), and considerably higher numbers and diversity of
HC-degrading bacteria were observed in rhizosphere soil as com-
pared to bulk soil (Fan et al., 2008; Cheema et al., 2009; Haritash
and Kaushik, 2009). As some secondary metabolites produced by
plant are structurally similar to HCs, consequently bacteria
residing in plant environments adapt to HCs (Kamath et al.,
2004; Purwanti et al., 2012). Therefore, enhanced HC remediation
by vegetation might be due to an increase in the population and
activity of HC-degrading bacteria in the rhizosphere (Weyens
et al., 2009c; Soleimani et al., 2010; Lu et al., 2010b).
3. HC remediation by plant–bacteria partnerships
For a long period, PGPB were largely applied in agriculture for
facilitating plants to uptake nutrients from the environment or
preventing plant diseases (Zhuang et al., 2007). The combined
use of plants and pollutant degrading and/or PGPB is relatively a
new concept in the field of remediation of contaminated soil and
water (Zhuang et al., 2007; Weyens et al., 2009c, 2009d; Glick,
2010; Afzal et al., 2012; Ahmad et al., 2012). Although phytoreme-
diation (use of plants alone) provides an ecologically and econom-
ically attractive technique for remediating soils contaminated with
HC (Pilon-Smits, 2005; Dzantor, 2007; Olson et al., 2008), one of
the major limitations of using this approach is the fact that many
plant species are sensitive to HCs (Huang et al., 2004; Chaudhry
et al., 2005) and even plants, which tolerate the pollutant usually
do not grow well and cannot effectively support HC degradation
(Glick, 2003; Germaine et al., 2009; Peng et al., 2009; Zhang
et al., 2012). The primary inhibiting factors are considered to be
toxicity of low molecular weight compounds and hydrophobic
properties that limit the availability of plants to absorb water
and nutrients (Tesar et al., 2002; Peng et al., 2009; Wyszkowski
and Ziólkowska, 2009). Additionally, in most polluted soils, micro-
bial population is decreased, so that there are not sufficient micro-
organisms either to degrade HCs or to improve plant growth and
development (Glick, 1995; Siciliano and Germida, 1998; Huang
et al., 2004; Zhang et al., 2012). To overcome this problem, interac-
tions among microbes and plants have received great consider-
ation because of the possible role of microbes on plant growth
promotion and degradation of HCs in contaminated soil (Weyens
et al., 2009c; Tang et al., 2010a; Fernández et al., 2011). Bacterial
inoculation enhances plant resistance to the contaminant stress
and increases their acclimation rate and biomass formation
(Muratova et al., 2009). In return, vegetation enhances the popula-
tion and HC degradation potential of applied microorganisms as
 Author's personal copy
1320 S. Khan et al. / Chemosphere 90 (2013) 1317–1332
the roots of growing plants can improve the soil structure (Susarla
et al., 2002; Abhilash and Yunus, 2011; Ma et al., 2011). Recently,
Cowie et al. (2010) reported that PGPB enhanced HCs removal from
the soil, mainly due to improving plant growth, and bacterial pop-
ulation and activities. Similarly HC-degrading bacteria enhanced
plant biomass production and HC degradation (Andria et al.,
2009; Yousaf et al., 2011; Afzal et al., 2012). By the synergistic ac-
tion of the plants and inoculated bacteria, rhizodegradation of HCs
exhibited more efficient as compared to microbial remediation and
phytoremediation (without bacterial inoculation) (Ho et al., 2007;
Gurska et al., 2009; Afzal et al., 2012).
Although some studies reported that fungi can colonize in dif-
ferent compartments of plant and can enhance HC degradation
(Gunderson et al., 2007; Alarcón et al., 2008; Soleimani et al.,
2010), bacteria are most important group capable of improving
phytoremediation of HCs from contaminated environment (Gurska
et al., 2009; Afzal et al., 2011; Wang et al., 2011; Yousaf et al.,
2011). These are ubiquitous in the environment and play an impor-
tant role in biodegradation of HC pollutants from the soil, water
and air (Rojo, 2009; Yergeau et al., 2009; Yousaf et al., 2010a; Ali
et al., 2012; Al-Awadhi et al., 2012). The bacteria carrying HC deg-
radation genes have been reported in the rhizosphere as well as
plant apoplast (Siciliano et al., 2001; Biryukova et al., 2007; Yousaf
et al., 2010a), although HC degradation activities of the EB have
been rarely addressed (Newman and Reynolds, 2005; Germaine
et al., 2009; Afzal et al., 2011; Yousaf et al., 2011). Highly diverse
HC-degrading bacteria carrying alkane mono oxygenase (alkB)
and cytochrome P450 alkane hydroxylase (CYP153) genes have
been isolated from the plant environment (Hamamura et al.,
2006; Kaimi et al., 2007; Yousaf et al., 2010a; Becerra-Castro
et al., 2011).
Recently, it is suggested that bacteria having pollutant-degrad-
ing and/or plant growth-promoting activities can play an impor-
tant role in enhancing plant growth and remediation of
contaminated soils and therefore essential, or at least important,
Bacteria
Hydrocarbon
degradation
Phytotoxicity and
evapotranspiration
reduction
Stimulation of plant
resistance mechanism
Endophytic bacteria
Rhizospheric bacteria
Bacteria
Hydrocarbon
degradation
Enhanced nutrients and
hydrocarbon availability
Plant growth promoting
hormones production
Fig. 1. Plant–bacteria partnerships for the remediation of hydrocarbon contaminated soil. Plants assist bacteria in the rhizosphere and endosphere by providing nutrients and
space, enrichment of hydrocarbon degrading bacteria, transforming hydrocarbons into less toxic forms, and inducing catabolic gene expression for hydrocarbon degradation.
Plant associated rhizo- and endophytic bacteria help the plant by decreasing toxicity of soil through direct mineralization of hydrocarbons, increasing availability of nutrients
(N, P, Fe), reducing phytotoxicity and evapotranspiration, producing plant growth promoting hormones and enhancing the bioavailability of hydrocarbons. = hydrocarbons,
. = rhizobacteria, = endophytes.
for certain phytoremediation methods (Weyens et al., 2009c,
2009d; Glick, 2010). Plant and bacteria interact with each other
to absorb, degrade, or remove toxic contaminants from polluted
soil and water. Bacteria that reside in different compartments of
plant can synthesize several compounds that assist the plants to
overcome stress, providing essential nutrients required for plant
growth and development, improving plant defence system against
pathogens, and stimulating contaminants degradation (Fig. 1). En-
hanced plant growth and biomass production by applying HC-
degrading and/or PGPB efficiently reduce the proportion of toxic
HC pollutants to the quantity of plant tissue, reducing plant stress
(Alkorta and Garbisu, 2001; Gurska et al., 2009; Afzal et al., 2011,
2012).
The effectiveness of plant–bacteria partnerships depends lar-
gely on the survival and metabolic activities of exogenous bacteria
carrying degradation genes required for the enzymatic breakdown
of organic pollutants. HCs are remediated from the soil mainly by
plant-associated bacteria and the efficiency of remediation is asso-
ciated with the survival of inoculated HC-degrading bacteria in the
rhizosphere and endosphere of the plant (Glick, 2003; Liste and
Prutz, 2006; Muratova et al., 2008; Yousaf et al., 2011). Neverthe-
less, it is also important to monitor gene abundance and gene
expression during phytoremediation of contaminated soil to get
stronger evidence about the persistence and functional activity of
the applied microorganisms (Juhanson et al., 2009). Quantitative
PCR (qPCR) has been used to monitor the presence of specific
organisms in an environment (e.g. the abundance of alkB gene in
the rhizo- and endosphere of plants vegetated in HC contaminated
soil) and to monitor defined functional activity (e.g. alkane-degrad-
ing alkB gene expression during phytoremediation of HC contami-
nated soil) (Andria et al., 2009; Panicker et al., 2010; Afzal et al.,
2011). Recently, studies regarding the abundance and expression
of alkB and CYP153 genes in rhizosphere, and shoot and root inte-
rior of plants vegetated in HC contaminated soil (Andria et al.,
2009; Afzal et al., 2011; Yousaf et al., 2011; Schulz et al., 2012),
Plant
Residency for
endophytic bacteria
Nutrients for
endophytic bacteria
Transformed products
(oxidized or reduced
form)
Hydrocarbons in plant
Hydrocarbons in soil
Plant
Provision of nutrients
Enrichment of
hydrocarbon degrading
bacteria
Induce catabolic genes
for hydrocarbon
degradation
 Author's personal copy
S. Khan et al. / Chemosphere 90 (2013) 1317–1332
indicated that bacteria carrying these genes are not only able to
colonize the rhizosphere and plant interior but are also metaboli-
cally active in HC degradation. Furthermore, these studies indicate
that survival and metabolic activities of HC-degrading bacterial
strains varied distinctly between different strains, plants species,
plant development stages and plant compartments. More recently,
greater numbers of HC-degrading bacteria possessing alkB and tol
genes were found at the vegetative growth stages of ryegrass
(L. multiflorum L.) (Nie et al., 2011).
Recently, it has been recommended that the use of bacteria hav-
ing both pollutant degrading as well as plant growth promoting
activities is superior than that the bacteria having only pollutant
degrading or plant growth promoting activities (Glick, 2010). PGPB
showing 1-aminocycloprpane-1-carboxylate (ACC) deaminase
activity can decrease ethylene amounts produced by plants under
stress and consequently reduce stress symptoms leading to im-
proved plant growth and development (Shah et al., 1998; Glick,
2003; Glick et al., 2007). Bacterial ACC deaminase activity en-
hances plant growth, particularly root growth and development,
which play an important role in the phytoremediation, as the root
exudates enhance bacterial survival and metabolic activities in the
rhizosphere (Juhanson et al., 2009). This particular tendency (ACC-
deaminase activity) of bacteria proposes that the application of
such bacteria can reduce the possible toxic effects of HCs on
growth and development of plants. More recently, we have re-
ported that bacteria possessing HC-degrading as well as plant
growth-promoting (ACC-deaminase) activities significantly en-
hanced more plant biomass production and HC remediation than
the bacteria having only HC degrading activity (Afzal et al., 2011,
2012; Yousaf et al., 2011). Plant and bacteria interact with each
other constantly and form close associations or symbiotic relation-
ships. For example, nitrogen fixing plant-associated bacteria can fix
the nitrogen from the atmosphere and provide to plants. Moreover,
some bacteria showed simultaneous N2 fixation and HC degrada-
tion leading to speculation that combined use of plants and such
bacteria may be a valuable option for the remediation of HCs from
N-limited soils (Foght, 2010). Inoculation of plants with bacteria
possessing both HC degrading and plant growth promoting activi-
ties, with both seed inoculation and soil method, has been success-
fully applied in the laboratory, greenhouse, and field for the
remediation of HC contaminated soil and water (Huang et al.,
2004; Kuiper et al., 2004; Gurska et al., 2009; Afzal et al., 2012).
Many studies evidenced that organic compounds released by
plant roots act as signaling agents and perform an important role
in plant–bacteria interactions (Badri et al., 2009; Xie et al., 2012).
Diverse bacterial population was observed in the rhizosphere and
endosphere of different plants vegetated in HC contaminated soil,
it might be due to the fact that different plants produce different
root exudates and chemicals in the rhizosphere and plant interior
(Siciliano et al., 2001; Smalla et al., 2001). However, in recent stud-
ies, it was reported that the population of HC-degrading bacteria
increased in the rhizosphere due to the presence of HCs in soil,
and their number in the total bacterial population increased as
well (Muratova et al., 2009; Pritchina et al., 2011). The major stim-
ulation factor was the HC, however, the stimulating effect of the
plant root exudates was also involved. Higher numbers of HC-
degrading bacteria were isolated from different compartments of
Italian ryegrass than birdsfoot trefoil (Yousaf et al., 2010a). These
bacteria showed higher diversity, particular in plant interior. Banks
et al. (2003) reported that the type of plant and level of HC contam-
ination may greatly affect rhizosphere microbial communities. It
was also reported that the rhizosphere microorganisms of alfalfa
was less inhibited by HC pollution and had a higher degradation
potential than the rhizosphere microflora of reed (Muratova
et al., 2003). Moreover, biodegradation potential of plant-
1321
associated bacteria, their persistence and metabolic activities are
affected by root exudates composition (Schulz et al., 2012).
During phytoremediation of contaminated soil, plant-
associated bacteria can promote plant growth by many different
mechanisms – by providing nutrients, plant growth hormones or
by antagonizing pathogens (Weyens et al., 2010c). Due to the pres-
ence of organic pollutants, especially HCs, the availability of min-
eral nutrients in the soil, including nitrogen, phosphorous and
iron can be limited, consequently plant growth is restricted (Kirk
et al., 2005; Kechavarzi et al., 2007; Nie et al., 2011). Zhou et al.
(2009) reported that the phosphorus and water amounts in HC
contaminated soil were important parameters for the phytoreme-
diation of HC pollutants from soil. Maximum HC degradation was
observed in treatments with high concentrations of phosphorous
and water contents, however, comparatively less degradation
was observed in treatments with lower phosphorus concentrations
and water contents. In other studies, addition of organic manure
and NPK fertilizer in HC polluted soils enhanced plant growth
and HC degradation (Bordoloi et al., 2012; Jidere et al., 2012; Song
et al., 2012), indicating that HC contaminated soils were deficient
in bioavailable nutrients. PGPB can solubilize the mineral nutrients
and enhance plant growth and remediation of organic contami-
nants. Some plant associated bacteria have nitrogenase enzyme
that catalyzes the reduction of atmospheric nitrogen to ammonia,
which can be utilized by plant. The presence of such plant associ-
ated bacteria could help plants to grow under nitrogen limitation
on contaminated land (Doty, 2008; Weyens et al., 2009b, 2009c,
2009d). Many plant associated-bacteria can enhance the solubility
of inorganic phosphates by synthesizing organic acids, some can
degrade organic compounds containing phosphates by releasing
enzymes phosphatases (Kim et al., 1997; Vessey, 2003). Further-
more, these bacteria can synthesize specific organic compounds,
siderophores, which reduce iron form Fe(III) to Fe(II), which are
preferably absorbed by plants (Katiyar and Goel, 2004).
Traditionally, the production of phytohormones was exten-
sively investigated to elucidate the different effects of PGPB on
plants, and most of studies explored the role of the phytohormone
auxin (Brown, 1974; Patten and Glick, 1996; de Garcia Salamone
et al., 2006). However, in the last two decades it has been observed
that many PGPB possessing the ACC deaminase activity (Glick,
2003; Blaha et al., 2006; Shaharoona et al., 2006; Nadeem et al.,
2007; Rodriguez et al., 2008), and this bacterial activity can reduce
the intensity of pollutant stress in a developing plant (Arshad et al.,
2007; Glick, 2010; Afzal et al., 2012).
Bioremediation efficiency is likely to be more limited by the
bioavailability of HCs rather than by the total number of HC-
degrading bacteria (Hamdi et al., 2007). Some plant-associated
bacteria can produce biosurfactants that can enhance the bioavail-
ability of HCs and may be useful for phytoremediation application
(Golubev et al., 2009; Pacwa-Plociniczak et al., 2011). Different
types of HC-degrading genes were observed in plant-associated
RB and EB (Rojo, 2009; Yousaf et al., 2010a). Although there are
some microorganisms that can mineralize HC contaminants (Cai
and Xun, 2002; Dai et al., 2003), individual species generally do
not have the capability to degrade a HC completely. However,
the consortium of bacterial strains having different degradation
pathways perform well in the rhizosphere of plants vegetated in
HCs contaminated soil (Macek et al., 2000; Kuiper et al., 2004;
Chaudhry et al., 2005; Yateem et al., 2007; Balcom and Crowley,
2010; Afzal et al., 2012).
HCs and other organic pollutants move into the rhizosphere
through transpiration stream (Harvey et al., 2002). The HCs uptake
by plant roots and their accumulation in roots and shoots is quite
low as compared to plant-promoted biodegradation in the rhizo-
sphere (Sheng et al., 2008a; Lu et al., 2011; Meng et al., 2011;
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1322 S. Khan et al. / Chemosphere 90 (2013) 1317–1332
Wang et al., 2012a, 2012b). However, HCs with low lipophilicity
seem to be transported into roots and then shoot before the rhizo-
sphere microorganisms can mineralize them. The HCs that are ta-
ken up by plants, reside in the plant tissues up to 2 d, EB seem to be
more suitable for the mineralization of these organic pollutants
(Weyens et al., 2009b, 2009d).
For efficient phytoremediation of organic soil contaminants, the
soil has to provide the appropriate environment for both the plant
and the associated microorganisms. Soil properties such as pH, par-
ticle size, texture, organic matter, cation exchange capacity and
structure affect not only plant growth and development, and sur-
vival, colonization and metabolic activities of inoculated bacteria
(Labana et al., 2005; Kaimi et al., 2007; Sun et al., 2010; Schulz
et al., 2012), but also the biodegradation of organic pollutants
(Kaakinen et al., 2007; Jung et al., 2008; Haritash and Kaushik,
2009). Furthermore, soil properties can influence the solubility
and bioavailability of HCs (Sijm et al., 2000; Chung and Alexander,
2002; Parrish et al., 2005). Humic substances and clay minerals are
strongly bound by HCs (Richnow et al., 1995). During bioremedia-
tion of organic pollutants, the quantitative analysis of functional
genes has provided an important tool for examining relationship
between specific bacterial population and the efficiency of biodeg-
radation processes (Piskonen et al., 2005; Salminen et al., 2008;
Baek et al., 2009; Panicker et al., 2010). Recently, we have observed
that soil types (sandy, loamy sand and loam) affect colonization,
gene abundance and metabolic activities (gene expression) of
two HC-degrading bacterial strains in the different compartments
of Italian ryegrass (Afzal et al., 2011). High colonization and gene
abundance were observed in loamy soils than other two soils by
both culture-dependent and independent techniques. Maximum
gene expression and HC degradation were observed in loamy soil
and were significantly higher compared to those in other soils. This
study indicates that soil type affects the bacterial colonization and
metabolic activities and consequently the biodegradation of organ-
ic pollutants. In another recent study, it was observed that the
abundance of HC-degrading genes and their functional structure
in the rhizosphere were influenced by the soil salinity (Zhong
et al., in press). Similarly, Wang et al. (2012a) reported that soil
types (red and alluvial) and compost amendment affect microbial
population, interactions among root exudates, microorganisms
and compost and hence pyrene degradation. Addition of organic
amendments enhances solubilization and bioavailability of HCs
that could facilitate phytoremediation of HC contaminated soils
(Cheng and Wong, 2006; Cheng et al., 2008; Ke et al., 2009;
Williamson et al., 2009; Wang et al., 2012a). Furthermore, it was
also reported that inoculation method (seed inoculation and soil
inoculation) affects bacterial colonization, plant growth and HC re-
moval from soil (Afzal et al., 2012). Among two tested methods,
soil inoculation was found more effective than seed inoculation,
indicating bacterial inoculation mode can affect phytoremediation
of HC contaminated soil.
4. RB with the capacity to enhance HC phytoremediation
RB are defined as the bacteria that live in close vicinity to the
root or on root surface, beneficial to host plant under certain con-
ditions. Microbial populations in the close vicinity of roots play an
important role for the proper functioning of the ecosystem. RB
with plant growth promoting potential have been traditionally ap-
plied in agriculture to enhance crop yields. However, potential
benefits of their use in the phytoremediation of organic and inor-
ganic pollutants from soil and water have been explored recently
(Hutchinson et al., 2004; Bashan et al., 2008; de-Bashan et al.,
2011; Rajkumar et al., in press). The use of RB capable of degrading
HC and/or promoting plant growth in combination with a specific
plant could provide a cost-effective, environmental friendly, effi-
cient and sustainable remediation technology for the 21st century
(Weyens et al., 2009c; Glick, 2010; Turan et al., 2012). Combined
use of plants and effective microorganisms helps in the prolifera-
tion of specific contaminant degrading microorganisms in the rhi-
zosphere (Kuiper et al., 2002; Singer et al., 2003; Rentz et al., 2005;
Smalla et al., 2006; Hartmann et al., 2008; Cébron et al., 2011). The
absence of HC-degrading bacteria in the rhizosphere can be over-
come by the inoculation of HC-degrading bacteria and/or PGPB to
plant through soil or seed inoculation (Normander and Hendriksen,
2002; Villacieros et al., 2005; Germaine et al., 2009; Muratova
et al., 2009; Afzal et al., 2012). This approach was found to be suc-
cessful in reducing the levels of HCs in polluted environments, and
consequently improving plant growth and development (Johnson
et al., 2004; Yousaf et al., 2010b; Afzal et al., 2011). Various plant
and microbial characteristics, involving a large number of the
genes of microbes, are needed for effective rhizosphere coloniza-
tion (Lugtenberg et al., 2001; de Weert et al., 2002; Kuiper et al.,
2004; Silby and Levy, 2004). Roots offer perfect attachment sites
for microorganisms, and also supply nutrients in the form of exu-
dates consisting of organic acids and amino acids, enzymes, sugars,
and complex carbohydrates (Shim et al., 2000). Plant roots are
known to release high amounts of photosynthetically derived or-
ganic compounds into the surrounding soil that play an important
role in attracting beneficial bacteria and forming mutualistic rela-
tionships. These organic compounds could be classified as exu-
dates, secretions, plant mucilages, mucigel, and root lysates
(Olson et al., 2003; Reichenauer and Germida, 2008). It has been re-
ported that following root exudation, HC-degrading bacteria, able
to aggressively colonize the root surface, become abundant. Conse-
quently, the number of HC-degrading bacteria in the close vicinity
of plant roots generally becomes higher than that observed in the
bulk soil (Kuiper et al., 2004; Hong and Cho, 2007). Low molecular
weight organic acids, amino acids and sugars released by plant
roots play an effective role in the interactions of plants with their
associated bacteria (Jones, 1998; Knee et al., 2001), and conse-
quently enhance bacterial degradation of soil contaminants. Plants
are also known to release several other organic compounds includ-
ing terpernes, flavonoids and some lignin-derived components
with chemical structures similar to those of the HCs. Such com-
pounds induce expression of HC-degrading genes in bacteria resid-
ing in the rhizosphere and hence bring about shifts in microbial
metabolism of HC (Tesar et al., 2002; Singer et al., 2003; Sun
et al., 2010). Moreover, the degradation of some organic
compounds (e.g. flavonoids) by RB produce organic intermediate
compounds, such as resorcinol, phloroglucinol phenylacetic acid,
cinnamic acid and protocatechuic acid (Shaw and Burns, 2004)
which are believed to be biodegraded through the b-ketoadipate
pathway (Parke et al., 2000). As many HCs are mineralized through
the same pathway, the RB adapted to metabolize the above men-
tioned compounds also degrade HCs (Kim et al., 2008). Similarly,
salycilate, which induces resistance in plants against pathogens,
also induces HC-degradation in some microorganisms (Chen and
Aitken, 1998).
Plant tolerance to HCs seems to be of crucial importance for the
recovery of soil health during phytoremediation of polluted soils
(Barrutia et al., 2011). Generally, HC-degrading RB may enhance
plant tolerance to HCs, present in the soil as pollutants, by degrad-
ing these organic compounds (Escalante-Espinosa et al., 2005).
Many studies have revealed that plants can select certain bacteria
in their rhizosphere by different processes and activities, such as
changing physicochemical and biological properties of the soil,
the root architecture, or releasing specific organic compounds that
attract beneficial bacteria and repels others (Hartmann et al.,
2009). Vegetation stimulated up to 5-fold rise in HC-degrading
bacterial population in diesel contaminated soils, indicating
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S. Khan et al. / Chemosphere 90 (2013) 1317–1332
positive association between plants and HC-degrading bacteria
during the remediation of contaminated soils (Mezzari et al.,
2011). More recently, it was observed that in diesel contaminated
soil, the roots of reed (Phragmites australis), bulrush (Scirpus tripu-
eter), galingale (Cyperus rotundus) and wildrice (Zizania latifolia)
enhanced the tolerance and biodegradation activity of soil micro-
organisms (Cao et al., 2012), although the extent of degradation
was higher in the soil vegetated with reed. In another study, Phil-
lips et al. (2012) reported that gene abundance and mineralization
of HCs by RB was significantly impacted by the composition of root
exudates, as certain compounds (acetate, alanine) were found to be
associated with increased mineralization capacity, alongside some
others e.g. malonate decreased mineralization. The presence of
these compounds within root exudates influences the success of
a given phytoremediation treatment of a HC contaminated soil.
Similarly, Toyama et al. (2011) reported that the benzo[a]pyrene
biodegradation in the rhizosphere of P. australis was stimulated
as the proportion of phenolic compounds in root exudates in-
creased. They conclude that bacteria–root exudate interactions
can enhance biodegradation of HCs. Shifts in the diversity of RB ob-
served due to HC contamination are also influenced by the plant
species (Balcom and Crowley, 2009). Many factors, such as pollu-
tant level in the soil, plant growth stage and plant species comple-
ment the effect of root exudates composition, resulting in
variations in the population of the RB (Phillips et al., 2006; Haichar
et al., 2008; Kidd et al., 2008). The quantity of root exudates also
depends on the properties of the soil and the bioavailability of min-
eral nutrients (Paterson and Sim, 2000; Bais et al., 2006).
The bacterial survival and colonization in the rhizosphere is ini-
tiated with attachment of bacterial strains with the roots of plants.
The attachment of bacteria with roots has been largely studied,
although, complete mechanism is still not clear, it might be due
to secretion of certain compounds by bacteria, such as surface pro-
teins, capsular polysaccharides, flagella and chemotaxis (Capdevila
et al., 2004; Rodríguez-Navarro et al., 2007). It is assumed that a
plant and its associated bacteria make a continuous communica-
tion of signals with each other to establish bacterial colonization
on the surface of roots. Many compounds that are released by
plants, particularly flavonoids, are known to play an important role
in exchange of signals and plant–microbe interactions (Steinkell-
ner et al., 2007). Similarly reactive oxygen species (ROSs), such as
superoxide anion ðO 2 Þ and hydrogen peroxide, play an important
role during plant–microbe interactions (Fester and Hause, 2005;
Baptista et al., 2007; Nanda et al., 2010). An oxidative burst often
takes place at the site of attempted invasion during the early stages
of plant–microbe interactions. Moreover, a second ROS production
can be observed during certain types of plant–pathogen interac-
tions, which triggers hypersensitive cell death. This second ROS
wave seems absent during symbiotic plant–microbes interactions.
This difference between these two responses is thought to play an
important signaling role leading to the establishment of plant–
microbes interactions.
Plants inhabiting polluted soil can serve as a source of microor-
ganisms that exhibit HC-degradation potential. Cohen et al. (2002)
observed that although Azolla pinnata was killed by low concentra-
tions of diesel, it released a diversity of HC-degrading microorgan-
isms. This study implies that at least some microorganisms
involved in the degradation of HCs are derived from plant-associ-
ated microbial community rather than from bulk soil. Consider-
ably, high population and diversity of bacteria are observed in
rhizosphere soil as compared to bulk soil, and since several root se-
creted compounds are structurally similar to HCs, these bacteria
are adapted to tolerate/degrade HCs. Furthermore, root penetration
in the soil transports HC-degraders into contact with the target
substrate (Leahy and Colwell, 1990; Cohen et al., 2004; Zhong
et al., in press). Also, pollutants, including HCs are transported from
1323
the bulk soil into rhizosphere through transpiration stream
(Weyens et al., 2009c). Roots also provide oxygen which is neces-
sary for the metabolic degradation of HCs. As HC-degrading bacte-
ria are associated with plant roots of most, but not all, plants in
HC-polluted soil are likely to have the ability to remediate HCs
(Daane et al., 2001; Siciliano et al., 2001). The application of com-
post was also observed to confer beneficial effects (Tesar et al.,
2002; Antizar-Ladislao et al., 2006; Williamson et al., 2009), which
are probably because of the input of nutrients and microorganisms
that participate in the breakdown of HCs (Yousaf et al., 2010b).
PGPR also assist host plant to overcome contaminant-induced
stress responses, thus providing improved plant growth and hence
HC degradation (Weyens et al., 2009c). Inoculation of Rhizobium
meliloti to alfalfa was reported to stimulate not only growth of
indigenous HC-degrading rhizosphere microorganisms but also
soil microbial activity and hence plant biomass production indicat-
ing that application of PGPR may be a promising phytoremediation
strategy for reclamation of agriculture soil contaminated with HCs
(Teng et al., 2011). Generally, production of plant growth-promot-
ing hormones is believed to be the major means of various effects
of PGPR on plants, and phytohormone auxin has been extensively
studied (Werner et al., 2001). However, many PGPR produce the
enzyme ACC deaminase (Shaharoona et al., 2006; Nadeem et al.,
2007; Yue et al., 2007), which decreases the level of stress in a
developing plant, due to the presence of contaminants (Glick,
2003; Arshad et al., 2007; Glick et al., 2007). Recently, Hong et al.
(2011) evaluated the effect of inoculation of a rhizobacterium Gor-
donia sp. S2RP-17 on phytoremediation of diesel-contaminated soil
by Z. mays, and found a positive correlation in HC degradation, ACC
deaminase activity and siderophore production. Recently, we have
reported that RB showing HC-degradation as well as ACC deami-
nase activity enhanced more plant biomass production and HC
degradation than those bacteria having only HC degrading capabil-
ity (Afzal et al., 2011, 2012). RB can also stimulate resistance sys-
tem in plants in order to overcome pathogens by producing
different organic compounds such as 2,3-butanediol, pyoverdine,
and lipopeptide surfactants (Badri et al., 2009; Raaijmakers et al.,
2009).
Association between plant and the inoculated bacteria plays an
important role not only in the rhizosphere colonization, plants also
depend on their associated bacteria to achieve efficient degrada-
tion of contaminants (Afzal et al., 2011; Hong et al., 2011; Yousaf
et al., 2011). However, this association can be influenced by other
rhizospheric microorganisms and environmental conditions. The
bacterial abundance in the rhizosphere has been shown to increase
not only the biodegradation of HCs and other organic pollutants,
but also to provide an ideal environment for horizontal gene trans-
fer and gene rearrangements (Yousaf et al., 2010a). For efficient
remediation of HCs from the soil, not only bacterial survival and
colonization have to be maintained in the rhizosphere, but the bac-
teria have to be metabolically active in HC degradation (Juhanson
et al., 2009; Afzal et al., 2011). The observations that inoculated
bacteria can overcome other soil microorganisms by producing
toxic chemicals, utilizing highly fast nutrients utilizations system,
or by physical exclusion are well documented (Lugtenberg and
Dekkers, 1999). However, it is of utmost importance to observe
the survival and colonization of inoculated RB in the rhizosphere
of plants vegetated in HC contaminated soils. Several studies have
been performed to observe survival and colonization of the inocu-
lated bacteria in the rhizosphere, mainly by labeling inoculated
bacterial strains with a marker gene, such as luxAB encoding bacte-
rial luciferase, gfp encoding green fluorescent protein, gusA encod-
ing b-glucuronidase, or other reporter genes (Wilson et al., 1995;
Segura et al., 2009; Afzal et al., 2012). Recently, we have labeled
inoculants bacterial strains with gusA10 marker gene in order to
monitor their survival and colonization in the rhizosphere of
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1324 S. Khan et al. / Chemosphere 90 (2013) 1317–1332

Table 1
Examples of successful remediation of hydrocarbons from soil by combined use of plants and rhizobacteria.

Plant used Rhizobacteria Bacterial characteristics Reference

Maize (Z. mays L.) Pseudomonas sp. UG14Lr Hydrocarbon degradation Chouychai
et al. (2012)
Italian ryegrass (L. multiflorum var. Taurus) Pantoea sp. strain BTRH79 Hydrocarbon degradation and ACC Afzal et al.
deaminase activity (2012)
Alfalfa (M. sativa L.) R. meliloti (strain ACCC 17519) Hydrocarbon degradation Teng et al.
(2011)
Maize (Z. mays L.) Rhizobacterium Gordonia sp. S2RP-17 Hydrocarbon degradation, ACC deaminase Hong et al.
and siderophore synthesizing activities (2011)
Sorghum (S. bicolor) Sinorhizobium meliloti Hydrocarbon degradation, auxin- Golubev
production et al. (2011)
Ryegrass (L. multiflorum) Acinetobacter sp. strain Hydrocarbon degradation Yu et al.
(2011)
Italian rye grass (L. multiflorum var. Taurus) and Pantoea sp. strain BTRH79, Pseudomonas sp. strain Hydrocarbon degradation Yousaf et al.
birdsfoot trefoil (L. corniculatus var. Leo) ITRH76 (2010b)
Winter rye (Secale cereale L.), alfalfa (M. sativa L.) Azospirillum brasilense SR80 Hydrocarbon degradation, indole-3-acetic Muratova
acid production et al. (2010)
Italian rye grass (L. multiflorum var. Taurus) Rhodococcus sp. strain ITRH43 Hydrocarbon degradation Andria et al.
(2009)
Sorghum (S. bicolor L. Moench) S. meliloti P221 Phenanthrene degradation, indole-3- Muratova
acetic acid production et al. (2009)
Maize (Z. mays L.) P. putida MUB1 Hydrocarbon degradation Chouychai
et al. (2009)
Annual ryegrass (L. perenne), tall fescue (F. Pseudomonas strains, UW3 and UW4 ACC deaminase production Gurska et al.
arundinacea, var. Inferno), barley (Hordeum (2009)
vulgare)
Rice (Oryza sativa L.) Acinetobacteria sp. Hydrocarbon degradation Li et al.
(2008)
Barley (H. sativum L.) P. putida KT2440 Hydrocarbon degradation Child et al.
(2007a)
Barley (H. sativum L.) Mycobacterium sp. strain KMS Hydrocarbon degradation Child et al.
(2007b)
Wheat (Triticum spp.) Pseudomonas sp. GF3 Phenanthrene degration Sheng and
Gong (2006)
Wheat (T. spp.) A. lipoferum sp. Hydrocarbon degradation and indole-3- Muratova
acetic acid production et al. (2005)
Common reed (P. australis) P. asplenii AC ACC deaminase production Reed and
Glick (2005)
White Clover (T. repens L.) R. leguminosarum Hydrocarbon degradation Johnson
et al. (2004)
Tall fescue grass (F. arundinacea) A. brasilense Cd, Enterobacter cloacae CAL2, P. putida Hydrocarbon degradation and ACC Huang et al.
UW3, P. putida, Flavobacterium sp., P. aeruginosa deaminase activities (2004)
Barley (H. sativum L.) P. fluorescens, P. aureofaciens Hydrocarbon degradation Anokhina
et al. (2004)
Barmultra grass (L. multiflorum) P. putida PCL1444 Naphthalene degrading bacteria Kuiper et al.
(2001)

Italian ryegrass during phytoremediation of diesel contaminated
soils. After 3 months inoculated bacterial strains were successfully
recovered from the rhizosphere soil as well as from root and shoot
interior (Afzal et al., 2012).
The list of plant-associated HC-degrading RB is very extensive.
Recently, many HC-degrading bacterial strains have been isolated
from the rhizosphere of different plants, and their HC-degrading
potentials are explored (Yousaf et al., 2010a, 2010b). Previously,
many other HC-degrading RB, isolated from different plants,
showed the capability to degrade a wide variety of HCs (Daane
et al., 2001; Kuiper et al., 2001; Tesar et al., 2002; Johnson et al.,
2004; Muratova et al., 2005). Some recent successful examples of
RB application for the phytoremediation of HC contaminated soil
are shown in Table 1.
5. EB with the capacity to enhance HC phytoremediation
EB colonize inside plant tissues and can form symbiotic, mutu-
alistic and commensalistic relationships with their host plant
(Ryan et al., 2008). After the first report that bacteria isolated from
the root interior of plants vegetated in HC contaminated soils
hosted genes encoding HC degradation (Siciliano et al., 2001), re-
cently, several studies were performed to isolate HC-degrading
EB from different plants vegetated in HC contaminated soils (Phil-
lips et al., 2008; Yousaf et al., 2010a, 2010b; Taghavi et al., 2011; de
Oliveira et al., 2012). These studies reveal that the population of EB
possessing HC-degrading genes is dependent on the presence and
concentration of HCs in the soil. The majority of HC-degrading EB
are the soil inhabitants. Before EB are able to show plant growth-
promoting effects, they may move to rhizosphere of the host plant
and then the rhizoplane. However, some of the bacteria residing on
rhizoplane can penetrate into plant roots, and some of them may
migrate to aerial plant parts (Compant et al., 2010). Some EB can
colonize rhizosphere soil as well as the plant endosphere (Sessitsch
et al., 2002; Germaine et al., 2009; Afzal et al., 2011, 2012). Fur-
thermore, EB can assist their host plant to overcome stress re-
sponse due to the presence of high levels of HC pollutants in soil,
and may improve plant growth and development by decreasing
ethylene levels with bacterial ACC deaminase activity (Glick,
2003; Sheng et al., 2008b; Yousaf et al., 2011). As in soil, many bio-
tic and abiotic factors can affect plant–bacteria interactions, the
use of EB for the remediation of HCs and other organic compounds
from soil and water has been studied (Doty, 2008; Ryan et al.,
2008; Weyens et al., 2009b, 2009d; Yousaf et al., 2011). Due to col-
onization in the internal tissues of the plant (Sessitsch et al., 2002),
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S. Khan et al. / Chemosphere 90 (2013) 1317–1332
EB face comparatively less competition for nutrients and space as
compared to RB, and are physically protected from adverse
changes in the environment (Schulz and Boyle, 2006). Isolation
and characterization of EB possessing organic pollutants degrada-
tion potential have been assessed, and EB that can mineralize sev-
eral organic contaminants including HCs have been isolated from
different plants (Siciliano et al., 2001; van Aken et al., 2004;
Germaine et al., 2006; Phillips et al., 2008; Yousaf et al., 2011).
A strong association exists between EB and RB and several EB
can also survive and colonize in the soil surrounding the roots,
where they can penetrate into their associated plant via the roots.
In strong plant–bacteria association, plants provide nutrients and
residency for EB. In return, EB support plant growth and biomass
production by direct or indirect ways. Direct plant growth improv-
ing approaches may include production of ACC deaminase for
reducing the stress in plants due to the presence of contaminants
(Arshad et al., 2007; Glick, 2010), nitrogen fixation, (James, 2000;
Doty, 2008), production of plant growth promoting hormones
(Asghar et al., 2004; Belimov et al., 2005), and modification of
sugar sensing systems in plants (Goddijn and Smeekens, 1998).
Recently, Dashti et al. (2009) reported that EB possessing HC-
degradation and nitrogen fixation capabilities enhance HC degra-
dation without adding any nitrogen source in HC contaminated
soils. This study suggests that use of EB having more than one ben-
eficial characteristic improves the phytoremediation process and
makes this technology more economical and environmentally
friendly. EB can also indirectly improve plant growth in HC con-
taminated soils by reducing the growth and activity of pathogens
through competition for nutrients and space (O’Sullivan and
O’Gara, 1992), through stimulation of plant resistance mechanisms
(Kloepper et al., 2004), synthesis of hydrolytic enzymes (Krechel
et al., 2002) and degradation of organic pollutants (Weyens et al.,
2009b, 2009d; Yousaf et al., 2011).
Siciliano et al. (2001) reported that bacteria exhibiting HC deg-
radation are more abundant among endophytic communities than
rhizospheric population of a plant vegetated in HC polluted soil.
This finding indicates that EB have a role in mineralizing the HCs.
They have the capability to enhance the efficiency of phytoremedi-
ation by degrading organic compounds in the soil and may en-
hance soil fertility through nitrogen fixation and phosphate
solubilization (Ryan et al., 2008). Poplar (Populus spp.) and willow
(Salix spp.) were found to be suitable tree species for both biomass
production and phytoremediation and some of their associated EB
were found capable of fixing nitrogen (Taghavi et al., 2005). Many
EB exhibit natural competence for degrading organic contaminants
or may introduce contaminant degrading genes in local bacterial
population by horizontal gene transfer (Phillips et al., 2008; Yousaf
et al., 2010a). Some EB have the capability to tolerate and mineral-
ize organic pollutants including HCs probably due to their expo-
sure to various compounds in the plant/soil niche. This natural
organic pollutant-degrading capacity of EB is being explored in or-
der to improve organic pollutant remediation potential of different
plants (Barac et al., 2004; Germaine et al., 2006; Phillips et al.,
2008; Weyens et al., 2009b, 2009d; Afzal et al., 2011; Yousaf
et al., 2011).
The population and diversity of EB are highly variable, depend-
ing mainly on the plant genotypes, plant development stage,
inoculums density, contaminant amount, and soil physicochemical
properties (Pillay and Nowak, 1997; Tan et al., 2003; Yousaf et al.,
2010a, 2010b, 2011; Becerra-Castro et al., 2011). During phyto-
remediation of diesel contaminated soil strain to strain variations
in colonizing capabilities of different EB have been found (Yousaf
et al., 2011; Afzal et al., 2012). A recent study by Yousaf et al.
(2010a) illustrates the diversity of EB found in different plants,
growing in diesel contaminated soils. Italian ryegrass hosted a high
number of HC-degrading EB belonging to diverse phylogenetic
1325
groups, however, birdsfoot trefoil did not host such large number
of EB. Comparably high population and diversity of HC-degrading
bacteria within the ryegrass as compared to birdsfoot trefoil might
be because grasses release alkanes in the rhizosphere (Marseille
et al., 1999) and also absorb alkanes from soil, and likely to be host-
ing bacteria able to degrade alkanes as sole carbon source. Simi-
larly Phillips et al. (2008) reported that plant species vegetated
in HC polluted soils hosted different EB, which may enhance ability
of plants to remediate HCs from contaminated soils. Recently,
metagenomic study reveal that EB isolated from the roots of rice,
vegetated in uncontaminated soil, showed HC degradation poten-
tial (Sessitsch et al., 2012). The studies of Tesar et al. (2002), Moore
et al. (2006) and Yousaf et al. (2010a) explained that within the di-
verse bacterial population found in Italian ryegrass, birdsfoot tre-
foil and poplar trees, several EB had the capability to improve
phytoremediation of HCs and other organic compounds. Variations
in sensitivity and tolerance levels among different plant species
and even cultivars to HC pollutants in soils might be attributed
to variations in endophytic bacterial population and activities.
Many studies reveal that between inoculated RB and EB, EB
showed high colonization and activities in different compartments
of plants, particularly inside plant tissues (Rosenblueth and
Martínez-Romero, 2006; Andria et al., 2009; Yousaf et al., 2010a;
Afzal et al., 2011, 2012). It was also reported that inoculation of
EB result in better survival of plants which might be due to plant
growth-promoting and/or HC-degrading activities. Recently, we
have observed that EB after inoculation in soil were not only capa-
ble of colonizing inside the plant but were also metabolically active
in the degradation of HCs. It was observed by quantifying the gene
abundance and gene expression in the root and shoot of Italian rye-
grass, alfalfa and birdsfoot trefoil vegetated in diesel contaminated
soil (Afzal et al., 2011; Yousaf et al., 2011). Our studies further re-
veal that three different endophytic strains of Enterobacter ludwigii
show different levels of gene abundance and expression in differ-
ent plants species, at different plant growth stages and even in dif-
ferent compartments of the plant. At early growth stages of the
plant, the level of gene abundance and expression is poorer in
the roots and shoots than rhizosphere, however, enhances in roots
and shoots at latter growth stages. This shows that E. ludwigii
strains initially colonize in the rhizosphere and move into the roots
and shoots at later plant growth stages.
It has been reported that the degradation of extremely water
soluble HC is oftenly not completed by the rhizosphere microor-
ganisms, resulting in accumulation in plant tissues and/or evapo-
transpiration into atmosphere (Weyens et al., 2009b, 2009d).
Recently, we have observed that EB with appropriate degradation
pathways are metabolically active in the root and shoot of plants
vegetated in diesel contaminated soils (Andria et al., 2009; Afzal
et al., 2011; Yousaf et al., 2011) indicating that the inoculated bac-
teria are involved in the degradation of HCs inside the plant. Sim-
ilarly, Taghavi et al. (2011) observe that EB enhance plant tolerance
and HC remediation from contaminated groundwater. As HCs
move through plant vascular tissues, HC-degrading EB colonizing
in these tissues can promote in planta degradation, resulting in re-
duced phytotoxicity and evapotranspiration (Weyens et al., 2009b,
2009d).
The migration of EB from rhizospheric soil into roots seems to
be through the transpiration stream (Compant et al., 2005). Some
soil bacteria can penetrate into roots and move into shoots, indi-
cating that these soil bacteria are a source for EB (Germaine
et al., 2004), however, it is also observed that some phyllosphere
bacteria may be a source of EB (Hallmann et al., 1997). Recently,
it is observed that plants may host different EB including bacteria
capable of degrading HCs. Yousaf et al. (2010a) isolated HC degrad-
ers, Pseudomonas, Arthrobacter, Enterobacter, and Pantoea species,
from the root and stem tissues of Italian ryegrass and birdsfoot
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1326 S. Khan et al. / Chemosphere 90 (2013) 1317–1332

Table 2
Examples of successful remediation of hydrocarbons from soil by combined use of plants and endophytic bacteria.

Plant used Rhizobacteria Bacterial characteristics Reference

Italian ryegrass (L. multiflorum var. Taurus) Pseudomonas sp. strain ITRI53,
Pseudomonas sp. strain
MixRI75
Italian ryegrass (L. multiflorum var. Taurus), birdsfoot E. ludwigii strains
trefoil (L. corniculatus var. Leo) and alfalfa (M. sativa
var. Harpe)
Italian rye grass (L. multiflorum var. Taurus) and birdsfoot Pantoea sp. strain ITSI10,
trefoil (L. corniculatus var. Leo) Pseudomonas sp. strain ITRI15
Wheat (Triticum sp.) and maize (Z. mays) Burkholderia cepacia strain
FX2
Poplar P. putida W619-TCE
Yellow lupine B. cepacia VM1468
Pea (Pisum sativum) P. putida
Italian rye grass (L. multiflorum var. Taurus) Pseudomonas sp. strain ITRI53
Wheat (Triticum sp.) and maize (Z. mays) Enterobacter sp. 12J1
Hydrocarbon degradation Afzal et al. (2011)
and Afzal et al.
(2012)
Hydrocarbon degradation and ACC deaminase Yousaf et al. (2011)
activities
Hydrocarbon degradation Yousaf et al.
(2010b)
Toluene degradation Wang et al. (2010)
Trichloroethylene degradation Weyens et al.
(2010c)
Trichloroethylene degradation and Ni resistance Weyens et al.
(2010a)
Naphthalene degradation Germaine et al.
(2009)
Hydrocarbon degradation Andria et al. (2009)
Pyrene degradation, indole acetic acid (IAA), Sheng et al. (2008a)
siderophore production and inorganic phosphate
solubilization

trefoil vegetated in HC contaminated soils. Similarly, Siciliano et al.
(2001) found endophytic HC degraders in tall fescue (Festuca arun-
dinacea) and rose clover (Trifolium fragiferum) vegetated at an
aged-HC polluted site, with up to 4% of culturable EB possessing
genes involved in HC degradation. As EB can transfer HC-degrading
genes to the indigenous bacterial population residing inside the
plant, there is increasing interest in the role of EB in phytoremedi-
ation application (Barac et al., 2004, 2009; Weyens et al., 2009d;
Yousaf et al., 2011).
The potential benefits of using EB to enhance organic pollutants
degradation are summarized by Newman and Reynolds (2005).
The most important benefit being where use of molecular biology
techniques is required to express a specific gene in the plant, EB are
easier to manipulate than plant. Furthermore, quantitative gene
expression of HC-degrading genes inside the plant could be a use-
ful monitoring technique for determining the potential of the phy-
toremediation technology (Afzal et al., 2011; Yousaf et al., 2011).
The distinctive environment of the interior plant supports the pol-
lutant-degrading bacteria with an aptitude to attain higher popula-
tion size due to less competition for nutrients and space. One major
benefit of using EB is that any toxic organic compound moved in
the plant may be mineralized inside plant tissues, thus decreasing
the phytotoxicity and removing pollutant toxic effects on herbivo-
rous fauna residing on or near polluted soils. For the effective
plant–endophyte partnerships for the remediation of HC-polluted
soils, HC-degradation potential of EB is an important requirement
(Nie et al., 2011). Although RB-assisted phytoremediation of HCs
has been extensively studied (Johnson et al., 2004; Peng et al.,
2009; Cai et al., 2010), reports on application of plant–endophyte
partnerships for the phytoremediation of HC-polluted soils are
comparatively less (Table 2).
6. Conclusions
Plant–bacteria partnerships can be exploited to enhance the
remediation of HC contaminated soils. However, for efficient HC
remediation, it is of primary importance that the inoculated HC-
degrading bacteria colonize the rhizosphere and plant interior in
order to mediate their effects on plant growth and contaminant
degradation. RB and EB showing HC-degradation and/or plant
growth promoting ACC deaminase activity are more effective in
enhancing plant growth and subsequently HC-degradation. The
bacterial ACC deaminase activity accelerates root growth, resulting
in better access to nutrients and water and thus faster initial
growth, which enable plants to counteract stress responses caused
by contamination. Consequently, high amount of nutrients re-
leased by well established plant enhances bacterial population
and activities to degrade HCs. Combined use of plant and effective
bacteria seems to be a more promising technique for the remedia-
tion of HC contaminated soil as compared to alone bioaugmenta-
tion (only use of microorganisms) and phytoremediation (only
use of plants).
7. Future prospects
The inoculation of plants with both RB and EB showing different
traits, such as HC degradation, ACC deaminase activity, nitrogen
fixation, enhancing nutrients and pollutants bioavailability, may
be a more efficient approach for the remediation of HC contami-
nated soils, but additional research is required to further elucidate
this strategy. Concerted efforts should focus on increasing the
numbers and diversity of beneficial bacterial populations, particu-
larly for those showing ACC deaminase activity and HC
degradation.
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