Accepted Manuscript

Title: Rhizoremediation of petroleum

hydrocarbon-contaminated soils: Improvement opportunities
and field applications

Authors: Imran Hussain, Markus Puschenreiter, Soja Gerhard,

Philipp Schöftner, Sohail Yousaf, Aijie Wang, Jabir Hussain
Syed, Thomas G. Reichenauer

PII: S0098-8472(17)30343-X
DOI: https://doi.org/10.1016/j.envexpbot.2017.12.016
Reference: EEB 3356

To appear in: Environmental and Experimental Botany

Received date: 10-8-2017

Revised date: 11-12-2017
Accepted date: 17-12-2017

Please cite this article as: Hussain, Imran, Puschenreiter, Markus, Gerhard, Soja,
Schöftner, Philipp, Yousaf, Sohail, Wang, Aijie, Syed, Jabir Hussain, Reichenauer,
Thomas G., Rhizoremediation of petroleum hydrocarbon-contaminated soils:
Improvement opportunities and field applications.Environmental and Experimental
Botany https://doi.org/10.1016/j.envexpbot.2017.12.016

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Review article

Rhizoremediation of petroleum hydrocarbon-contaminated soils: Improvement

opportunities and field applications
Imran Hussain1, 2, Markus Puschenreiter3, Soja Gerhard1, Philipp Schöftner1, Sohail Yousaf4, Aijie
Wang5, Jabir Hussain Syed6,7*, Thomas G. Reichenauer1*
1
AIT Austrian Institute of Technology, Department of Energy, Business Unit Environmental

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Resources and Technologies Tulln, Austria
2
Department of Molecular Systems Biology, Faculty of Life sciences, University of Vienna,

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Austria
3
Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Austria

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4
Department of Environmental Sciences, Quaid-I-Azam University Islamabad, Pakistan
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Key laboratory of Environmental Biotechnology, Chinese Academy of Sciences Beijing, China

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6
Department of Meteorology, COMSATS Institute of Information Technology, Park Road Tarlai
Kalan 45550, Islamabad
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Department of Civil & Environmental Engineering, The Hong Kong Polytechnic University,
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Hong Kong
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Corresponding Authors:

*Thomas G. Reichenauer, PhD

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Tel: +43505503545
Fax:+43505503452
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Thomas.Reichenauer@ait.ac.at
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Graphical Abstract:

Highlights:

 Documented available data on rhizoremediation and its applications.

 Rhizoremediation of TPH-impacted soils has demonstrated encouraging results.


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Variety of soil amendments can enhance the remediation efficiency.
 There is an urgent need to search out suitable plant and microbe pair with efficient soils
amendments.

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Abstract
The remediation of petroleum hydrocarbon-contaminated soils has remained a challenging issue
for environmental scientists and engineers. Within the scope of bioremediation; rhizoremediation

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has emerged as the most suitable method for petroleum-impacted soils. It can be promoted by the
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proper selection of suitable plant-microbe combinations and its overall efficiency can be enhanced
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by adding suitable soil amendments. This review article summarizes the available knowledge on
rhizoremediation with respect to plant selection, inoculation with specific microbial strains
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(hydrocarbon-degrading as well as plant growth-promoting microbes) and a variety of soil

amendments for enhancing the remediation efficiency. A large set of organic and inorganic soil
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amendments has been presented for the discussion on potential increase of rhizospheric effects.
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Finally, we discussed a unique multi-process phytoremediation system (MPPS) for the remediation
of petroleum-polluted soils along with its field applications.
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Keywords: petroleum hydrocarbons; rhizoremediation; soil amendments; multi-process

phytoremediation system; filed applications
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Table of Contents:
1. Introduction
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2. Rhizoremediation: a synergistic form of plant and microbe actions

2.1. Rhizosphere and rhizosphere effect (RE)
2.2. Root exudation as an ecological driver in the rhizosphere
2.3. Mechanisms of TPH degradation stimulated by root exudates
2.3.1. Direct degradation

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 2.3.2. Enhanced pollutant bioavailability
2.3.3. Structural analogy and co-metabolism
2.3.4. Stimulation by energy and nutrient flow
2.4. Selection of plants for rhizoremediation
2.5. Enhancing the rhizoremediation of petroleum-polluted soils
3. Soil amendment for enhancing phytoremediation

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3.1. Stimulation by adding chemical fertilizer, manures, or bulking agents
3.2. Compost addition

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3.3. Biochar addition
3.4. Inoculation of soil/seeds with plant-growth-promoting and hydrocarbon-degrading

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rhizobacteria
4. Multi-process phytoremediation system (MPPS)

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5. Conclusions

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1. Introduction
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Pollution associated with crude oil and its refined products is of great concern worldwide due to
the threats it poses for human and ecosystem health, soil structure and ground-water quality
(Alexander, 2000; de Boer and Wagelmans, 2016; Sammarco et al., 2016). Total petroleum
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hydrocarbons, commonly termed TPH (crude oil, gasoline, creosote and diesel), are one of the most
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common groups of persistent organic pollutants (Huang et al., 2004a, 2005). TPH enter the
ecosystem – mainly soil and water – through leakage of underground storage tanks (LUST), oil
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spills, transportation processes and industrial processes (Aisien et al., 2009). This group comprises
gasoline range organics (GRO) and diesel range organics (DRO). The former includes small chain
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alkanes (C6-C10) such as benzene, toluene, ethylbenzene and xylene (BTEX), and the latter
consists of long chain alkanes (C10-C40) and highly hydrophobic chemicals such as polycyclic
aromatic hydrocarbons (Kamath et al., 2004).
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Accidental large-scale oil spills and their associated pollution are present throughout the world in
significant amounts (Van Hamme et al., 2003; Atlas and Bragg, 2009; Atlas and Hazen, 2011).
Extensive soil pollution with TPH results in extreme hydrophobic conditions and infertile soils,
ultimately leading to reduced plant and microbial biomass (Hutchinson et al., 2001a; Ramadass et
al., 2015; Liu et al., 2016). Worldwide research by many groups have pinpointed the drastic and
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deleterious effects of hydrocarbon pollution on soils (Gaskin and Bentham, 2010a; Kim et al.,
2010; Bramley-Alves et al., 2014; Shahsavari et al., 2015; Shahzad et al., 2016). In addition,
hydrocarbon pollution may also severely affects plants, bacteria, earthworms and other associated
soil biota (Hentati et al., 2013; Ramadass et al., 2015). Recent studies clearly underline that TPH
pollution greatly impacts soils and their associated environmental matrices.

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This urgently calls for the effective removal of petroleum hydrocarbons from the environment. The
extremely complex and toxic nature of crude oil and its products, however, complicates cleanups.

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Both physical and chemical methods have been employed, including excavation, thermal
desorption, soil vapor or stream extraction, incineration, soil washing/flushing, volatilization,

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solidification/stabilization, supercritical fluid (SCF) oxidation and encapsulation (Abhilash et al.,
2009; Peng et al., 2009; Walls, 2010; Khan et al., 2014). These methods are considered to be

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essential for speedy cleanup and to prevent oil spreading (Dave et al., 2011; Dixit, 2016).
Nonetheless, these methods have disadvantages because they are (i) disruptive to the environment
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(ii) expensive due to high energy consumption and (iii) have poor public acceptability (Kaimi et
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al., 2006). Among biological methods, bioremediation and phytoremediation are promising
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technologies that can offer both engineering and ecological benefits (Alkorta and Garbisu, 2001b;
Chaudhry et al., 2005; Bramley-Alves et al., 2014; Grace Liu et al., 2011a).
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This technology has recently become a promising alternative method for the remediation of
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petroleum-polluted soils because it offers ecological, cost-effective and natural aesthetic benefits.
Plant-based remediation technologies use a variety of plant processes for environmental cleanups.
These processes include phytoextraction, phytovolatilization, phytostabilization,
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phytotransformation and rhizoremediation (Chaudhry et al., 2005). All biological methods are less
labor-intensive and potentially offer complete mineralization of crude oil to CO2 and H2O
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(McGuinness and Dowling, 2009). However, the application of biological methods is limited
because cleanup and remediation take more time. Additionally, the applicability of biological
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methods can be limited by contaminant distribution, soil nutritional status, soil water pH,
temperature and oxygen supply (Chatterjee et al., 2010). In Table 1 and Table 2, we have briefly
described the processes and mechanisms involved in bioremediation and phytoremediation of
TPH-polluted soils and ground water with reference to some recent studies.

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The main objective of this review article was to briefly summarize different biological approaches
available for remediation of TPH-impacted soils with emphasis on rhizoremediation technologies.
The structure of this review article consists of systematic and well-defined outline on
rhizoremediation, its influencing factors and available opportunities for its improvement at
laboratory, greenhouse and field scale levels. The subsequent focus of this review article will be
on combination of different phytoremediation technologies presented as multi- process

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phytoremediation system.

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2. Rhizoremediation: a synergistic form of plant and microbe actions
Use of selected green plants and their associated microorganisms to detoxify and clean-up

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environmental contaminants is termed phytoremediation (Thijs et al., 2016). This technology may
use the natural potential of selected plants and microbes for transformation, extraction,

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sequestration, assimilation as well as degradation of organic and inorganic type of pollutants. This
broaden scope of phytoremediation can be obtained by utilization various natural processes of
plants such as phytoextraction, N
rhizofilteration, phytostabilization, phytovolatilization,
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phytotraspiration and rhizodegradation. These processes have been reviewed very precisely in
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many review articles (Kooh et al., 2017; Truu et al., 2015; Ali et al., 2013; Alkorta and Garbisu,
2001a), and are summarized in Table 2. In this review article, we have discussed rhizoremediation
with focus on petroleum hydrocarbon degradation. The peculiar mechanisms involved in
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detoxification of organic pollutants e.g., petroleum hydrocarbons were summarized by Sanderman

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(1992) as “green liver concept” (Truu et al., 2015; Reichenauer and Germida, 2008). Parameters
such as physico-chemical characteristics of pollutants, environmental conditions, availability of
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hydrocarbon degradative strains and plant characteristics are responsible for overall success of
phytoremediation trials in TPH-impacted soils (Truu et al., 2015; Sessitsch et al., 2013;
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Vangronsveld et al., 2009).

The plant-stimulated bioremediation of organic pollutants by rhizospheric microorganisms is

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termed rhizoremediation. It is also referred to as rhizodegradation, rhizosphere bioremediation and

microbe assisted phytoremediation technology (MAPT). Rhizoremediation, a major mechanism
for phytoremediation of petroleum-polluted soils, mainly focuses on stimulating the population of
degrading microorganisms through the plant rhizospheric effects (Truu et al., 2015; Merkl et al.,
2004, 2005b; Newman and Reynolds, 2004). The rhizosphere is the extremely active area around
roots (1-2 mm) influenced by plant activity (Brink, 2016; Dzantor, 2007; Liu et al., 2014). In the
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rhizosphere, roots can provide a favorable microenvironment and a carbon source for hydrocarbon-
degrading microbes. In return, microorganisms enhance plant growth by providing plant nutrients,
protection against plant pathogens, and contaminant degradation (Macek et al., 2000; Qixing et al.,
2011). It is well established that rhizoremediation by grasses, trees and other annual species helps
in dissipation of TPH from petroleum-polluted soils (Cook and Hesterberg, 2013; Gaskin et al.,
2008; Liu et al., 2012; Smith et al., 2006; Soleimani et al., 2010a). Table 3 provides a summary of

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recent rhizoremediation studies (2009-2017) on TPH removal by different plants.

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2.1. Rhizosphere and rhizosphere effect (RE)
By definition, the rhizosphere is a vigorous habitat around root-zone inside soils that extends 1-2

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mm away from plant -roots and typically contains an increased (10-100 times) number of microbes
compared to bulk soil (Hinsinger et al., 2005; York et al., 2016). The stimulated increase of
microbial biomass in the rhizosphere is an attribute of the “rhizosphere effect” caused by physical

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and chemical effects of plant roots (Truu et al., 2015; Nie et al., 2010). The synergistic roles of
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plant roots and microorganisms strengthen the RE. Due to this RE, organic compounds are
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degraded here by stimulated microbial biomass and activity (Nie et al., 2010). Plant roots release a
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variety of photosynthetically derived organic compounds into the rhizosphere that may act as
carbon sources for fungi and bacteria. Up to 20% of the total fixed carbon by plants may be released
in the rhizosphere (Han et al., 2016). In return, microorganisms enhance plant growth by providing
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plant nutrients, protecting against plant pathogens and degrading contaminants (Chaudhry et al.,
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2005; Macek et al., 2000; Martin et al., 2014). Microorganisms also support root growth and
decrease the production level of stress hormones (Dams et al., 2007).
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2.2. Root exudation as an ecological driver in the rhizosphere

Root exudation is the most important factor affecting microbial drift in the rhizosphere and is
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considered a potential driving force for stimulated rhizoremediation (Martin et al., 2014; Phillips
et al., 2012). At the rhizosphere, the tremendous (huge 5-10x) increase in microbial biomass is a
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function of plant roots, which provide substrate for microbial growth and metabolism (Hinsinger
et al., 2005). Root-released compounds can be classified into four distinct groups based on their
mode of production. These include passive exudates, secondary plant metabolites (SPMs),
mucilages (from root tip and epidermal cell) and lysates (from senescent tissues and roots).
Technically, root exudates can also be categorized into water-soluble and insoluble fractions. The
soluble fraction consists of low-molecular-weight carbohydrates, amino acids, organic anions and
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secondary metabolites, whereas organic polymers (sloughing cells and root debris) are included in
the list of the insoluble fractions (Martin et al., 2014; Walker, 2003; Walker et al., 2003). All these
compounds are used as an energy and carbon source by soil microbes during their metabolic
processes (Chaudhry et al., 2005). TPH biodegradation can also be stimulated by the physical
effects of plant roots such as soil aeration and the provision of sites for microbial attachment
(Martin et al., 2014). Two key mechanisms (operated) by root exudates are responsible for the

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distinct community shifts in contaminated soils: alteration of microbial catabolic gene expression
and selection of specific microbial strains. Generally, it is well established that root exudates play

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a distinctive role in shaping rhizosphere microbiome in petroleum and nitro aromatic polluted soils
(Thijs et al., 2016; Sutton et al., 2013; Berg and Smalla, 2009; Siciliano et al., 2003). Many studies

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revealed that host plants shape the microbial community by secreting specific root exudates in
result of selective responses to contaminants, pathogen attack and resource utilization (Neal et al.,

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2012; Berendsen et al. 2012). Experiments showed that plant root exudates can induce

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biodegradation of petroleum in TPH-polluted soils (Baetz and Martinoia, 2014; Phillips et al.,
2012). Artificial addition of plant root exudates can initiate and enhance biodegradation of TPH
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(Rentz et al., 2005). Adding root exudates, however, negatively affect TPH dissipation (Phillips et
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al., 2012). The secretion of root exudates from plant roots depends on environmental factors and
the growth stage of plants. Other biological factors like the presence of arbuscular mycorrhizal
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fungi (AMF), regions of root secretion and plant species also are important for root exudation
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during TPH remediation. Interestingly in rhizospheric region, the production of bio-surfactant by

plants roots and rhizospheric bacteria may enhance the bioavailability and uptake of organic
pollutants (Truu et al., 2015). This aspect can create extra benefits in weathered/aged contaminated
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soils those are generally less responsive as compared to freshly spiked soils (Bezza and Chirwa,
2017; Devin et al., 2017). Poor solubility of TPH molecules in water and their adverse effects on
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plant growth limits the scope of phytoremediation. Therefore, it is well established that using plant
and microbes in pair has more positive results on remediation trials as compared to use of each
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partner alone (Xun et al., 2015). To exploit the plant and microbe partnership, use of endophytic
and rhizospheric microbial strains with suitable plants got huge intention these days (Thijs et al.,
2016). The fact that vegetative soils have higher (10-100 times) microbial number and diversity as
compared to bulk soils (Hinsinger et al., 2005; Wenzel, 2009) support the argument that use of
rhizospheric microbes as partner with plants for successful and speedy remediation trials. Very
importantly, in the rhizospheric region, the presence of specific organic compounds (terpenes,
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flavonoids and phenolic) having structural analogy with petroleum hydrocarbons can induce
expression of degrading genes and their abundance. This phenomenon of enhanced catabolic gene
expression has been successfully used in mineralization, sequestration as well as degradation of
organic pollutants (Dubey and Fulekar, 2013) includes TPH (Andria et al., 2009).

Primarily, the use of rhizobacteria was very common in agriculture for reducing stress, promoting

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growth and stimulating yield productivity by improving specific plant to microbe interactions
(Bhattacharyya and Jha, 2012; Saleem et al., 2007: Narasimhan et al., 2003). This concept gained

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special attention for utilization in remediation trials for improved phytoremediation efficiency of
many organic xenobiotics (Yang et al., 2009). Recent studies demonstrate the potential application

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of plant and rhizobacteria partnerships for the clean-up of TPH impacted soils (Abhilash et al.,
2013; Brink, 2016; Singh et al., 2016). From the above discussion, it can be concluded that plant-

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microbe interactions often have mutual benefits for plants and rhizobacteria (Kuiper et al. 2001).
Rhizobacteria utilize organic contaminants as a carbon and energy source for their cell functioning
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and life cycle, whereas plants favor the survival of rhizobacteria by providing residency (Kuiper et
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al., 2004). Additionally, plants can change the rhizosphere area to favorable microenvironment by
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providing root exudates, organic acids, cations/anions and biogenic surfactants. Rhizobacteria
capable of synthesizing bio-surfactants have been shown to enhance the bioavailability of organic
pollutants by releasing the pollutant from soil particles, and thus their degradation (Whitfield
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Åslund et al., 2010). These bio-surfactants can be helpful for desorption of many organic
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xenobiotics from soil particles. To date, rhizosphere engineering gained interest by modifying this
microenvironment with nutrient adjustment, exudates regulation and insertion of transgenic strains
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(Abhilash et al., 2009; Aken and Doty, 2009; Eapen et al., 2007).

2.3. Mechanisms of TPH degradation stimulated by root exudates

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In natural soil systems, degradation of organic contaminants by both ways (aerobic and anaerobic)
is an important pathway of bacterial remediation (Boopathy, 2004). But in case of TPH, the rate
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and extent of anaerobic bioremediation is narrow and decrease especially when soils are polluted
with recalcitrant hydrocarbons (Grishchenkov et al., 2000). That is why, rhizoremediation of
hydrocarbons is widely accepted scientific mechanism in TPH-impacted soils for enhanced and
speedy trials (Thjis et al., 2016). Notably, in TPH-impacted soils most of microbes has facultative
nature (survive under both oxic and anoxic conditions). When plant roots interact with microbes in
TPH-impacted soils, roots provide microaerophilic conditions. Plant root exudates; low molecular
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weight organic acids (malic acid, succinate and ascorbate), provide substrate for microbial
metabolism as well as adjust the pH of soils. Therefore, degradability and availability of
hydrocarbons to microbes increases. There are four possible ways by which root exudates can
enhance the biodegradation of petroleum-contaminated soils. These include direct degradation by
plant enzymes, enhancement of pollutant bioavailability, co-metabolic process and stimulation by
energy/nutrient flow (Martin et al., 2014).

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2.3.1. Direct degradation

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Enzymatic breakdown mediated by microbes is the principal mechanism of hydrocarbon removal.
Plant root exudates can directly influence TPH removal by secreting extracellular enzymes such as

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laccases and peroxidases (Martin et al., 2014). Additionally, AMF play an important role in the
dissipation of TPH molecules by providing extracellular enzymes (Xun et al., 2015).

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2.3.2. Enhanced pollutant bioavailability

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Plant root exudates can enhance pollutant bioavailability by increasing their solubility thus making
them more available for microbial attack. For example, lipophilic substances (rhamnolipid, Tween
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80) can increase the solubility of TPH and increase the probability for their degradation (Read et
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al., 2003). Low molecular weight (LMW) aliphatic carboxylates (citrate and oxalate) also can
increase TPH bioavailability by promoting their desorption from soil matrices (Cai et al., 2016).
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2.3.3. Structural analogy and co-metabolism

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Many plant root exudates in general and secondary plant metabolites in particular are structurally
similar to various TPH, especially aromatic hydrocarbons (Singer, 2006). These structural
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similarities (analogy) can enhance TPH degradation by stimulating co-metabolic processes. Co-
metabolism is defined as the oxidation of a non-growth substrate in the presence of another other
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growth substrate and is regarded as a main mechanism behind TPH degradation (Kuiper et al.,
2004).
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2.3.4. Stimulation by energy and nutrient flow

The most significant role of root exudation is to provide a constant source of energy and nutrients
to microorganisms, which increases microbial activity and further enhances TPH degradation. In a
single year, plants can secrete 10-40% of their photosynthetic products into the soils; this may be
equal to 4500 kg C ha-1 and 15-60 kg N ha-1 (Alkorta and Garbisu, 2001b; Gerhardt et al., 2009;

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Martin et al., 2014). LMW root exudates are a fundamental source of energy and nutrients in the
rhizosphere.

2.4. Selection of plants for rhizoremediation

Although rhizoremediation uses the natural potential of plants and their associated microbes, it can
be enhanced by using suitable plant-microbe pairs (Afzal, 2010; Afzal et al., 2014; Chaudhry et

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al., 2005; Tang et al., 2010a; Wenzel, 2009). Plants’ tolerance to specific contaminants and their
extensive root systems (which promote TPH-degrading microbes) are two preconditions for

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rhizoremediation (Adam and Duncan, 1999, 2002). Both of these characteristics vary on inter- and
intra- species levels (Liste and Felgentreu, 2006a; Liste and Prutz, 2006; Siciliano et al., 2002).

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Therefore, the selection of suitable plant species and genotypes plays an important role in the
development of remediation methods for TPH. Different types of plants including grasses,

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legumes, herbs, medicinal plants, ornamentals and trees have been extensively studied about the
dissipation of petroleum hydrocarbons. Each type of plant has specific advantages over the others.
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For example, the fibrous root system of grasses (family Poaceae) has the advantage of a higher
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surface area, which enhances the contact between pollutants and degrading microbes and also
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provides supplementary access to fine pores (Gaskin et al., 2008; Gaskin and Bentham, 2010a). In
most rhizoremediation studies on removing petroleum hydrocarbons, grass species were used.
Kaimi et al. (2006) presented a case study on the root development of 12 selected grasses in
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petroleum-contaminated (2% w/w) soils. Total measured root lengths of Southern crabgrass,
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Bermuda grass and Italian ryegrass were 1398 cm, 858 cm and 185 cm, respectively. In addition
to having high root biomass, grasses can provide large amounts of soluble organics and secondary
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plant metabolites (SPMEs) in the plant rhizosphere, which also supports pollutant degradation
(Chaudhry et al., 2005; Martin et al., 2014; Singer, 2006).
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Legumes (family Fabaceae) comprise about 18,000 species, and have a variety of methods for
nutrient acquisition, plant defense and reproduction. These can be used to fix atmospheric nitrogen
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and restore the nitrogen pool in a wide range of terrestrial environments. Legumes, therefore, have
competitive advantages over other plant species in low-nitrogen soils because, they can obtain
additional nitrogen through symbiotic relationships with N-fixing rhizobia (Wenzel, 2009). Hall et
al. (2011) reviewed the role of the Fabaceae in phytoremediation and concluded that this family
exhibits a wide range of adaptations to petroleum-contaminated soils. Ornamental plants are an
important type of higher plants, that can be used in the phytoremediation of petroleum-polluted
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soils (Liu et al., 2008, 2010, 2012; Zhang et al., 2010). An additional benefit associated with
ornamental plants is the minimal risk for introducing contaminants or their metabolites into the
food chain. Few studies are available on the role of ornamental plants in the phytoremediation of
petroleum-polluted soils, calling for more efforts to explore the potential of these plants. The use
of medicinal and herbal plants for phytoremediation purposes is uncommon. Some authors have
suggested using medicinal plants in petroleum-polluted soils not only to remedy the contaminated

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environment but also to produce valuable drugs. Table 4 portrays the above-mentioned plant types
used in different phytoremediation (rhizoremediation) studies on petroleum hydrocarbons.

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2.5. Enhancing the rhizoremediation of petroleum-polluted soils

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Phytoremediation is included in the list of priority in-situ methods for the remediation of TPH-
polluted soils. However, the application of plants alone may have limited remediation efficiency.

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To reduce soil treatment time and improve phytoremediation remediation efficiency, this
technology can be coupled with different approaches. These include addition of inorganic
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(nutrients, fertilizers, surfactant) and organic soil amendments (compost, biochar), soil or plant
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(seed) inoculation with endophytic or rhizospheric microbial strains, the application of transgenic
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microbes or plants, and integrated approaches.

3. Soil amendment for enhancing phytoremediation

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In literature, a large database of organic and inorganic amendments is available for enhancing the
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overall phytoremediation efficiency. Compost, agro-industrial waste, biochar, humic substances

and biosolids are listed as organic amendments while lime and zerovalent iron grid are considered
as inorganic soil amendments (Wiszniewska et al., 2016; Fernández et al., 2012). Recently,
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Wiszniewska et al. (2016) provided an extensive review on natural soil amendments for improved
phytoremediation of polluted soils. Natural soil amendments are believed to provide variety of
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benefits such as nutrients (NPK) balance, enhanced microbial activity, stimulate soil quality
(hydrolases and oxidoreductases) and aggregation (Mench et al., 2010). Additionally, presence of
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natural soil amendments enhances plant survival, diversity and biomass production as well as
biodegrading activity of microbes (Huang et al., 2013; Wiszniewska et al., 2013; Lamichhane et
al., 2012). Moreover, the use of humic substances (humic acid, fulvic acid and humine) and crude
plant extracts or exudates can initiate positive effects on plant growth of phytoremediation plants
in organic contaminants polluted soils (Wiszniewska et al., 2016; Huang et al., 2013; Park et al.,
2011) especially petroleum polluted soils (Guo et al., 2017a, b, Jiang et al., 2017). However, limited
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information is available on long term influence of soil amendments on other soil organisms during
remediation experiments and merits further research. Table 5 summarizes different soil
amendments used to stimulate the phytoremediation or rhizoremediation of TPH-contaminated
soils.
3.1. Stimulation by adding chemical fertilizer, manures, or bulking agents
The importance of nutrients in petroleum-contaminated soils has long been recognized because

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they support a variety of microbial processes. Macronutrients such as nitrogen (N), phosphorus (P)
and potassium (K) are required for amino acid production and energy transport (e.g. adenosine

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triphosphate). Meta-analysis of microbial biomass indicates that carbon (C), N and P are present in
a ratio of 106:16:1, (Redfield et al., 1963). Often, contaminated soils have a low nutrient status and

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may require nutrient additions for sufficient microbial and plant growth, which ultimately favors
the dissipation of contaminants. To estimate how much nutrient amendment is required, the so-

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called Redfield ratio (106:16:1) is commonly cited as an optimal C/N/P target. Palmroth et al.

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(2002) and Vaajasaari and Joutti (2006) reported that soil amendment with NPK fertilizer
stimulates the rhizoremediation of petroleum hydrocarbon by up to 65%, while addition of
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municipal bio-waste compost has 60% removal efficiency over a period of 39 months. Hutchinson
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et al., (2001b) also explained that optimal remediation of petroleum hydrocarbons was obtained by
fertilization leading to a C: N: P ratio of 100:2:0.2. Vegetation establishment plus fertilization led
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to a reduction of the more recalcitrant fractions in petroleum-polluted soils (White et al., 2006).
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Results from Tanee and Kinako (2008) demonstrate that bio-stimulation with inorganic fertilizer
led to the highest hydrocarbon dissipation, whereas organic manure proved to be less effective.
Ryegrass produced more root biomass than any other species when grown in inorganic fertilizer or
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hardwood sawdust + inorganic-fertilizer-amended soil (White et al., 2003a). Similarly, Cartmill et

al. (2014) described that applying the controlled release fertilizer (CRF) overcame growth
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impairment by stimulating photosynthesis, chlorophyll content and the bacterial population in

hydrocarbon-contaminated soils.
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Regarding levels of fertilizer application, Merkl et al. (2005a) suggested medium dose of NPK
application (600 mg N/P/K kg−1 soil) for phytoremediation with B. brizantha in nutrient-poor
loamy sand (18.4% oil dissipation after 22 weeks). Nonetheless, other amendments such as broiler
litter, paper mill sludge or hardwood sawdust can be used to enhance the phyto- and bioremediation
of diesel-contaminated soils (White et al., 2003). In another study reported by the same research

12
group, it was shown that the addition of broiler litter led to the greatest reduction in TPH in all
planted treatments after 14 weeks (White et al., 2006). To increase the aeration in aged
contaminated soils, bulking agents such as chopped bermudagrass hay, sawdust and vermiculite
have been added yielding positive results in dissipating petroleum hydrocarbons (Rhykerd et al.,
1999). Regarding synergistic interactions of microbes and nutrients, Arslan et al. (2014) reported
that bacterial inoculation improved plant growth and that hydrocarbon degradation further

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stimulated by nutrient supplement. Their results showed an enhanced rhizospheric effect by
increased root colonization and metabolic activity of inoculated stains. Another recent report on

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the combined application of microbes and nutrients described very similar results (Shahzad et al.,
2016). They showed 59% more TPH dissipation in soils amended with nutrients versus un-treated

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soil planted with maize. Fu et al. (2012) studied the removal of selected PAHs in aged contaminated
soils by alfalfa and organic fertilizer. Organic fertilizer enhanced PAHs dissipation by 11.6%

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compared to the unfertilized and unplanted treatment. That study agrees well with Choi and Chang

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(2009), who reported a positive correlation between the nitrogen fertilizer level and petroleum
hydrocarbons degradation. In another study, Coulon et al. (2005) reported the dissipation of TPH
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and PAHs by the combined action of temperature and nitrogen fertilizer. That study showed 77-
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95% loss of the alkane fraction; PAHs dissipation was 80% under optimal conditions at 10 °C and
fertilizer added.
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Apart from inorganic fertilizers, various bulking agents and organic fertilizer/manures can also
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stimulate the rhizoremediation efficiency. These bulking agents include wood chips, sawdust, hay,
brans, straw and bark from different plants (Rhykerd et al., 1999; Vasudevan and Rajaram, 2001).
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These low-density bulking agents improve the remediation rate by stimulating microbial activity
by increasing soil porosity and oxygen diffusion (Rhykerd et al., 1999).
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3.2. Compost addition

Compost is known as soil conditioner. It is used as an organic amendment in soil because it contains
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high loads of microorganisms and nutrients, which improve soil quality, promote plant growth and
stimulate the degradation of various organic contaminants in soil like TPH and PAHs (Beesley et
al., 2010).

Application of compost that serves as source of xenobiotic-degrading microbes such as bacteria

and fungi, can enhance the degradation of contaminants into less toxic substances (White et al.,

13
2003). In several studies composting was used to remediate soil contaminated with TPH. Sasek et
al. (2003) reported that using mushroom compost; 37-80% of PAHs were removed from soil in
100 days. Soil amendment with compost removed 40% of TPH compared to non-amended soil,
which removed only 9% (Ofek et al., 2014). Amendments with sewage sludge, cattle manure and
compost enhanced the removal efficiency of PAHs from 35.5% to 44.9% and 25.5% to 42.3%,
respectively while control obtained only 5.6% removal rate (Zhang et al., 2012). Choi and Chang,

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(2009) reported the highest degradation percentage of TPH in soil amended with three-year-old
compost, i.e. (41.1%). By using compost and biopiles of different parent material, hydrocarbon

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removal was increased by up to 99% (Van Gestel et al., 2003).

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3.3. Biochar addition
Biochar is a carbonaceous, fine-grained, porous material prepared by thermal decomposition of

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organic materials under oxygen-limited conditions (Cantrell et al., 2012). Biochar’s originated
from variety of parent’s materials has been used extensively in the remediation of organic and
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inorganic environmental pollutants. These parent materials may include agricultural and forestry
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byproducts, industrial derivatives, animal/poultry wastes and sewage sludge or slurries
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(Wiszniewska et al., 2016). In the published literature, agricultural byproducts such as straws (rice,
maize, wheat), seed cakes, nut shells and grasses has been used extensively. While forestry
byproducts comprise of wood chips, wood pallets, bamboo sticks, willow wood and hardwood.
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Industrial byproducts that has been used as BC parent material are bagasse (sugarcane), algae
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biomass and fruits/vegetables pulp. Lastly, animal/poultry manure as well as sewage sludge has
been also used as popular parent material for biochar preparation (Lehmann et al., 2006; Spokas et
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al., 2009; Tang et al., 2013). Particularly, different BC with their parent materials that has been
reported for the remediation of petroleum hydrocarbons are listed in the Table 6.
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Adding biochar to degraded soils improves soil properties. For example, it enhances soil fertility,
nutrient retention and oxygen supply (Tang et al., 2013). Biochar has been used to improve soil
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properties (fertility, moisture contents, nutrient retention) as well as to remediate heavy metals
contamination by surface adsorption, precipitation, partitioning, and sequestration (Beesley et al.,
2011; Tang et al., 2013). Microbial growth and biomass can be promoted by various physical and
chemical effects mediated by biochar. These effects are due to the presence of additional reactive
surfaces, soil tensile strength, bulk density, soil pH, water holding capacity and change in porosity
(Lehmann et al., 2011). Biochar can also function as shelter for microbes in the soils (Ameloot et
14
al., 2013). The large surface area, porosity and binding capacity can retain PAHs and the nutrients
provide a good habitat for microbes. Biochar enhances soil aeration, thus increasing microbial
populations (Bruun et al., 2008). Denyes et al. (2012) reported that biochar addition to soils also
improves plant growth and physiology (chlorophyll content and shoot to root biomass). Biochar
amendments also increase the soil pH by accumulating basic cations (Rees et al., 2014).

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Biochar is recognized as universal sorbent because of its strong potential to bind environmental
contaminants, and it has long been used as a sorbent for both organic and inorganic pollutants

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(Ahmad et al., 2014). Different functional groups (O-containing carboxyl, hydroxyl and phenolic
groups) in biochar effectively bind contaminants in soil (Uchimiya et al., 2011). The sorption

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properties of biochar are influenced by various factors like pyrolysis temperature, residence time
and parent material (Ahmad et al., 2014). Low-temperature biochar has been used for remediation

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purposes because it does not strongly absorb the contaminants (compared with activated carbon)
and thus facilitates degradation (Ren et al., 2016). Initially, biochar and other carbonaceous
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materials were used to reduce the bioavailability of organic contaminants (Beckingham and Ghosh,
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2017; Hale et al., 2012; Kołtowski et al., 2017). Sorption can reduce the short-term risk but may
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not be optimal for remediation purposes. Recently, biochar has been applied to stimulate
phytoremediation efficiency by increasing plant growth, and water holding capacity and by
improving soil structure (Cao et al., 2016; Lyu et al., 2016; Sohi et al., 2010).
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Qin et al. (2013) studied TPH removal after biochar addition as a soil amendment. They reported
a 92.6% removal of saturated hydrocarbons from petroleum-contaminated soil. Amendment with
sewage sludge biochar at 2%, 5% and 10% levels reduced PAHs by 31%, 39% and 43%,
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respectively (Zielińska and Oleszczuk, 2016). Currently, a novel technology has been developed
in which biochar is used as an immobilized carrier for selected microorganisms, thus enhancing
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the biodegradation. The immobilized-microorganism technique (IMT) was reported by Chen et al.
(2012), who concluded that biochar can promote bioremediation of contaminated soils and has
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more benefits over conventional carriers such as wood chips, bamboo leaves, pineapple needles
and wheat straws. In another study, Zhang et al. (2016) demonstrated that immobilization of a salt-
tolerant strain (HRJ4) with biochar as a carrier material showed higher TPH degradation. This calls
for further exploring other hidden benefits of biochar for TPH remediation (e.g. by selecting
suitable biochar and plant types).

15
3.4. Inoculation of soil/seeds with plant-growth-promoting and hydrocarbon-degrading
rhizobacteria

Phytoremediation (use of plants alone) of TPH provides a green alternative to different exhaustive
techniques for remediation of petroleum-polluted soils (Olson et al., 2007). Limitations for using
this system are:

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 Many plant species are sensitive to hydrocarbons; even tolerant species of plants neither
grow well nor support degradation

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 Deficient microbial populations (lower species diversity as well as reduced biomass) to
support plant growth as well as microbial degradation of petroleum hydrocarbons

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To overcome these challenges and enables a realistic application of this technology, a unique
phytoremediation system (having plant-growth-promoting rhizobacteria (PGPR) was proposed for

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hydrocarbon-polluted soils (Huang et al., 2005; Gurska et al., 2009). Initially, PGPR were mainly
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used in agriculture to facilitate nutrient uptake and protection against various plant diseases
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(Zhuang et al., 2007). Application of this technology (combined use of plant-growth-promoting
and hydrocarbon-degrading microorganisms) is a relatively new concept in the field of
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hydrocarbon remediation of soils and groundwater (Afzal, 2010; Weyens et al., 2009b). Microbial
strains can enhance plant resistance to contaminant stress and stimulate the production of their
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biomass (Muratova et al., 2012). In return, vegetation can improve the soil structure, that ultimately
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leading to abundant populations of microbes around roots (Susarla et al., 2002b). PGPR showed
promising results by supporting plant growth and health even under unfavorable soil and pollutant
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conditions (Fester et al., 2014; Hou et al., 2015; Gerhardt et al., 2015, 2017)

Many genera of bacteria and fungi are known to promote plant growth in diesel-impacted soils.
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Among bacteria, approximately 70 oil-degrading genera have been identified so far, whereby
Pseudomonas, Bacillus, Acinobacter, Rhodococcus and Arthrobacter are the most prominent
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genera that are reported to promote plant growth. Regarding fungi, arbuscular mycorrhizal fungi
(AMF), basidiomycetes, white-rot and litter-decomposing fungi have been shown to promote plant
growth under hydrocarbon stress (Hernández-Ortega et al., 2012; Xun et al., 2015). This
technology provides a rapid rate of rhizoremediation within an acceptable time frame (2-3 years).
Bacterial inoculation prior to seed sowing with PGPR can increase plant resistance to TPH, salt,
trace metals and other environmental stresses (Hou et al., 2015). In return, this yields a vigorous
16
plant growth and more root biomass. The result is an active rhizospheric effect by providing greater
proliferation of both naturally occurring and deliberately introduced microbial strains (Ma et al.,
2011; Song et al., 2016). The significant increase in roots and microbial biomass in soils provides
a sink for rapid metabolism of TPH within the active rhizosphere zone (Brink, 2016; Leigh et al.,
2002). Additionally, rapid root development in contaminated soils improves soil structure and
nutrient uptake (Abed et al., 2015; Susarla et al., 2002b).

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Cowie et al. (2010) reported PGPR-enhanced rhizoremediation of TPH by simultaneous

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enhancement in plant growth and microbial populations. Similarly, petroleum-hydrocarbon-
degrading-bacterial strains enhanced plant growth as well as pollutant dissipation from soils

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(Yousaf et al., 2010). The synergistic use of plants and PGPR, yields significant results on different
scale of studies (greenhouse to field). For example, Ho et al. (2007) reported the promotion of

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PAHs degradation in the rhizosphere of tall fescue with a specific interaction of PGPR strains (P.
fluorescens 5RL). Similarly, Glick (2003) also suggested that bacteria exhibiting both plant-
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growth-promoting activities as well as hydrocarbon-degrading potential are more suitable for
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rhizoremediation than those having only one of the above mentioned characteristics. Many other
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studied have demonstrated that rhizoremediation rate and efficiency can be stimulated by
inoculating seeds with PGPR strains (Abed et al., 2015; Gurska et al., 2009; Hou et al., 2015;
Shahzad et al., 2016; Xun et al., 2015). Some studies also reported on the role of fungi and their
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colonization in different plant parts during the rhizoremediation of organic pollutants. Hernández-
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Ortega et al. (2012) underlined the role of AMF during the phytoremediation of diesel-
contaminated soils. TPH degradation in the rhizosphere of Melilotus albus was enhanced by better
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physiological responses and more root colonization.

Despite the above-mentioned soil amendments for improving phytoremediation efficiency, several
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other techniques are also available which are recently reviewed by Gerhardt et al., 2017. These
include use of proper agronomic practices, omics technologies (genomics, transcriptomics,
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proteomics, metabolomics and metagenomics), transgenic as well as somaclonal variants and

mutant lines (Gerhardt et al., 2017; Kidd et al., 2015; Parihar et al., 2015). Recently, a new concept
engineering the metaorganisms was proposed by Thijs et al. (2016) for efficient utilization of
ecological and predictive modelling for improving phytoremediation. In contaminated
environment, host plant selects specific microbes from pool of microbial communities having
degradative potential and subsequently utilized that microbes for degradation (Hassan et al., 2013,
17
2014). The detailed mechanisms how the host plant can do this is missing and merits further
research.

Thijs et al. (2016) have explained this phenomenon by comparing the establishment of microbiome
development in rhizospheric/endospheric region with gut microbiome development. They
proposed a competition-driven ecological model (adapted from Scheuring and Yu, 2012), which

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explained the driving forces may play direct or indirect role for increasing the abundance of plant
growth promoting and pollutant degradative microbiome. Four distinctive strategies were proposed

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in the above-mentioned model which may be utilized in redirecting the microbiome in the
rhizospheric region of contaminated soils; 1). select the right and unique host plant that define the

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required microbial community structure and function. This tool will help to modify plant-assisted
microbial communities. Thus, for improving phytoremediation efficiency and biomass yield,

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specific plant based selection strategies (for elevated microbiome functions) can be utilized. 2).
optimize plant growth and biodegradation rate by changing the quality and quantity of root
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exudates. This will be done by stimulating the microbial colonization with a so-called technique
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called rhizoengineering. Specific steps of rhizoengineering are plant breeding (of specific plant
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traits) and genetic modification in both plants and microbes. 3). modification of driving forces such
as competition for resource exploitation and cooperative interactions (co-existence, mutualism and
symbiosis) can be done for enhancing biodegradation efficiency. 4). feeding the supply lines by
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isolation and culturing of unique degradative microbial strains, their inoculation as single strains
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or consortia, time point for inoculation and utilization of horizontal gene transfer ability of
microbes.
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4. Multi-process phytoremediation system (MPPS)

Rhizoremediation comprises plants and microbes as its integral parts. This means it has potential
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disadvantages associated with both partners. In some cases, it is difficult to establish sufficient
plant biomass because most plants are sensitive to contaminants (petroleum hydrocarbon) or to the
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conditions (at or in the contaminated site) for effective remediation measures (Siciliano and
Germida, 1998). In other cases, a decrease in microbial biomass in contaminated soils resulted in
deficient microbial communities both for supporting plant growth and for hydrocarbon
degradation. A variety of additional mechanical and biological methods are available for
remediation of petroleum-polluted soils. Frequent disadvantages associated with these techniques
are sub-optimal process rates and long treatment times. To overcome these challenges, a multi-
18
process phytoremediation system (MPPS) was proposed by Huang et al. (2004, 2005). This
approach is based on combining three prominent processes, i.e. land farming (aeration and
photooxidation), bioremediation (inoculation of PGPR) and phytoremediation (use of plants only).
This synergistic application of multiple techniques stimulates remediation processes by exploiting
the benefits of each component.

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When applying MPPS, two steps may be considered for complete removal of petroleum
hydrocarbons. The first one – soil pretreatment by proper agronomic practices such as tillage –

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ensures proper mixing of soil layers to homogenize “hot-spots” and allows aeration for
photochemical degradation of low molecular petroleum hydrocarbons. Appropriate levels of

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organic and inorganic fertilizer may be added to improve soil tilth, seed germination and plant
growth. The second key component of MPPS is to inoculate seeds/plants with PGPR to both

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improve plant tolerance and promote hydrocarbon degraders. With MPPS, a mixture of plants (e.g.
grasses with legumes) can yield more root biomass and support different microbial communities
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in the rhizosphere (Wenzel, 2009). MPPS can be applied to a wide range of contaminated soils. To
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date, MPPS for TPH-contaminated soils has been developed with the following advantages.
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 Rapid and sustainable means of remediation due to its complementary processes

 PGPR favors plant growth and microbial community developments by reducing
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contaminant and environmental stresses

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 Effective mineralization of TPH because of abundant roots and microbial biomass

 PGPR copes with ethylene production in plants in harsh environmental conditions
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(contaminant and salt stress, cold, drought, poor soil quality)

 High remediation rates within acceptable time frames
 MPPS can be designed for different TPH-contaminated soils because each soil has a unique
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set of features.

Until now, MPPS has been used for the effective removal of PAHs, TPH, salt and metals from
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different contaminated soils in greenhouse and field conditions (Gerhardt et al., 2015; Gurska, et
al., 2009). Huang et al. (2004) reported on a synergistic use of different remedial methods to
dissipate PAHs. Their results showed that the effectiveness of a single method was limited, and
that the combination of three remediation methods (termed MPPS) considerably improved
phytoremediation performance.
19
Another study by the same research group (Huang et al., 2005) described the effectiveness of MPPS
over land farming and phytoremediation alone. In a four-month trial period with TPH-
contaminated soil from the Imperial oil land farm site, MPPS removed twice as much TPH as land
farming alone, 50% more than bioremediation alone and 45% more than phytoremediation alone.
After another four-month trial period, MPPS removed 90% of all TPH in the contaminated soil,
whereas phytoremediation alone could remove only 50%. MPPS showed promising results in field

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studies as well. In a three-year field trial, a PGPR-enhanced phytoremediation technology helped
remediate a site heavily contaminated with recalcitrant petroleum hydrocarbons (Gurska et al.,

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2009). The authors reported a TPH remediation from 130 g/kg soil to 50 g/kg in a three-year period.
In addition to this, MPPS also remediated the recalcitrant fraction (F3 and F4) from these

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contaminated soils. Plant shoot and root biomass was significantly increased by applying PGPR.
Recently, another study, Macci et al. (2013) reported on a combination of natural attenuation and

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phytoremediation (combination of different plant species) as a real-scale phytoremediation

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technology. Their results showed significant reduction in heavy metals and hydrocarbon contents
by 20-40% and 40%, respectively. In that study, plants were selected for their rapid growth
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capability and suitability to local climatic conditions.
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Despite the proven effectiveness of rhizoremediation (in broader prospectus phytoremediation) in

TPH-impacted soils, its exploitation as practically feasible and full-scale technology is still lacking.
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Recently, Gerhardt and team (Gerhardt et al., 2017) present research and knowledge gap between
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academic, development sector and industrial organization which may be overcome for better
utilization of this green and sustainable technology (Stephenson et al., 2014; Conesa et al., 2012;
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Onwubuya et al., 2009; Rodemeyer, 2005). They also suggested different ways to promote
phytoremediation acceptance by government and industrial sectors for utilizing the broader concept
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of phyto-management.

5. Conclusions
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There is an urgent need to develop low-cost, efficient, sustainable and eco-friendly techniques for
the remediation of petroleum hydrocarbon-contaminated soils. Biological remediation techniques
do have benefits over physical and chemical methods due to the ecological and technical drawbacks
of conventional approaches. These techniques have gained special interest because they offer
environmentally safe and sustainable ways for TPH remediation as compared to conventional
methods. Utilization of microbes and plants for remediation (bioremediation/phytoremediation)
20
are typically associated with slow rates of remediation due to the low nutrient availability and
pollutant toxicity in TPH-impacted soils. Rhizoremediation, the synergistic form of bioremediation
and phytoremediation is a scientifically proven, preferred remediation technology for TPH-
impacted soils. It improves the overall performance and rate of remediation by synergistic
interactions of plant roots and microbes. The overall performance and efficiency of
rhizoremediation can be enhanced by selecting suitable pairings of plants and microbes, by adding

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organic and inorganic soil amendments, and by subsequently applying agronomic practices. All of
the mentioned strategies can be combined in a multi-process phytoremediation system. This system

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offers many scientific and ecological benefits and has been proven to be an effective remediation
measure in many field studies. Rhizoremediation of TPH-impacted soils has demonstrated

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encouraging results in different experiments and field trials. Nonetheless, there is need to search
out suitable plant and microbe pair with efficient soils amendments such as inorganic fertilizer,

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nutrients, surfactants, biochar, compost and organic amendments as well as cost effective and

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efficient application rates. Subsequently results from laboratory or greenhouses should be
replicated at field levels to broaden the scope of rhizoremediation.
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Acknowledgments
This work was partly funded by Austrian Institute of Technology Vienna, Austria, Department of
Health and Environment under the umbrella of business unit Environmental Resources and
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Technologies. Imran Hussain is gratefully acknowledging the Higher Education Commission

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(HEC) of Pakistan for financial support during my whole stay of four year for my doctoral degree
in Vienna.
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Table 1: Biological remediation technologies available for treatment or remediation of variety of organic contaminants in soils and groundwater

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Techniques Description Target pollutants Medium References

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Natural attenuation (NA) Transformation, immobilization or reduction of pollutants to les toxic BTEX, Chlorinated Soils and Mulligan and Yong,
compounds by means of natural processes without human intervention. solvents 2004; Smets and

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(Considered partially Mechanisms involved in the process of NA are physical (dilution, dispersion, groundwater Pritchard, 2003
biological method) and adsorption), chemical (volatilization) and biological (microbial

A
breakdown).

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Bioremediation The degradation of organic pollutants into carbon dioxide, and water or into Chlorinated solvents, Soils, surface Chaudhry et al., 2005;
a transformed product by the action of biological agents under natural petroleum hydrocarbons, water and Chen et al., 2012,
conditions. Bioremediation is included in the list of the top ten technologies pesticides and underground 2015
for improving human and environmental health and is considered as low-cost, chlorinated biphenyls water
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efficient, environmental friendly and sustainable solution.

Bioaugmentation The addition of enriched competent microbial cultures or the addition of Petroleum hydrocarbons Soils and Azad et al., 2014; Cai
genetically engineered bacteria with specific catabolic capabilities for and Pesticides waste water et al., 2016; Mancera-
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stimulating the performance of microorganisms in contaminated medium. López et al., 2008;

This approach is totally based on the concept of soil activation. Soil activation Tyagi et al., 2011
means the cultivation of the biomass (microorganisms) from a fraction of
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contaminated soils and subsequently its use as an inoculum for

bioaugmentation.
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Biostimulation Stimulation of microbial degradation f pollutants by adjusting Petroleum hydrocarbons  Leigh et al., 2015
environmental parameter or addition of rate limiting nutrients or and Pesticides
biosurfactants or biopolymers.
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Bioventing Bioventing can be described as adding oxygen or enhanced ventilation to Chlorinated compounds Soils and Chein et al., 2010;
solid voids in contaminated soils by pumping in order to enhance the growth and VOCs groundwater Khan and Zytner,
of aerobic microorganisms. This increased air flow and enhanced oxygen 2013)
supply are supposed to improve microbial metabolism of concerned
pollutants.
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Biopiles This treatment involves the piling of contaminated soils into piles or heaps Volatile organic soils Baker and Moore

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and then simulating aerobic microbial activity by aeration and subsequently contaminants (VOCs), (2000)
addition of minerals, nutrients, and moisture may improve its performance. non-halogenated VOCs,
halogenated VOCs and
pesticides

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N
A
M
ED
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A
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Table 2: Phytoremediation techniques/processes used to treat various pollutants with specific plants, including recent case studies (1999-2016)

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Techniques Description Target pollutants Plants used References

Phytoextraction Extraction of pollutants in plant- Trace elements, Hyperaccumulator plants such as Ali et al., 2013; Robinson et al.,

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harvestable parts (e.g. shoots) radionuclides, precious Brassica 2006)
elements including gold,

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nickel and thallium

Rhizofilteration Plants used for treatment of polluted Polluted streams and Aquatic macrophytes and ferns (Ball, 2004; Favas et al., 2016;

A
water wastewaters Hinchman et al., 1996)
contaminated with

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metals, radionuclides

Phytostabilization Reducing the bioavailability of Metals and Shrubs, herbs, grasses, legumes (Frérot et al., 2006; Huang et al.,
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pollutants using plant roots radionuclides and trees 2016)

Phytovolatilization Conversion of pollutants to volatile Volatile organic Brassica plants and willows (Limmer and Burken, 2016)
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forms and further release into compounds (VOCs) and

atmosphere radionuclides

Phytodesalination Removal of salt from saline and saline- Salts Halophytic plants such as Suaeda (Fatehi Pouladi et al., 2016; Rabhi
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sodic soils fruticose and Hordeum vulgare et al., 2010)

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Phytodegradation Degradation of organic pollutants by TNT, MTBE, TCE and Grasses, trees (Al-Baldawi et al., 2015; Newman
enzymatic activities TPHs and Reynolds, 2004)

Rhizoremediation Degradation of organic xenobiotics in TPHs, PAHS, PCBs, Grasses, legumes, ornamental (Abhilash et al., 2013; Gartler et al.,
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rhizosphere by bacterial and fungal DDTs, pesticides and plants, cash crops and trees 2014; Maqbool et al., 2012; Pilon-
activities many others Smits, 2005)
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Table 3: Selected rhizoremediation trials (2009-2016) for TPH-contaminated soils with focus on organic and inorganic soil amendments for its improvement

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Plant used Amendment used Initial pollutant level Dissipation efficiency References

Alfalfa Organic fertilizer PAHs 12.4 mg/kg soil, 11.6% removal compared to non-vegetated (Fu et al., 2012)

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benzo[a] pyrene 1176 control
(Medicago sativa) µg/Kg

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Wheat Fungal strains PAHs 78.2 mg/kg soil 13% dissipation compared to non-vegetated (Ingrid et al., 2016)
(Rhizophagus control

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(Triticum estivum) irregularis)

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Medik AMF such as 7500 mg/kg soil 17.7% TPH degradation compared to control (Hernández-Ortega et al., 2012)
Glomus Zac-19
(Melilotus albus)
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Alfalfa Sewage sludge and 20-35% more degradation compared to control (Zhang et al., 2012)
cattle manure
(Medicago stiva)
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Italian ryegrass Endophytes strains 1-2% diesel w/w soil Strain ITRH43 showed better colonization at 2% (Andria et al., 2009)
such as diesel concentration
(Lolium multiflorum) Pseudomonas
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ITRI53 and
Rhodococcus
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ITRH43

Maize Inorganic fertilizer 30% oily sludge 59% higher degradation in combined treatment (Shahzad et al., 2016)
and bacterial
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(Zea mays) consortia

Italian ryegrass Inorganic nutrients 15 g/kg soil 85% degradation of TPH in inoculated and (Arslan et al., 2014)
plus endophytes higher levels of nutrients
(Lolium multiflorum)

Italian ryegrass Controlled release 3000, 6000 and 15,000 85% degradation at 3000 mg/kg, 66 % at 6000 (Cartmill et al., 2014b)
(Lolium multiflorum) fertilizer (CRF) mg/kg soil mg/kg and 73% in 15,000 mg/kg level
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Cymbopogon ------------------------- 10000 mg/kg soil 50-88% reduction of TPH levels in planted (Gaskin and Bentham, 2010b)

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ambiguus, Brachiaria --- treatments
decumbens,
Microlaena stipoide

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Salix purpurea Endophytic strain Pyrene 0.1 ppm in water 70% reduction in pyrene (Khan et al., 2014)
Pseudomonas putida

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PD1

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Vetiveria zizanioides Wheat husk, 252 g/kg soil 72% TPH reduction in treated versus 33.4% in (Nanekar et al., 2015)
microbial consortia control

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and nutrients

Rottb Hask Nutrients 80,000 mg/kg soil 86.5% reduction after 360 days (Basumatary et al., 2012)
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(Cyperus brevifolius)

Ornamental plants ------------ 10,000 mg/Kg soil 50% reduction in TPH load (Liu et al., 2012)
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Echinacea purpurea

Tall fescue PGPR inoculum TPH 4407 mg/kg soil more than 50% reduction of aliphatic (Hou et al., 2015)
hydrocarbons
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(Festuca
arundinacea)
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Annual ryegrass PGPR inoculum 130 g /kg soil 50% reduction of TPH in treated versus 20% in (Gurska et al., 2009)
control
Tall fescue and
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Fall rye

Fescue species Endophytic fungi 4700 mg/kg soil PAHs removal 80-84%, TPH 64-72% (Soleimani et al., 2010b)

Brassia scoparaia 2-3% oil 31% reduction of TPH in vegetated soils (Moubasher et al., 2015)
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Table 4: Comprehensive guide regarding plant selection in different phytoremediation/rhizoremediation trials of TPH-contaminated soils with unique plant

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characteristics and their types

Plant type Common name Technical name Special features Case studies

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Native/Wild Tall fescue Festuca arundinacea Grasses are/have: easy growing, extensive fibrous (Adam and Duncan, 2002, 1999, Afzal et
grasses root system, robust growth, generally more al., 2012, 2012; Al-Baldawi et al., 2015;
Fescue Vulpia myuros

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resistant to environment stresses, hydrocarbon Andria et al., 2009; Arslan et al., 2014;
tolerance and degrading abilities suitable for Ball, 2004; Basumatary et al., 2012; Binet
Canary grass Phalasris arundinacea

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growing in diverse climatic conditions. et al., 2000; Cai et al., 2016; Fu et al.,
2012; Gaskin et al., 2008; Gaskin and
Italian ryegrass Lolium multiflorum

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Bentham, 2010b; Gurska et al., 2009; Hou
Tropical pasture grass Brachiaria brazantha et al., 2015; Huang et al., 2005, 2004b;
Kaimi et al., 2006; Liste and Prutz, 2006;
Nut grass Cyperus rotundus P.-W. G. Liu et al., 2014; Merkl et al.,
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2005a, 2004; Moubasher et al., 2015;
Mullumbimby couch Cyperus brevifolius Muratova et al., 2012; Nanekar et al.,
2015; Peng et al., 2009; Rezek et al., 2008;
Winter rye Secale cereale
Soleimani et al., 2010b; Tang et al., 2010b;
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Common reed Phragmites australis Yousaf et al., 2010)

Yard grass Eleusine indica

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Seep wood Suaeda glauca

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Sea lavender Limonium bicolor

Saltbush Atriplex centralasiatica

A

Fescue Festuca pratenis

Meadow grass Poa pratensis

Tussock grass Poa foliosa

Ryegrass Lolium perenne

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Highland bent Agrostis castellana

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Sheep fescue Festuca ovina

Bulrush Scirpus grossus

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Sea rush Juncus maritimus

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Elephant grass Pennisetum purpureum

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Bermuda grass Cynodon dactylon

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Crab grass Digitaria sanguinalis

Lemon scanted grass Cymbopogon ambigus

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Signal grass Brachiaria decumbens

Weeping grass Microlaena stipoide

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Khas kheas Vetiveria zizanioides

Sedge Carex stricta

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Legumes White clover Trifolium rapens Legumes are: the third largest angiosperm family (Fan et al., 2008; Gartler et al., 2014; Hall
of plants; they can grow in a wide range of et al., 2011; Ho et al., 2007; Joner and
Pea Pisum sativum habitats and low-nutrient soils; can provide Leyval, 2003; Liste and Felgentreu, 2006b;
nitrogen to depleted soils because they can fix Palmroth et al., 2002; Pichtel and
A

Common vetch Vicia sativa

atmospheric nitrogen. Liskanen, 2001; Smith et al., 2006; White
et al., 2003a; Yan et al., 2015)
White mustard Sinapsis alba Symbiotic nitrogen fixation (SNF) can make
legumes a candidate for ideal plants to colonize
Clover trifolium pratense
and restore the quality and health of N-limited
Balck medik Medicago lupulina environments.
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Red clover Trifolium pratense

SC
Alfalfa Medicago sativa

Oil seed rape Brassica napus

U
Smooth brome Bromus inermis

N
Milk weed Astragalus adsurgens

A
Kadzu Pueraria labota

M
Centrosema Centrosema brasilianum

Wild groundnut Calopogonium mucunoides

ED
Fodder burnet Sanguisorba minor

Birdsfoot trefoil Lotus carniculatus

PT

Agronomic Sorghum Sorghum bicolor (Adam and Duncan, 1999; Copaciu et al.,
crops 2016; Gurska et al., 2009; Hong et al.,
Maize Zea mays 2011; Jung et al., 2010; Kaimi et al., 2006;
Muratova et al., 2009, 2012; Shahsavari et
E

Flax Limum usitatissimum

al., 2015; Shahzad et al., 2016; Shirdam et
CC

al., 2008; Tang et al., 2010b)

Wheat Triticum estivum

Barlay Hordeum vulgare

A

Rice Oryza sativa

Spinach Spinacia oleracea

Mung bean Vigna radiata

Suddan grass Sorghum sudanense

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Cotton Gossypium hirsutum

SC
Ornamental Four O'clock flower Mirabilis jalapa (Grace Liu et al., 2011b; Inckot et al.,
plants 2011; Liu et al., 2008, 2012; R. Liu et al.,
Galega Galega orientalis 2014; Peng et al., 2009; Smith et al., 2006;

U
Suleiman and Bhat, 2003; Yan et al., 2015;
Fire Phoenix
Zhang et al., 2010)

N
Cornflower Centaurea cyanus

A
Snapdragon Antirrhinum majus

M
Barberton daisy Gerbera jamesonii

Blanket flower Gaillardia aristata

ED
Purple coneflower Echinacea purpurea

Devil's beggarticks Bidens frondosa

PT

Sensitive plant Mimosa pilulifera

Shrubs and Jatropha Jatropha curcas (Cook and Hesterberg, 2013; Dadrasnia
trees and Agamuthu, 2013; Euliss et al., 2008;
E

Mulberry Morus rubra Fatima et al., 2015; Jagtap et al., 2014;

Khan et al., 2014; Kumar et al., 2013;
CC

Pine Pinus sylvestris

Moore et al., 2006; Palmroth et al., 2002;
Tischer and Hübner, 2002; Walker et al.,
Poplar Populus deltoides
2003; Zalesny et al., 2005)
A

Pine Pinus dessiflora

Chinese thuja Thuja orientalis

Willow salix alba, exigua

Black willow Salix nigra

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Black alder Alnus glutinosa

SC
Black locus Robinia preudoacacia

Song of India Dracaena reflexa

U
Yellowwood conifer Podocarpus polystachyus

N
A
M
ED
E PT
CC
A

Table 5: Summary of organic, inorganic and chemical amendments used to stimulate rhizoremediation efficiency in TPH-contaminated soils
Type of amendment Specific name of
supplements
Inorganic and organic Solution of KH2PO4 and
fertilizer microbial consortia
Oleophilic fertilizer
Slow release fertilizer like
Chitosan and Osmocote
M
NH4Cl and
NaH2PO4 mixture
ED
Microbial consortia and
nutrients
PT
Organic fertilizer
E
Sewage sludge and cattle
manure
CC
Inorganic fertilizer
Tea leaves, potatao skin and
soya cake
A
Cow dung , poultry manure
and pig waste
PT
RI
SC
Contaminant type and Main findings References
level
Crude oil 5.4% 7.4% biodegradation efficiency for weathered Trindade et al., 2005
U
contaminated soils after 41 days trail
TPHs 2500, 5000, Fertilization enhanced TPH removal and lipase Margesin et al.,
N
10000,20000 mg/Kg soil activity after 38 days 2007
Arabian light crude oil Osmocote enhances metabolic activity and Xu et al., 2005
A
3.5 w/w% microbial biomass. It also stimulates
biodegradation of hydrocarbons (99.7 %).Chitosan
was failed to enhance all parameters during while
remediation trail (56 days)
TPH 55.2 ppm The addition of nutrients resulted in 20% increase Abed et al., 2015
in oil remediation rates. Additional bacterial
consortia lead to a further increase of only 1%.
TPH 15 g/Kg soil Bacterial inoculation improved plant growth and Arslan et al., 2104
hydrocarbons degradation that was further
stimulated by nutrient supplement.
PAHs 12.4 mg/Kg soil, 11.6% removal as compared to non-vegetated Fu et al., 2011
benzo[a] pyrene 1176 control
µg/Kg
20-35% more degradation as compared to control Zhang et al., 2012
30% oily sludge 59% more dissipation of TPHs in soils as Shahzad et al., 2016
compared to un-treated maize.
5% and 15% diesel oil A total of 81% and 42% oil loss was recorded in Dadrasnia and
soil amended with soy-cake at 5% and 15% oil Agamuthu, 2014
pollution, respectively.
0.5%, 2.5% and 5% Addition of organic wastes led to reduction in the Adesodun and
spent oil and metals levels of the metals and total hydrocarbon. Mbagwu, 2008
Effectiveness indicates that poultry manure
followed by pig wastes were better nutrients
supplement for immobilization of these metals
within the soil and in stimulating microbial
degradation of this waste oil.
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Tea leaves, potatao skin and 2.5% and 1 % diesel fuel Diesel fuel disappeared more rapidly in the soil Dadrasnia and

SC
soya cake amendment with Soya cake than all others. TPH Agamuthu, 2013
loss was 84% and 91% for P. polystachyus
amended with organic wastes in 2.5% and 1% oil,
respectively.

U
Bulking agents Manure and sand or hay Diesel at 2 v/v % soil Use of sand as a bulking agent gives highest TPH Coles et al., 2009
removal rate (96%) by using clean Ottawa sand

N
sand, compost, kitchen TPH 130000 mg/Kg The reduction of TPH after 373 days of treatment Kriipsalu et al., 2007
waste and shredded waste DM was 62, 51, 74 and 49% respectively as

A
wood amendments
Chopped bermudagrass hay, TPH 10w/w % DM Tillage and adding bulking agents enhanced Rhykerd et al., 1999
sawdust, vermiculite and remediation of oil-contaminated soil. TPH removal

M
forced aeration content was 90% in the tilled-hay and t 77% for
tilled-vermiculite treatments
Hardwood sawdust, TPH 7.9 and 8.8 % Broiler litter resulted in increased soil nitrogen and White et al., 2008
ED
peppermill sludge, broiler phosphorus but reduces germination. Highest
litter and inorganic nutrients degradation was observed in inorganic fertilizer

Coffee beans TPH 58000 mg/Kg Soil The highest TPH removal (63%) was obtained Roldan-Martin et al.,
with a soil/ cofeebean ratio of 98:2 over 15 days, 2007
PT

corresponding with the highest rates of microbial

respiration and the greatest increases in bacterial
and fungal counts.
E

Surfactant and OSE11 and Brij70 Diesel fuel 10336 mg/ Commercial product caused the higher reduction Diaz et al., 2012
commercial products Kg of TPH (49.4%) as compared to control. Brij700
CC

did not improve biodegradation in any of the

studied conditions
Biosurfactant (PES-51) 5100 mg/Kg Average reduction of 70% in the semi volatile Tumeo et al., 1994
petroleum hydrocarbon were observed. Diesel
range petroleum hydrocarbons were completely
A

removed to levels below the detection limit.

Natural gums and synthetic TPH 321000 mg/Kg Ionic surfactants showed removal rates a 78.51 % Hernández-Espriú et
surfactants where control was 30%. Guar gum and al., 2012
locust bean gum showed efficiencies of 54.38 %
and53.4% respectively.

Biosurfactant produced by
Ochrobactrum intermedium
Biochar Wheat straw biochar
produced at 450 °C under
anaerobic conditions for 1 h
using a slow pyrolysis
process
M
Wheat straw biochar
produced at 300 °C for 6 h
ED
anaerobic conditions using
a slow pyrolysis process
A wheat straw biochar was
produced under oxygen
limited conditions at 500 °C
PT
for 4 hr.
E
Bulrush straw biochar was
used in the current study.
CC
The mentioned biochar was
produced from the pyrolysis
of dried bulrush straw under
low-oxygen and controlled
A
pressure (for 3 h at 300°C)
Biochar was produced from
rice straw at 500°C using a
slow pyrolysis under limited
oxygen conditions
PT
RI
Diesel oil 20 w/w % The isolate degraded up to 40% of the Bezza and Chirwa et
SC
hydrophobic long chain aliphatic and polycyclic al., 2015
aromatic hydrocarbons in liquid cultures with 4%
(v/v) crude oil sludge while the biosurfactant
amended microcosm showed up to 70%
U
degradation of the most hydrophobic components
of the petroleum sludge in 3 weeks
TPH 7719 ± 113 mg/kg The removal of TPHs in impacted-soils was higher Han et al., 2016
N
in vegetated treatments with ryegrass while
biochar alone and in combination showed less
A
degradation efficiency. The removal rate of
planted treatment was 53.13%, while biochar
showed only 34.14% as compared to non-
vegetative control that was 36.23%.
Phe and BaP were 100 The wheat straw biochar and associated biochar Cao et al., 2016
and 50 mg/kg amendments showed inhibitory effects on
Phenanthrene removal while they give positive
results for the removal of Benzo(a)pyrene
Hexachlorobenzene HCB removal was stimulated by the joint action of Song et al., 2016
(HCB) ryegrass in biochar amended soils especially in the
region of rhizosphere (2mm). The current
enhanced removal of pollutants was supposed to
occur by increased bioavailable fraction of
pollutant as well as higher microbial activities.
TPH = 9.62 g/kg After 56 days, the total degradation efficiency of Wang et al., 2017
TPH was greater in biochar (46.92%) and biochar
plus nutrients amended soils (51.35%) as
compared to control soils (28.2%). These figures
regarding degradation of TPH from this study
confirms the unique advantages of adding biochar
alone and in combination for remediation of TPH-
impacted soils.
TPH =16300 mg/kg After the entire course of experiment (180 days), Qin et al., 2013
it’s concluded that soil amended with rice straw
biochar showed better improvement in pollutant
removal as compared to non-amended control.
Notably, when biochar was added at 80th day the
TPH concentration decreased to below the
threshold limit of hydrocarbons in Chinese soils
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Biochar of two different PAHs Amendment with sewage sludge biochar at 2%, Zielińska and

SC
types originated from parent 5% and 10% levels reduced PAHs by 31%, 39% Oleszczuk, 2016
material of sewage sludge and 43%, respectively
was used in the current
study. The pyrolysis

U
conditions for preparation of
these two biochar were 500
°C and 700 °C respectively

N
under limited oxygen
conditions

A
Compost The white button mushroom PAHs= 610 mg The degradation of individual PAHs was in range Sasek et al. 2003
(Agaricus bisporus) compost PAHs/kg DM of soil of 20–60% at the end of 54 days of composting
used in the current study followed by further increase of PAH removal (37–

M
was ordered from a 80%) after another 100 days of maturation
commercial compost
producing company. It
ED
consists of wheat straw,
chicken manure and gypsum
The mixed bio-waste Diesel fuel 6 g/Kg fresh Up to 85% reduction in diesel content was Van Geste et al.,
consists of vegetable, fruit matter observed in composting treatments while only 2003
and garden waste was used 35% reduction was noted in soils at room
PT

for composting and then temperature. Specifically, 99% reduction in TPH

subsequently used in this contents was reached in lab scale compost bin.
current study
Sewage sludge and compost TPH =10,000 mg/kg After 30 days of incubation, the compost only Namkoong et al.,
E

were used in multiple ratios treatment showed 94.6% TPH removal as 2002
compared to un-amended soil have showed only
CC

64%. Notably, 1:0.5 and 1:1 of contaminated soils

to compost showed 97-98% reduction in
hydrocarbons contents
Urban immature compost TPH=521 g/kg in acidic The results regarding TPH removal in all the Asgari et al., 2017
A

was added as an amendment sludge treatments showed a prominent reduction (48%).

to acidic sludge in multiple Mixing ratio of (acidic sludge to compost) 1:5
ratios showed highest removal while 1:100 gives lowest
rate of reduction.

Compost, biochar and
nutrients solution mixed
with chemical amendments
like Detained Igepal CA-720
Hydrocarbon Oat plants (Avena sativa)
degrading was cultivated for 60 days
microorganisms and that were inoculated with
Plant Growth PGPR (Acinetobacter sp.)
Promoting plus AMF (Glomus
M
Rhizobacteria (PGPR) intraradices)
Tall fescue (Testuca
ED
arundinacea) inoculated
with two PGPR strains
(Klebsiella sp. D5A and
Pseudomonas sp. SB)
PT
Tall fescue inoculated with
E
halotolerant and alkaline
tolerant plant growth-
CC
promoting rhizobacteria
A
Alkane-degrading and plant
growth-promoting bacteria
were used with carpet grass
(Axonopus affinis)
PT
RI
Naphthalene =50 mg/kg The results showed (after 61 days), that the Chirakkara and
SC
Phenanthrene =100 biochar and compost addition showed positive Reddy, 2015
mg/kg and mixed heavy correlation with plant growth parameters.
metals Especially, the best growth (115% increase) was
observed for sunflower plants having compost
U
amended soils. Hydrocarbons removal were
enhanced in the presence of all studies
amendments especially compost.
N
TPH =5 and 10 g/kg The results demonstrated that TPH-impacted soils Xun et al., 2015
inhibit the growth of mentioned plants, however
A
the use of PGPR and AMF strains reduce
phytotoxicity as well as increase remediation
potential of plants. After 61 days of experiment,
the TPH removal rate was 49.73 % as compared to
non-vegetated control soils.
AHs = 4000 mg/kg and Plant growth as well as hydrocarbons (both Hou et al., 2015
PAHs =500 mg/kg aliphatic and polycyclic) degradation rate was
maximum when plant were inoculated with PGPR
strains. The results showed more pronounced
effects on higher molecular fractions (C21–C34)
of AHs and PAHs. The results of the current study
also showed specific effects of some bacterial
strains as compared to their diversity on
phytoremediation.
TPH= 16920 mg/kg In the current study, a total of 115 PGPR strains Lie et al., 2014
were isolated and identified. Among them one
unique isolate named as D5A showed prominent
characteristics related to plant growth promotion
under extreme conditions. Later in a pot trial,
inoculation of tall fescue with this strain showed
increased removal of TPH by 16% as compared to
phytoremediation alone.
Diesel oil 10 g/Kg of The results of the current study demonstrated that Tara et al., 2014
Soil inoculation of selective grasses with hydrocarbon-
degrading as well as PGPR strains showed more
positive effects in phytoremediation experiments.
This approach having both characteristics of
degradation plus growth promotion is more
effective for remediation of TPH-impacted soils.
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Table 6: Biochar type (s) with their specific parent material (s) used to remediate variety of petroleum hydrocarbons

SC
Biochar (BC)type Parent material (s) Hydrocarbons Case studies
remediated

U
Agricultural Dried rice straw, wood and Phenanthrene Feng and Zhu, 2018
byproducts bamboo powder
Wheat straws that were air- TPHs Han et al., 2016

N
dried, milled and grounded
Wheat straws Phenanthrene Cao et al., 2016

A
Benzo[a]pyrene

Forestry Pine wood Naphthalene, Chirakkara and Reddy,

M
byproducts Phenanthrene and 2015
Mixed heavy
metals
ED
Ponderosa Pine wood and PAHs Keiluweit et al., 2012
lignin-rich
Animal waste Poultry manure and litter TPHs and heavy Barati et al., 2017
metals Venegas et al., 2015
Meat and bone-meal TPHs Karppinen et al., 2017
PT

Pig manure TPHs Ren et al., 2016

Sewage sludge Sewage sludge PAHs Zielińska and Oleszczuk,
E

2016
Sewage sludge PAHs Oleszczuk et al., 2012
CC

Wet sewage sludge PAHs Waqas et al., 2014

A