Accepted Manuscript Title: Combined Application of Biochar, Compost, and Bacterial Consortia with Italian ryegrass Enhanced Phytoremediation of Petroleum Hydrocarbon Contaminated Soil Authors: Fida Hussain, Imran Hussain, Aqib Hassan Ali Khan, Yousaf Shad Muhammad, Mazhar Iqbal, Gerhard Soja, Thomas Gerhard Reichenauer, Zeshan, Sohail Yousaf PII: DOI: Reference:
S0098-8472(18)30722-6 https://doi.org/10.1016/j.envexpbot.2018.05.012 EEB 3449
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
31-12-2017 11-5-2018 12-5-2018
Please cite this article as: Hussain, Fida, Hussain, Imran, Khan, Aqib Hassan Ali, Muhammad, Yousaf Shad, Iqbal, Mazhar, Soja, Gerhard, Reichenauer, Thomas Gerhard, Zeshan, , Yousaf, Sohail, Combined Application of Biochar, Compost, and Bacterial Consortia with Italian ryegrass Enhanced Phytoremediation of Petroleum Hydrocarbon Contaminated Soil.Environmental and Experimental Botany https://doi.org/10.1016/j.envexpbot.2018.05.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Combined Application of Biochar, Compost, and Bacterial Consortia with Italian ryegrass Enhanced Phytoremediation of Petroleum Hydrocarbon Contaminated Soil
Fida Hussaina,e,1, Imran Hussainb,c,1 Aqib Hassan Ali Khana, Yousaf Shad Muhammadd, Mazhar Iqbala, Gerhard Sojab, Thomas Gerhard Reichenauerb, Zeshanf, Sohail
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Yousafa*
a
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Department of Environmental Sciences, Faculty of Biological Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan b
AIT Austrian Institute of Technology GmbH, Environmental Resources and Technologies, Konrad-
Department of Ecogenomics and Systems Biology, University of Vienna, Althanstrasse 14, 1090
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c
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Lorenz-Straße 24, 3430 Tulln, Austria
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Vienna, Austria d
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Department of Statistics, Faculty of Natural Sciences, Quaid-i-Azam University, 45320, Islamabad.
Pakistan
Department of Biological Sciences, School of Environmental Sciences, Kangwon National
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e
University, Gangwon, South Korea. f
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Institute of Environmental Sciences and Engineering (IESE), School of Civil and Environmental
Engineering (SCEE), National University of Sciences and Technology (NUST), Islamabad 44000,
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Pakistan.
Both authors contributed equally to this paper
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*Correspondence Sohail Yousaf (PhD), Department of Environmental Sciences, Faculty of Biological Sciences Quaid-i-Azam University, 45320 – Islamabad, Pakistan E-mail:
[email protected],
Phone: +923455142400 Running Title: Combined Application of Biochar, Compost, and Bacterial Consortia Enhance Phytoremediation
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Highlights Biochar and compost amendment enhanced rhizosphere effect
TPHs Rhizoremediation is improved by bacterial consortia and organic
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amendments
The organic amendments improved plant growth and bacterial count in
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Compost is a rich source of bacteria in the rhizosphere
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rhizosphere
Abstract
Petroleum hydrocarbons are extensively utilized in petrochemical industries and cause soil
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deterioration during exploration, transportation, refining and making petroleum products. We hypothesized that the combined use of compost, biochar and bacterial consortia as soil
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amendments may enhance the rhizoremediation potential of ryegrass by strengthening the plant rhizospheric effect for efficient total petroleum hydrocarbon removal. The present study
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focused on phytoremediation of hydrocarbons in spiked contaminated soil amended with biochar (5% v/v), and compost (5% v/v). Spiked soil was inoculated with consortia of four hydrocarbon degrading bacterial strains (Pseudomonas poae, Actinobacter bouvetii, Stenotrophomonas rhizophila and Pseudomonas rhizosphaerae). The spiked soil was
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prepared by spiking agricultural soil with 3.4% (w/w) of crude oil. Italian ryegrass (60 seeds pot-1) were sown and plants were harvested after 75 days. The highest hydrocarbon removal (85%) was observed in spiked soil amended with compost, biochar and consortia. Bacterial inoculation with biochar and compost showed significantly higher hydrocarbon degradation as compared to all other treatments. Highest TPHs degrading bacteria (5.74×107 cells g-1 of soil) were observed in rhizosphere of spiked soil amended with
compost, biochar and consortia. The organic amendments improved plant growth and bacterial count in rhizosphere which resulted in higher removal of hydrocarbons. We concluded that plant-microbe interactions together with the organic soil amendments offer an emerging trend for remediation of hydrocarbons. Rhizoremediation is a green solution to overcome the quandary of total petroleum hydrocarbon contamination in soil.
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Key Words: Biochar, Compost, Consortia, Italian ryegrass, Rhizoremediation
1. Introduction Pollution associated with petroleum and its refined products is of great concern due to its damages for human and ecosystem health, soil structure and ground water quality (Alexander, 2000; Jain et al., 2011; Meagher, 2000). Total petroleum hydrocarbons (TPHs) describe the sum of hydrocarbons that are contained in crude oil or any product produced from it like gasoline, creosote or diesel. TPHs belong to the most common groups of persistent organic pollutants. Toxic and deleterious effects of TPHs on the ecosystem are
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linked with their persistence in the environmental matrics like soil, water and atmosphere (Huang et al., 2005). Leakage of underground storage tanks, accidental oil spills, extraction
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losses and transportation losses are frequent causes of soil and sediment pollution with TPHs (Khan et al., 2016a; Margesin et al., 2007). Extensive soil pollution with petroleum hydrocarbons results in extreme harsh surroundings, produces hydrophobic conditions and infertile soils that ultimately lead towards less plant and microbial growth (Hutchinson et al.,
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2001; Jain et al., 2011). The drastic and deleterious effects of oil spills on soils are of great
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concern worldwide and have been pointed out by several researchers (Khan et al., 2016b;
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Obida et al., 2018). Petroleum products alter the soil structure by reducing its porosity and destruct its aggregates. They also affect physicochemical properties of soil like pH, total
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exchangeable cation capacity, alkaline cation saturation degree, oxygen content and humidity. This disrupts biological equilibrium and impairs plant cultivation (Bramley-Alves
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et al., 2014; Gurska et al., 2009; Shahsavari et al., 2015; Shahzad et al., 2016). Trends of developing innovative technologies for the removal of TPHs have been explored in
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the recent years. Among these technologies, phytoremediation is the most eco-friendly, economically sustainable, cost-effective technology and offers ecological and natural aesthetic benefits. The use of plants and associated rhizospheric microbial populations to
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degrade organic pollutants is becoming a promising method for remediation of petroleum polluted soils (Bisht et al., 2015; Germaine et al., 2015; Zhuang et al., 2007). In a broader view, phytoremediation (as part of bioremediation) is also included in the list of “Top 10
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technologies for improving human health in developing countries” (Daar et al., 2012). TPHs degradation by plant roots and its associated microbes has been reported as rhizoremediation and it’s the principal mechanism responsible for TPH removal by plants (Chaudhry et al., 2005; Pilon-Smits, 2005). Rhizoremediation efficiency and overall dissipation of TPH removal can be improved by strengthening the plant rhizospheric effect. Microbial biomass and activity is stimulated in the rhizosphere because roots excrete significant amounts of
sugars, amino acids, organic acids, hormones, vitamins, mucilage and other substances together with sloughed-off root cap (Jones et al., 2009; Kawasaki et al., 2016). Compost addition in TPH contaminated soils is the source of xenobiotic-degrading microbes which can degrade contaminants into less toxic substances as well as improves soil quality, soil aeration, and promote plant growth (Beesley et al., 2010). The addition of biochar to contaminated soils results in improvement of soil properties like soil fertility, nutrient retention, water holding capacity, and oxygen supply as well as remediate contamination by
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surface adsorption, precipitation, partitioning, and sequestration. Microbial growth and
biomass can be promoted by the addition of biochar as it also provides shelter for microbes in
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the soil (Tang et al., 2013). Denyes et al., (2013) reported that biochar addition to soils improved plant growth and physiological developments (Chlorophyll content, shoot and root biomass). Similarly, augmentation of microbial consortia (selected microbial strains) is also well documented to promote the vigor and growth of plants as well as hydrocarbon
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degradation, thereby improving the phytoremediation potential (Alisi et al., 2009; Chen et al.,
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2013; Tyagi et al., 2011).
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To the best of our knowledge, no report has been published yet regarding the combined application of biochar, compost and selected microbial strains to enhance rhizospheric effect
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for improved performance of TPHs phytoremediation system. We hypothesize that the combined use of above mentioned three soil amendments may enhance the rhizoremediation
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potential of ryegrass. This can be done by strengthening the plant rhizospheric effect for efficient dissipation of TPHs. The objectives of this research work were to enhance the
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tolerance and remediation potential of ryegrass by organic soil amendments i.e. biochar, compost and microbial consortia in TPHs spiked soil. Secondly, to examine the influence of
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organic soil amendments (alone and in combination) on the rhizospheric effect of ryegrass. 2. Materials and methods 2.1. Soil preparation and spiking with TPH
The clean soil (without petroleum contamination) was collected from agricultural fields of
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Quaid-i-Azam University, Islamabad, Pakistan. The soil samples were homogenized to prepare one bulk sample. For the greenhouse experiment, soil was air-dried (at room temperature for 15 days), sterilized and ground to a final particle size of 2 mm. Soil samples were analyzed for moisture content, soil pH and electric conductivity (EC), soil texture, nitrogen, phosphorous, potassium (NPK) content, organic matter and total organic carbon (TOC), using standard methods (Estefan et al., 2013), before and after the amendments of biochar and compost. The spiked soil was prepared by spiking agricultural soil (textural class
loam) with 3.4% (w/w) of crude oil; this percentage was selected after analyzing soil samples collected from an oil refinery where historically contaminated soil with TPH has been found (Hussain et al., unpublished data). The spiked soil was left undisturbed for 2 weeks to achieve equilibration and was subsequently used for pot filling. The soil stabilization method was adopted to maintain soil equilibrium after volatilizing all the volatile fractions of petroleum hydrocarbons from the spiked soil (Afzal et al., 2012). We adapted this method because the focus of this work was non-volatile fraction of TPHs in the soils.
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2.2. Soil amendments with biochar and compost
The spiked soil was amended with biochar (5% v/v) and compost (5% v/v). The
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concentrations of amendments (biochar and compost) were selected on the basis of already published literature (Bielská et al., 2017; 2018, Zhang et al., 2016). Green garden waste biochar was obtained from AIT Austrian Institute of Technology, Tulln, Austria. Biochar was produced from green garden waste residues under low pyrolysis temperature (500 °C) for 120
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min in the rotary furnace (Frišták et al., 2015). For obtaining uniform grain size distribution,
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biochar was ground and subsequently sieved to 0.5–2 mm. Compost was obtained from
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Horticulture Department of National Agriculture Research Center, Islamabad, Pakistan and was prepared from the green garden waste material. Additionally, no nutrients or chemical
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fertilizer were added after soil spiking with diesel oil. It has been proved from previous research that soil nitrogen level of 0.05-0.08 is enough to support plant growth in diesel
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impacted soils (Maqbool et. al. 2012). The detailed characteristics and physicochemical properties of biochar and compost are presented in supplementary Table S1 and Table S2.
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2.3. Microbial consortia preparation
Pre-isolated diesel-degrading bacterial strains Pseudomonas poae, Actinobacter bouvetii, Stenotrophomonas rhizophila and Pseudomonas rhizosphaerae (Accession numbers
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KT758715, KT758716, KT758718, and KT758719, respectively) were characterized for biochemical activities (Supplementary Table S3) and used for microbial consortia (MC) preparation. Selected bacterial strains were also capable of bio-surfactants production (Khan
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et al., 2017). Briefly, each bacterial culture was refreshed on nutrient agar plates overnight at 37 °C. Broth culture of each bacterial strain was prepared in nutrient broth containing 2% crude oil, incubated at 200 rpm for 24 hours at 37 °C. After incubation, the bacterial cell suspensions were washed three times with sterilized saline solution (0.9 % NaCl) by centrifugation at 8000 g for 15 min and resuspended in sterilized saline solution, cell numbers were maintained at 108 cells ml-1 (Khan et al., 2016a). An equal amount of each resuspended
bacterial suspension (1:1:1:1) was added to obtain consortia suspension, for the inoculation of soil. 2.4. Experimental setup Seeds of Italian ryegrass (Lolium multiflorum) were collected from Farm dynamic Pvt. Ltd., Lahore, Pakistan. Seeds were surface sterilized by soaking in 5% sodium hypochlorite solution for 2 min, then in 70% ethanol for 2 min and then washed thrice with sterilized distilled water (Yousaf et al., 2010). The spiked soil was amended with biochar (5% v/v) and
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compost (5% v/v) in the respective treatments, including a control without biochar and
compost. Pots with dimensions 15 × 7 × 7 cm were filled with spiked soil (500 g pot-1) and
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subsequently placed in the greenhouse. Sixty surface sterilized seeds per pot were sown. One
week after seed germination, each pot was inoculated with 50 ml of inoculant suspension (108 cells ml-1) by evenly spreading the suspension on to the surface of soil in the whole pot. Bacterial suspensions were cultivated in nutrient broth at 30 ºC, centrifuged and resuspended
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in 0.9 % (w/v) NaCl containing one of the strains described above and for consortia four
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strains together. For control treatments, spiked soil was treated with equal volume of 0.9 % NaCl instead of inoculum suspension. Plants were grown at 25 ºC in the greenhouse (16 h
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light ⁄ 8 h dark) and moisture was maintained by adding equal amount (20 ml) of distilled
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autoclaved water on alternate days. Pots were placed in plastic saucers to avoid leaching and covered with 12 mm thin layer of quartz grains to prevent drying out of surface and soil
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damage due to drop effect. The experimental plan followed a completely randomized design with following treatments, each has three replicates.
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T1) Spiked soil (SS)/Non vegetative control (NV) T2) SS + Italian Ryegrass (IR)
T3) SS + IR + Microbial Consortia (MC)
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T4) SS + IR + Biochar (BC) T5) SS + IR + BC + MC
T6) SS + IR + Compost (CM)
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T7) SS + IR + CM + MC T8) SS + IR + BC + CM T9) SS + IR + BC + CM + MC Plants were harvested 75 days after sowing; soil samples were collected for analysis of TPH content. Plant growth parameters were also measured. 2.5. Plant and soil analysis 2.5.1. Plant parameters
Italian ryegrass seed germination was calculated for every 24 hours up to one week after seed sowing. The seed germination percentage was determined by using percentage germination, that is the number of seeds germinated per number of seeds sown, multiplied by 100 (Wang & Zhou, 2005). The above-ground plant parameters like shoot length, fresh and oven-dried (80°C) biomass were determined according to Merkl et al. (2004). Before harvesting, plant physiological development was examined by measuring chlorophyll contents, using a handheld automated chlorophyll meter (SPAD-502, Minolta, Japan). The measurements were
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taken on adaxial surface of 3 leaves of each plant and values were averaged (Markwell et al., 1995). Similarly chlorophyll florescence parameter and performance index (PI) were also
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analysed using a chlorophyll flourimeter (Hansatech, pocket PEA) (Schoedl et al., 2013). 2.5.2. Soil analyses 2.5.2.1. Colony forming units
Colony forming units in rhizospheric soil were calculated by dilution spread plate method on
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nutrient agar containing 2% crude oil (Xue et al., 2004). Briefly, rhizospheric soil samples
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were collected by gently shaking and abrading the roots. Rhizosphere soil samples (1 g) were
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serially diluted and 100 µl of 10-1 to 10-4 dilutions were spread on nutrient agar containing 2% crude oil. The plates were incubated at 30 °C for 24 hours and colonies on the plates were
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calculated.
2.5.2.2. Total petroleum hydrocarbons concentration
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2.5.2.2.1. Extract preparation
To determine the range of TPH in soil samples, the method used was ISO,16703:2004 E. In
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current experiment, soil was analysed in duplicate (at 0 and 75 days) to determine TPH contents. Five gram soil per sample (two sub sampels) was weighed in a beaker and oven dried for 24 hours at 105 °C. Then, 10 ml acetone was added and mixed well. Five ml RTW-
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Std. (retention time window standard solution which contains Heptane with 30 mg L-1 nTetracontane and 30 µl L-1 Decane) was added, followed by shaking for 1 hour. The mentioned RTW was added as an internal standard to the extraction solution that
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subsequently was used at the same concentration to every sample for extraction. After shaking, distilled water was added and samples were centrifuged with 9000 rpm (SORVALL® centrifuges RC 5C plus). Subsequently, an aliquot of supernatant was collected by a glass pipette and transferred into a separate 15 ml glass vial. The collection of supernatant was done twice for each sample. Lastly, for clearing obscures in the aliquot, Florisil (magnesium silicate) was also added in each extract. 2.5.2.2.2. Quantification of TPH
After extraction of soil samples, the fractionation was done on silica gel column and injected into the GC-MS (model Hewlett Packard 5890 series II) gas chromatograph equipped with a HP 5972 mass-selective detector and a HP automatic liquid sampler. Operating in the SIM mode selected as ion monitoring mode. Peripheral standards were prepared from Shell Diala oil i.e., a transformer oil standard used for the product used at the Pacific North West site. The two internal standards were naphthalene-d8 99.2% atom % D, Inc., Middlesex, Protocol Analytical, NJ and n-hexadecane-d34 99 atom % D, Protocol. The two alternate standards
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were anthracene-d10 99.5% atom % D, Protocol and n-tetracosane-d50 98.2% atom % D, Protocol. A HP-1 (60 m × 0.25 mm × 0.25 µm film thickness) cross-linked column of methyl
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silicone gum was used. The detector temperatures and injector were set at 300 and 280 °C respectively. The initial temperature was kept at 50 °C for 1 min then increased to 110 °C at
10 °C min-1 to 270 °C at 3 °C min-1 and to 300°C at 15 °C min-1 and held at that temperature for 10 min. A 2 µl aliquot was inoculated in the split less mode with a 1 min purgative off
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where Helium was the carrier gas at 0.9 ml min-1. The MS, with selection of full scan mode,
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was scanned from 50 to 550 amu at 0.9 scan s-1. The quantification of TPH in a sample from
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the same GC run was led via total chromatographic area counts based on internal standards (ISTD) and external standards (ESTD) both. Three amalgamation approaches were used to
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obtain the total area of a total ion chromatogram; (a) manual addition along the lowest point baseline; (b) sum of resolved individual composites after addition with the lowest point
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baseline held on; (c) sum of resolved individual composites after addition with the baseline recognized by MS ChemStation addition. The suitability of the gas chromatographic system
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for the resolution of n-alkanes and for the detector response was verified according to the ISO-procedure. Quantification was done with an external standard method using a mixture of diesel purchased from a local gas station and additive-free lubricating oil (HELCOM Inter
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comparison Lubricating Oil). Chromatograms were integrated manually and the soil TPH concentration determined using a linear regression approach. For determining the range of TPHs (C10-C40), the stored samples (2 ml) GC vials were put
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into an auto sampler unit of a gas chromatograph coupled with flame ionization detector (FID). Analyses were performed using a Hewlett Packed 5890 GC-FID having automatic injector 7673. Column having 30 m*0.25 mm dimension coated with 0.25 µm 5% phenyl 95% methyl polysiloxane stationary phase (DB-5) was used for hydrocarbon analysis. Helium was used as carrier gas while GC oven programmed as 60º C for one minute, 20º C min-1 up to 340º C and then finally 10 minutes at 340º C. TPH dissipation was reported in terms of percentage. This mentioned dissipation of TPH was calculated by using formula:
100 × [(Ci − Cf)/Ci], where Ci stands for the initial (after soil stabilization) soil TPH concentration, while Cf was the concentration of soil samples after 75 days of rhizoremediation trial. All the results regarding TPH concentration in every samples were presented on the basis of dry matter (DM) soils. 2.6. Statistical Analysis All statistical analyses were performed using SPSS and Microsoft Excel 2013. Firstly, the data were analyzed for homogeneity of variance (Levene's test) then subsequently to analysis
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of variance (ANOVA), and significant differences between means were determined by Tukey test (p < 0.05). Multiple linear regression (MLR) analysis was performed to define the
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relation between the added microbial consortia and root biomass (Root fresh mass – Root dried weight) to TPHs dissipation. For regression, data was min-max normalized for the prevention of large numeric ranges dominating those with small numeric range, to reduce the potential bias into the data values exactly (0 for least and 1 for highest). In order to find the
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most common groups of treatments towards the studied parameters of plant and soil, a
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dendogram was constructed using Ward Linkage of SPSS.
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Results
3.1. Soil physico-chemical parameters before and after the amendments
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The initial parameters (before and after the addition of biochar and compost are given in Table 1 (Initial period). The addition of organic amendments showed changes in every
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parameter before and after amendments. Addition of compost resulted in highest increase in EC (2.75 dsm-1). The addition of biochar and compost showed highest total nitrogen (0.09
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%), available phosphorus (44.33 mg kg-1), potassium concentration (77.28 mg kg-1), organic matter (1.92 %), and total organic carbon (1.11 %) (p = 0.05). Final soil parameters after plant harvesting are also presented in Table 1 (Final period). The
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increase in soil N levels at the end of experiment corresponds to the respective soil amendments, such as biochar, compost and their combinations. The increase in soil N levels of control soils correspond to the addition of nitrogen via plant roots in the forms of root
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exudates and rhizodeposits (Dijkstra et al., 2013). Highest EC was observed in soil amended with both biochar and compost (3.05 dsm-1). No change in soil texture was observed. The highest total N 0.58 and 0.68 %, at the end of experiment was observed in soil amended with compost and with biochar and compost, respectively. The available phosphorus, potassium and organic carbon were in the following orders Control < BC < CM < BC + CM, with highest available phosphorus at 42.28 mg kg-1,
potassium at 86.67 mg kg-1 and organic carbon at 3.46 %. Highest total organic carbon was 2.02 % and 1.89 %, for biochar and compost and only compost amended soils. 3.2. Plant parameters Germination rates (%) of Italian ryegrass in different treatments are shown in Figure 1. Significantly highest germination rate was noted for T9 (87.07 %). While T2, with 57.13 %, resulted in least germination rate. The results of Italian ryegrass physiological parameters are presented in Table 2. Italian ryegrass produced more biomass when treated with compost,
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biochar and bacterial inoculum. Plant physiological parameters were most improved in T9,
with maximum fresh shoot biomass of 20.13 g, dry shoot biomass 10.05 g, fresh and dry root
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biomass of 5.5 and 2.7 g, respectively. The height of Italian ryegrass aerial parts was
statistically highest in T9 which is 2.25 times higher (30.11 cm) than the lowest (13.33 cm) in spiked soil (T2). The total chlorophyll content, chlorophyll florescence parameter and performance index were significantly highest in T9 as compared to other treatments (Figure
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2). Chlorophyll content was 36.13 (SPAD value), chlorophyll florescence parameter of
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photosystem II 0.69 Fv/Fm, performance index, 1.38. While, lowest total chlorophyll content
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(2.76) 30.11, chlorophyll florescence parameter (0.16 Fv/Fm) rand performance index (0.21) were observed in T2, with a percentage reduction of 92.3, 76.8 and 84.7.
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3.3. Soil parameters
The highest microbial count (5.74 * 107 cells g-1 soil) was found in the rhizosphere of T9
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(Table 3) having combination of all amendments i.e. plants along with biochar, compost and microbial consortia. Combination of any of two amendments (biochar, compost or microbial
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consortia with IR) showed higher microbial count as compared to control but have nonsignificant results with each combination (T4, T5 and T6). The lowest CFUs were observed in treatments T1, T2, and T3 respectively.
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Regarding TPH concentration (at harvesting time), vegetated soil was more efficient in degradation as compared to non-vegetated soil (Figure 4). The highest TPH (85%) removal was observed in variant BC+CM+MC while lowest TPH degradation was reported in NV
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(11%). Presence of only Italian ryegrass (IR) supported TPH remediation by 47%. While addition of amendments in the presence of IR such as BC, CM and MC showed dissipation percentage of 65%, 70%, and 73%, respectively. Additionally, the combination of two amendments in the presence of IR showed vigorous TPH dissipation with BC+CM, BC+MC and CM+MC (75%, 82% and 84% respectively). Interestingly, the treatments CM+MC, BC+MC and BC+CM+MC showed non-significant difference with each other while significant differences as compared to their respective control.
Multiple regression predicted that for the TPHs dissipation, root biomass and rhizospheric CFUs are good predictors of TPHs dissipation as they have high multiple correlation coefficient (R) of 0.75. These variables statistically significantly predicted TPHs dissipation, F (2, 24) = 15.773, p < 0.0005, R2 = 0.568. Both variables added statistical significant to the prediction, p < 0.05. Using root mass and CFUs the following equation elaborate the TPHs dissipation; 𝑇𝑃𝐻𝑠 𝑑𝑖𝑠𝑠𝑖𝑝𝑎𝑡𝑖𝑜𝑛 = 0.59 − (0.113 × 𝐶𝐹𝑈𝑠) − (0.707 × 𝑅𝑜𝑜𝑡 𝑀𝑎𝑠𝑠)
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Dendogram was construed, using Wards method that was based on all studied parameters (Figure 3). This was done to find and explain the most common groups of treatments towards
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TPHs dissipation. Dendogram with four distinct groups was constructed using Ward Linkage of SPSS. The clearest distinction was the treatment in which IR was cultivated with biochar, compost and microbial consortia, it stood out as Group 4 (G4 in Fig. 3), and this group also showed highest TPHs dissipation and improved IR physiology and high rhizospheric CFUs.
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Treatments with compost, microbial consortia and biochar and compost, have similar results
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for IR physiology with very little variation in TPHs dissipation rate. Group 2 (G2) represent
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the least improved plant growth, but the biochar dissipation was better compared to IR only treatment. Group 3 represented the cluster of treatments in which soil amendments were done
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individually with microbial consortia. Treatments in G3 have improved IR physiology with increased rhizospheric CFUs, and reduced TPHs, but they are statistically lower than that of
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G4.
4. Discussion A worldwide research trend is trying to find innovative and novel approaches for
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bioremediation of persistent organic pollutants. Progress has been made in the domain of rhizoremediation of petroleum hydrocarbons. Moreover, the phytoremediation efficiency can
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be increased via stimulating rhizospheric effect by synergistic use of plant, microbe and organic amendments (Chaudhry et al., 2005; Ijaz et al., 2016; Lladó et al., 2013). This research work is one of such contributions that intend to facilitate the understanding of
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enhanced rhizodegradation of TPHs contaminated soil by using Italian ryegrass along with different organic amendments i.e. biochar, compost, and combination of TPHs degrading bacterial strains. It is well-known that soil contaminated with TPHs undergoes changes in physico-chemical properties that affect carbon availability, aeration and essential nutrients (NPK) availability hence limiting microbial and plant growth (Wang et al., 2011; Wang et al., 2012). In the present study, we investigated the effect of soil organic amendments on different soil
parameters and plant growth. Both, initial and final concentrations of NPK were found significantly higher (2 to 3 times) in soil co-amended with biochar and compost (Table 1). The higher availability of NPK resulted in improved rhizoremediation of hydrocarbons and also supported the growth of soil microbial community, that was suppressed in the presence of TPHs contamination (Nwaichi et al., 2015). Similarly, co-application of biochar and compost also improved the total organic carbon and organic matter in soil, in both initial and after treatment (Table 1). Soil organic matter is reported to improve soil fertility by
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improving the soil water holding capacity, reducing nutrient leaching and improving soil porosity and aeration (Macci et al., 2015), thus enhancing plant growth and microbial
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population. The addition of compost and biochar results in positive synergetic effect,
increased availability of nutrients, improved water holding capacity and pore structure of soil because of highly porous origin of biochar. Biochar also prevents nutrient leaching in soil (Atkinson et al., 2010). The treatments amended with biochar and compost separately,
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improved soil physico-chemical parameters, but co-amendment of biochar and compost
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showed significantly higher improvements. TPHs contaminated soils are characterized with
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limited amount of nitrates and phosphates compared to uncontaminated soil (Yousaf et al., 2011). Additionaly, it also poses stress to plant due to hydrophobicity which interrupts water
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and soil interaction, resulting in leaching of water and nutrients to lower soil horizons (Jain et al., 2011). We found that the addition of biochar, compost and microbial consortia resulted in
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significantly positive effects on growth parameters (shoot length, shoot and root biomass) of Italian ryegrass in TPHs contaminated soils (Table 2). While spiked soil without any
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amendment showed negative impact on plant growth. Further, spiked soil co-amended with biochar and compost, and augmented with microbial consortia resulted in highest germination rates, chlorophyll content, chlorophyll florescence parameter and performance indexes
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(Figure 1 and 2).
The addition of biochar individually did not show significant improvement in plant physiological parameters and biochemical characters (Table 2, Figure 1 and 2). This may be
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due to low biodegradability of biochar in comparison with compost, resulting in low availability of nutrients from biochar. Though, biochar improved soil porosity and pH, the low nutrient availability caused lower plant biomass (Warnock et al., 2007). Compost on the other hand resulted in statistically increased soil organic matter and total organic carbon (Table 1), that in turn resulted in higher plant physiological parameters compared to biochar (Table 2, Figure 1 and 2). The addition of microbial consortia in combination with biochar and compost resulted in further improvements in plant physiological parameters. The addition
of microbial inocula along with organic additives improved the release of nutrients (N, P, and K) (Bonanomi et al., 2017). We observed similar effects of microbial augmentation along with organic amendments on Italian ryegrass growth and physiological parameters. The above mentioned stimulation in plant vigour and physiological parmaters may have increased root biomass, subsequently leading towards higher microbial counts in rhizospheric soils (Table 3). This plant induced enhancement in microbial polulation resulted in favorable microenvironemmnt. Organic amendments addition to TPHs contaminated soils resulted in
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higher (3-5 times) root biomass and microbial counts as compared to plants alone and plants
with biochar treatment. This was due to higher nutrient availability in all amended conditions
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except biochar alone. The compost treatment stimulated the indigenous microbial population,
as compost added organic carbon, and released nutrients slowly upon degradation. The compost also contains high amounts of microorganisms which are able to degrade organic substances (Sasek et al., 2003; Ren et al., 2017), while, microbial inoculum augmentation
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was directly linked with an increase in rhizospheric CFUs.
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Plant vigour, root biomass and microbial population are three most prominent factors while
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evaluating the rhizoremediation efficiency in TPHs contaminated soils. Higher plant tolerance to TPHs contamination and its improvement by providing suitable amendment
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(alone or in combination) may respond to the higher root turnover and pronounced rhizospheric effect. Root turnover can be considered as injection system in rhizoremediation
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that promote rhizospheric effect by providing substrate for microbial growth (Leigh et al., 2002). In return, microbes degrade TPHs contaminants by using hydrocarbons as energy
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source. This type of enhancement in microbial population generally and TPHs degrader population specifically is referred as rhizospheric effect (Basumatary et al., 2012). The concentration of TPHs dissipation in vegetated soils was higher as compared to non-
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vegetated soil, during the course of rhizoremediation trial. The enhancement of plant growth and root biomass due to action of different organic amendments decreased contaminant load in all vegetated treatments (Figure 4). Presence of Italian ryegrass stimulated dissipation of
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TPHs (35% more), as compared to non-vegetated soil. This decrease in hydrocarbons load supports the argument that root development is an important factor for the success of rhizoremediation. This provides an evidence of joint venture of plant and microorganisms (Liu et al., 2014) in the rhizosphere of plants to degrade TPHs. It is already reported, that during phytoremediation trials plants can interact with microbes especially in rhizospheric zone (Khan et al., 2013). Moreover, plants can also support degradation of TPHs by improving microbial population, soil physiochemical properties, humification and adsorption
of pollutant in rhizosphere (Al-Mansoory et al., 2015). Collectively, plants and microbes in the rhizosphere zone can act as “cleaning agent” in petroleum contaminated soils (Gaskin and Bentham, 2010). Many previous studies have reported that presence of plants stimulated hydrocarbon removal significantly as compared to non-vegetated soils (Merkl et al., 2005, Muratova et al., 2009; Peng et al., 2009; Ribeiro et al., 2014). Amendments like biochar, compost and microbial consortia with Italian ryegrass resulted in increased TPHs remediation of 55%, 60% and 62%, respectively more than non-vegetated soil. This increase in TPHs
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removal rate may correspond to the stimulation of microbial population with biochar and compost amendments (Lim et al., 2016).
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Biochar has been used in remediating variety of inorganic and organic contaminants in soils. The mechanism for organic contaminants (included TPHs) remediation is increased sorption by the processes of adsorption, partition and sequestration (Beesley et al., 2010). Additionally, biochar can support plant growth by providing essential nutrients and can
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change microbial community structure. Remediation of TPHs and PAHs by biochar was
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reported widely where sorption of recalcitrant molecules is considered as main mechanism
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(Bushnaf et al., 2011; Zhang et al., 2013; Cao et al., 2016). In some studies biochar was used as stimulating agent (Qin et al., 2013) and carrier for selected microbial strains during
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remediation trails (Costa et al., 2014; Zhang et al., 2016). In a more recent study (Song et al., 2016), a novel biochar-plant tandem approach was used to understand the mechanism of
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rhizoremediation stimulation by biochar addition. They concluded that firstly recalcitrant organic molecules are adsorbed to biochar, then root exudates may help in desorption that
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subsequently will be available to degrader microbial community in the rhizosphere. The findings from current study are in accordance with Song et al., 2016, and Zhang et al., 2016. It is widely reported that TPHs contaminated soils are nutrient and organic matter deficient,
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that is why adding compost can improve soil quality (Bastida et al., 2016) and foster the activity and growth of microbes (Hickman and Reid, 2008) capable of hydrocarbon degradation. Additionally, compost amendment to soil increases soil nutritional status,
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biological and enzymatic activity and aeration (Roca- Perez et al., 2009; Lou et al., 2017). That is why, composting has a great potential in bioremediation of TPHs contaminated soils (Namkoong et al., 2002). The current study demonstrated the stimulated rate of degradation (60% more than control) in TPHs contaminated soils while amended with compost (5%). Our results portray the same picture as previous from literature (Scelza et al., 2007; Zhang et al., 2012; Bastida et al., 2016).
Combine application of plants with any of two amendments (BC+CM or BC+MC or CM+MC) showed higher TPHs removal percentage of 75%, 82% and 84% respectively. Whereas, IR with all amendment combinations (BC+CM+MC) showed the highest TPHs removal (85%). This supports the fact that these amendments play a vital role to modify micro-environment around roots that subsequently result into higher degradation. The mechanisms that support this higher degradation of TPHs in integrated way are explained as (1) biochar and compost provide scarce nutrients, aeration and improve soil quality in TPH-
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impacted soils (2) better nutrients, oxygen availability and plant tolerance to contaminant
itself strengthened the root establishment and proliferation in contaminated soils (3) roots
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released photosynthetically fixed carbon and organic anions in their close vicinity (rhizosphere) (4) compounds released by roots act as carbon source for microbes as well as
promote pollutant desorption hence bioavailability (5) enhancement in microbial population by integrated use of plants and amendments supported by variety of root exudates (6) higher
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microbial population creates enhanced rhizospheric effect (prime important factor for
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influencing rhizoremediation efficiency) (Beesley et al., 2010; Khan et al., 2016; Montiel-
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Rozas et al., 2016). 5. Conclusions
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Plant-microbes interactions in correspondence with the soil amendments offer an emerging trend of remediation technologies for a number of persistent organic pollutants especially
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TPHs. Rhizoremediation is a green solution to overcome the quandary of TPHs contamination in soil. By improving the plant-microbe associations, time required for
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successful phytoremediation can be minimized. We concluded that the combined application of biochar, compost and hydrocarbons degrading bacteria had positive impact on enhancing rhizospheric effect. The root biomass is the main contributing factor for higher rhizospheric
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effect. The stimulated rhizospheric effect not only improved plant performance against TPHs contamination but also enhanced degradation percentage. Though, individually each of these amendments significantly improved the plant’s tolerance and increased TPHs dissipation rate,
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the combined use of organic amendment and synergistic augmentation of microbial consortia is more advantageous.
Conflicts of Interest: The authors have no financial disclosures or conflicts of interest
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plant colonization. J. Appl. Microbiol. 109, 1389–1401. Zhang, H., Tang, J., Wang, L., Liu, J., Gurav, R. G., & Sun, K. (2016). A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant
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Corynebacterium variabile HRJ4 and biochar. J. Environ. Sci. 47, 7-13.
Zhang, H., Tang, J., Wang, L., Liu, J., Gurav, R.G., Sun, K., 2016. A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium
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variabile HRJ4 and biochar. J. Environ. Sci. 47, 7–13.
Zhang, J., Lin, X., Liu, W., Wang, Y., Zeng, J., Chen, H., 2012. Effect of organic wastes on the plant-microbe remediation for removal of aged PAHs in soils. J. Environ. Sci. 24, 1476–1482. Zhang, X., Wang, H., He, L., Lu, K., Sarmah, A., Li, J., Bolan, N.S., Pei, J., Huang, H., 2013. Using biochar for remediation of soils contaminated with heavy metals and organic pollutants. Environ. Sci. Pollut. Res. 20, 8472–8483.
Zhuang, X., Chen, J., Shim, H., Bai, Z., 2007. New advances in plant growth-promoting
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rhizobacteria for bioremediation. Environ. Int. 33, 406–413.
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Figure 1. Percentage germination rates of Italian ryegrass in different conditions. (BC = Biochar, CM = Compost, MC = Microbial consortia, BC+CM = Biochar + Compost, BC+MC = Biochar + Microbial consortia, CM+MC = Compost + Microbial consortia, CM+BC+MC = Compost + Biochar + Microbial consortia and NA= No amendments)
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Figure 2. Italian ryegrass’s a) Chlorophyll content, b) Chlorophyll florescence parameter, and c) Performance Index. (BC = Biochar, CM = Compost, MC = Microbial consortia, BC+CM = Biochar + Compost, BC+MC = Biochar + Microbial consortia, CM+MC = Compost + Microbial consortia, CM+BC+MC = Compost + Biochar + Microbial consortia and NA= No amendments)
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Figure 3. Dendogram using Ward Linkage exhibit the grouping of treatments towards all the studied parameters. (BC = Biochar, CM = Compost, MC = Microbial consortia, BC+CM = Biochar + Compost, BC+MC = Biochar + Microbial consortia, CM+MC = Compost + Microbial consortia, CM+BC+MC = Compost + Biochar + Microbial consortia and NA= No amendments)
BC+CM+MC
a
CM+MC
a
BC+MC
a
BC+CM
b
MC
bc
CM BC
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c d
NA NV
f 0
20
40
60
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TPH desipation (%)
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e
80
100
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Figure 4. Stimulation of degradation percentage of TPH influenced by different soil amendments. Data was generated from GC-MS analysis of soil samples collected after 75 days of remediation trial. They are presented as percent of TPH removed relative to the soil that contains (3.7g Kg-1 DM soil). Error bars presents standard deviation for three replicates. Different letters on each bar showed statistical significant difference between treatments by the result of one way ANOVA fallowed by Tukey test as post hoc analysis. (BC = Biochar, CM = Compost, MC = Microbial consortia, BC+CM = Biochar + Compost, BC+MC = Biochar + Microbial consortia, CM+MC = Compost + Microbial consortia, CM+BC+MC = Compost + Biochar + Microbial consortia and NA= No amendments, NV = Non vegetative)
Table 1. Initial and final physico-chemical parameters of soil Period
Treatme
pH
nt
EC (dsm-1)
Total
Availabl
nitrogen
eP -1
(%)
(mg kg )
Potassiu m
Organic matter
(mg kg1
)
TOC1
(%)
(%)
Control (without
1.24
7.15
0.06
14.70
59.96
amendme
±0.09d
±0.04b
±0.01c
±0.10b
±0.15c
1.51
7.42
0.07
15.49
69.83
1.44
±0.02c
±0.09b
±0.00bc
±0.04c
±1.76b
±0.01b
2.75
7.22
0.08
40.73
72.67
nt)
±0.16
BC + CM
a
2.29 ±0.05
±0.03
a
7.14 b
±0.04
±0.00
ab
±1.40
0.09 b
±0.00
b
44.33 a
±0.16
77.28 a
1.29
7.44
0.11
27.68
(SS)
±0.07b
±0.04a
±0.03c
±0.48d
1.40
7.45
0.40
32.12
1.28
CM
±0.05 3.05 ±0.06
±0.05
0.58
a
7.41 a
±0.07
±0.04
a
0.68
a
±0.07
a
±0.44 39.38
±0.48
b
42.28 ±0.48
±0.05a 1.11 ±0.03a
2.13
1.23
±2.52d
±0.01d
±0.10c
±2.00
c
80.33 ±1.53
±1.15
2.79 ±0.06c 3.26
b
86.67 a
1.92 ±0.04a
1.04
59.67
72.00
c
1.79 ±0.05a
±0.00b
a
±0.06
1.61 ±0.09b 1.89
b
3.46 ±0.04a
±0.09a 2.02 ±0.11a
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BC + CM
7.47 b
±0.05
b
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Final
±0.06
a
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±0.06
b
±1.28
a
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Control
BC
±1.53
b
±0.08b
0.85
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CM
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BC
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Initial
0.73
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1.26 ±0.09c
In treatment column (BC = Biochar, CM = Compost, MC = Microbial consortia and NA= No amendments) Total organic carbon
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Numbers in columns followed by different letters are significantly different according to The Tukey-test (p=0.05) No statistical test performed
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Table 2. Italian ryegrass physiological parameters under different treatments Fresh shoot biomass (g)
Dry shoot biomass (g)
Fresh root biomass (g)
Dry root biomas (g)
NA
1.10 ±0.11f
0.27 ±0.01g
1.13 ±0.15d
0.42 ±0.03g
BC
4.10 ±0.26e
1.83 ±0.06f
1.53 ±0.35d
0.54 ±0.05fg
CM
7.22 ±1.34d
3.42 ±0.35e
1.77 ±0.61cd
0.57 ±0.04ef
MC
5.21 ±1.00de
2.20 ±0.08f
2.00 ±0.56bcd
0.67 ±0.02de
BC + CM
10.50 ±1.00c
5.08 ±0.04d
2.33 ±0.40bcd
0.74 ±0.05d
BC + MC
12.67 ±1.04c
5.84 ±0.10c
2.93 ±0.74bc
0.91 ±0.04c
CM + MC
16.25 ±1.82b
7.64 ±0.58b
3.13 ±0.25b
1.84 ±0.04b
CM + BC + MC
20.13 ±0.61a
10.05 ±0.21a
5.50 ±0.30a
2.70 ±0.09a
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Treatments
Cumulative length of shoots and leaves in each pot
N
a
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In treatment column (BC = Biochar, CM = Compost, MC = Microbial consortia and NA= No amendments)
Numbers in columns followed by different letters are significantly different according to The Tukey-test (p=0.05)
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Similar small letters in superscript of the same column are not significant
Table 3. Rhizospheric microbial abundance and residual TPHs under each treatment CFU
TPH
(cells g-1 of soil)
(g)
NVC
1.20 * 102 ± (8.50 * 10) e
3.26 ±0.09 a
NA
7.80 * 104 ± (2.88 * 102) e
1.95 ±0.03 b
BC
5.55 * 105 ± (2.08 * 103) e
1.29 ±0.02 c
CM
2.57 * 106 ± (3.51 * 104) d
1.09 ±0.04 d
MC
2.67 * 106 ± (2.00 * 104) d
0.98 ±0.02 de
BC + CM
2.83 * 106 ± (1.52 * 104) d
0.91 ±0.02 e
BC + MC
3.92 * 107 ± (2.64 * 105) c
0.64 ±0.03 f
CM + MC
4.12 * 107 ± (8.96 * 105) b
0.57 ±0.09 f
CM + BC + MC
5.74 * 107 ± (2.64 * 105) a
0.55 ±0.03 f
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Treatment
In treatment column (BC = Biochar, CM = Compost, MC = Microbial consortia, NVC= Non- vegetative control and NA= No amendments) Significantly highest mean is “a” column wise in superscript, followed by later alphabets for lower means
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Similar small letter in superscript of the same column are not significant
