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Journal of Hazardous Materials 179 (2010) 891–894

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journa l homepage: www.e lsev ier .com/ locate / jhazmat

hytoremediation of soil contaminated with used lubricating oil usingatropha curcas

. Agamuthua, O.P. Abioyea,∗, A. Abdul Azizb

Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Chemical Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

r t i c l e i n f o

rticle history:eceived 8 February 2010eceived in revised form 19 March 2010ccepted 19 March 2010vailable online 25 March 2010

eywords:

a b s t r a c t

Soil contamination by used lubricating oil from automobiles is a growing concern in many countries,especially in Asian and African continents. Phytoremediation of this polluted soil with non-edible plantlike Jatropha curcas offers an environmental friendly and cost-effective method for remediating the pol-luted soil. In this study, phytoremediation of soil contaminated with 2.5 and 1% (w/w) waste lubricatingoil using J. curcas and enhancement with organic wastes [Banana skin (BS), brewery spent grain (BSG) andspent mushroom compost (SMC)] was undertaken for a period of 180 days under room condition. 56.6%

atropha curcasrganic wastessed lubricating oilontaminated soilhytoremediation

and 67.3% loss of waste lubricating oil was recorded in Jatropha remediated soil without organic amend-ment for 2.5% and 1% contamination, respectively. However addition of organic waste (BSG) to Jatropharemediation rapidly increases the removal of waste lubricating oil to 89.6% and 96.6% in soil contaminatedwith 2.5% and 1% oil, respectively. Jatropha root did not accumulate hydrocarbons from the soil, but thenumber of hydrocarbon utilizing bacteria was high in the rhizosphere of the Jatropha plant, thus sug-gesting that the mechanism of the oil degradation was via rhizodegradation. These studies have proventhat J. curcas with organic amendment has a potential in reclaiming hydrocarbon-contaminated soil.

. Introduction

Contamination of soil by organic chemicals (mostly hydrocar-ons) is prevalent in oil producing and industrialized countriesf the world, but pollution of soil by used lubricating oil iscommon phenomenon in every major city across the globe.

his may pose a great threat to the environment and humaneing at large. Different treatment methods have been employedo reclaim contaminated soil. Phytoremediation, a strategy thatses plant to degrade, stabilize, and/or remove soil contami-ants [1] can be an alternative green technology method foremediation of hydrocarbon-contaminated soil. It offers an envi-onmentally friendly, cost-effective and carbon neutral approachor the remediation of toxic pollutant in the environment2].

Phytoremediation has now emerged as a promising strategy

or in situ removal of many contaminants [3–5]. Microbe-assistedhytoremediation, including rhizoremediation appears to be par-icularly effective for the removal and/or degradation of organicontaminants from contaminated soil [1]. Furthermore, accord-

∗ Corresponding author. Tel.: +60 379674631.E-mail address: bisyem2603@yahoo.com (O.P. Abioye).

304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2010.03.088

© 2010 Elsevier B.V. All rights reserved.

ing to Palmroth et al. [6], root exudates from plants do help todegrade toxic organic chemicals and acts as substrates for soilmicroorganisms in the soil which directly results in increased rateof biodegradation of the organic contaminants.

Different types of plants have been found useful for phytotreat-ment of soil contaminated by hydrocarbons. Alfalfa and horseradish was found to reduce concentration of kerosene-based jet fuelby 57–90% in 5 months [7]. Peng et al. [8] observed 41.61–63.2%total petroleum hydrocarbons (TPH) removal by Mirabilis jalapaL. in 127 days. Euliss et al. [9] found out that Carex stricta, Pan-nicum virgatum and Tripsacum dactyloides significantly reducedTPH by 70% after 1 year of growth. Studies by various authorsshow Jatropha curcas as a potential plant for remediation of heavymetals-contaminated soil. The plant (J. curcas) has been impli-cated in remediation of soil contaminated by heavy metals (Al,Fe, Cr, Mn, Ar, Zn, Cd and Pb) due to its bioaccumulation potential[10–12].

In this study J. curcas was selected due to its hardiness, its char-acteristics as non-edible plant which can grow in tropical areas andits commercial viability for the production of biodiesel, therefore

the objective of this study is to determine the potential of J. curcas inremoving hydrocarbons from soil and to investigate effects of dif-ferent organic amendments on the ability of Jatropha in removinghydrocarbons. In addition, the mechanisms of removal of hydro-carbon will be determined.

8 zardous Materials 179 (2010) 891–894

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Table 1Experimental design.

Treatment Details of treatment

A 4 kg soil + 2.5% oil + 10% BS + Jatropha plantB 4 kg soil + 2.5% oil + 10% BSG + Jatropha plantC 4 kg soil + 2.5% oil + 10% SMC + Jatropha plantD 4 kg soil + 2.5% oil + Jatropha plantE 4 kg soil + 2.5% oil onlyF 4 kg autoclaved soil + 2.5% oil + 0.5% NaN3

G 4 kg soil + 1% oil + 10% BS + Jatropha plantH 4 kg soil + 1% oil + 10% BSG + Jatropha plantI 4 kg soil + 1% oil + 10% SMC + Jatropha plantJ 4 kg soil + 1% oil + Jatropha plant

TP

B

92 P. Agamuthu et al. / Journal of Ha

. Experimental

.1. Sample collection

The soil samples used for the study were collected from a nurs-ry section in Sungai Buloh, Kuala Lumpur, Malaysia. The J. curcaseedlings (3 weeks old) were provided by Dr John of the Nilainiversity College, Nilai, Malaysia. Used lubricating oil was col-

ected from Perodua car service centre, Petaling Jaya, while therganic wastes were collected from different locations; banana skinBS) was collected from IPS canteen, University of Malaya, brew-ry spent grains (BSG) was collected from Carlsberg brewery, Shahlam, Selangor and spent mushroom compost (SMC) was collected

rom Gano mushroom farm, Tanjung Sepat, Selangor.

.2. Physicochemical property determination of soil and organicastes

Nitrogen content of soil used for phytoremediation and organicastes was determined using the Kjeldahl method, while phospho-

us, iron, aluminum and arsenic contents were determined usingnductively coupled plasma-optical emission spectroscopy (ICP-ES optima 4100 DV, PerkinElmer, USA) after acid digestion in aicrowave oven. The pH was determined with pH meter (HANNAI 8424) on 1:2.5 (w/v) soil/distilled water after 30 min equilibra-

ion. Triplicate determinations were made.

.3. Soil preparation

Four kilogram (4 kg) of sieved (2 mm) soil was contaminatedith 2.5 and 1% (w/w) of used lubricating oil and thoroughly mixed,

% (w/w) of different organic wastes (BS, BSG and SMC) werelso mixed separately with the oil-contaminated soil. Plastic bagsere filled with the soil–oil–organic waste mixture and allowed

o stabilize for 4 days before transplanting the seedlings into theontaminated soil. Control treatment consisting of bags of the plantithout used lubricating oil or organic wastes was also set up. Addi-

ional control treatment comprising of autoclaved soil containing.5% (w/w) NaN3 was also set up to determine non-biological lossf waste lubricating oil from the soil. All the treatments were set upn triplicate at room temperature (28 ± 2 ◦C) with 24 h fluorescentamps. The plants were moderately watered every 2 days with tap

ater to prevent leaching from the plastic bags. The appearance ofhe plants in response to the oil in soil was monitored to determinef there is phytotoxicity of the oil to the plants. The design of thexperiment is as shown in Table 1.

.4. Sampling

Soil samples were taken within the rhizosphere zone of Jat-opha from each plastic bag every 30 days for analysis of totaletroleum hydrocarbon (TPH), pH, and hydrocarbon utilizing bac-erial (HUB) counts. At the completion of the experiment (180 days),he plants were uprooted. The soil samples were collected from

able 2hysicochemical properties of soil and organic wastes used for phytoremediation.

Parameters Soil BS

Nitrogen (%) 0.6 ± 0.02Phosphorus (mg kg−1) 32.1 ± 2.5 2Moisture content (%) 10.1 ± 1.3 7pH 6.8 ± 0.5Fe (mg kg−1) 76.3 ± 6.2Al (mg kg−1) 193.6 ± 9.1As (mg kg−1) 0.4 ± 0.01

SG: Brewery spent grain, BS: Banana skin, SMC: Spent mushroom compost.

K 4 kg soil + 1% oil onlyL 4 kg autoclaved soil + 1% oil + 0.5% NaN3

the rhizosphere of each plant and analyzed for total bacterial andhydrocarbon utilizing bacterial counts. The root was rinsed thor-oughly with tap water and the plant dry matter was determinedafter drying at 50 ◦C for 48 h. The root tissue was extracted withdichloromethane in a Soxhlet extractor for 12 h to determine if theroots absorb the hydrocarbon from soil. The extracts were analyzedfor hydrocarbons using gas chromatography with a mass-selectivedetector (GC/MSD) HP-6890 in scan mode. The GC was equippedwith cross-linked 5% phenyl methyl siloxane capillary column; HP-5MS. Helium was used as carrier gas. The temperature program wasstarted at 40 ◦C and raised by 10 ◦C/min until 300 ◦C, which wasmaintained for 8 min.

HUB counts in the soil was determined by plating a seriallydiluted 1 g of the soil on oil agar (OA) [1.8 g K2HPO4, 4.0 g NH4Cl,0.2 g MgSO4·7H2O, 1.2 g KH2PO4, 0.01 g FeSO4·7H2O, 0.1 g NaCl, 20 gagar, 1% (v/v) used lubricating oil in 1000 mL distilled water, pH7.4], and incubated at 30 ◦C for 72 h. The colonies on each platewere counted and recorded as colony forming unit per gram of soil(CFU/g). The pure culture of the bacterial isolates was identified byGram staining technique and API 20NE for Gram negative bacteriaand BBL Crystal rapid identification kit for Gram positive bacteria.

The total extents of used lubricating oil biodegradation insoil were determined by suspending 10 g of soil in 20 mL ofdichloromethane in a 250 mL capacity Erlenmeyer flask. Aftershaking for 1 h on an orbital shaker (Model N-Biotek-101), thesolvent–oil mixture was filtered using Whatman number 4 filterpaper into a beaker of known weight and the solvent was com-pletely evaporated. The new weight of the beaker (now containingresidual oil) was recorded. Percentage biodegradation of used oilwas calculated using the formula of Ijah and Ukpe [13]:

% biodegradation

= weight of oil (control) − weight of oil (degraded)Weight of oil (control)

× 1001

Statistical analysis of the data was carried out using one-wayANOVA with SPSS Statistics version 17.0.

G BS SMC

1.0 ± 0.1 0.4 ± 0.01 0.5 ± 0.030.6 ± 2.0 21.2 ± 1.4 22.5 ± 1.81.8 ± 3.5 38.5 ± 2.8 62.3 ± 4.16.7 ± 0.5 7.0 ± 0.3 5.6 ± 0.3

– – –– – –– – –

P. Agamuthu et al. / Journal of Hazardous Materials 179 (2010) 891–894 893

Table 3Dry mass of Jatropha plants used for phytoremediation.

Treatment Weight (g)

Leaves Stems Roots

A 1.45 ± 0.1 4.10 ± 0.25 0.60 ± 0.01B 6.18 ± 0.23 8.79 ± 1.2 2.22 ± 0.03C 0.74 ± 0.01 3.71 ± 0.18 2.09 ± 0.02D 1.23 ± 0.13 2.72 ± 0.12 1.28 ± 0.1E 3.93 ± 0.8 6.99 ± 1.1 2.54 ± 0.25F 12.27 ± 1.2 15.89 ± 2.11 3.96 ± 0.12G 2.67 ± 0.14 5.25 ± 0.7 3.52 ± 0.34H 2.68 ± 0.35 4.88 ± 0.6 3.01 ± 0.61I 8.77 ± 1.2 13.06 ± 1.4 3.53 ± 0.14

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, soil + 2.5% oil + BS; B, soil + 2.5% oil + BSG; C, soil + 2.5% oil + SMC; D, soil + 2.5% oilnly; E, soil + 1% oil + BS; F, soil + 1% oil + BSG; G, soil + 1% oil + SMC; H, soil + 1% oilnly; I, control soil i.e. without oil contamination.

. Results and discussion

.1. Physicochemical properties of soil and organic wastes usedor phytoremediation

The physicochemical properties of soil and organic wastes usedor phytoremediation as shown in Table 2 revealed that the soil hadow nitrogen content (0.6%), phosphorus content of the soil was2.1 mg kg−1. Of the organic wastes used, BSG had higher amountf nitrogen (1.02%) compared to BS (0.4%) and SMC (0.5%). The soillso contains certain heavy metals like Fe, Al and As.

.2. Response of plants to the oil

The appearance of the Jatropha plants in response to differentoncentration of oil was monitored throughout the 180 days of thexperiment; no plant death was recorded in all the treatments ofoil contaminated with 1% waste lubricating oil, however somef the plants showed signs of phytotoxicity such as yellowing ofeaves, stunted growth compared with control, the signs are in line

ith the findings of Vouillamoz and Mike [14]. Plants in soil con-aminated with 2.5% used lubricating oil showed high symptoms ofhytotoxicity with death of at least one Jatropha plant recorded inach treatment (data not shown). This results shows that Jatrophalants can tolerate minimum degree of exposure to hydrocarbons.ry mass of the Jatropha plants in each treatment was determinedt the end of 180 days as shown in Table 3.

.3. Loss of used lubricating oil in soil contaminated with 2.5%nd 1% oil

The percentage loss of used lubricating oil in soil treatment con-aminated with 2.5% and 1% oil are shown in Figs. 1 and 2. The

ig. 1. Percentage biodegradation of waste lubricating oil in soil contaminated with.5% oil. Bars indicates standard error (n = 3).

Fig. 2. Percentage biodegradation of waste lubricating oil in soil contaminated with1% oil. Bars indicate standard error (n = 3).

percentage loss of used lubricating oil at the end of 180 days in soilcontaminated with 2.5% and 1% oil ranged from 11.6% to 89.6% and14.8% to 96.6%, respectively in all the different treatments. Contam-inated soil treated with BSG recorded the highest loss of oil (89.6%and 96.6%) in 180 days followed by soil treated with BS (82.1% and90.1%) in 2.5% and 1% contaminated soil, respectively. The contam-inated soil containing only Jatropha plant, without organic wastestreatment recorded 56.6% and 67.6% oil loss while control soil with-out Jatropha plant showed 36.9% and 51% oil loss in 2.5% and 1%contaminated soil respectively at the end of 180 days. 11.6% and14.2% oil loss in soil contaminated with 2.5% and 1% oil may bedue to non-biological factors like evaporation; this was recordedin autoclaved soil treated with sodium azide after 180 days. Highloss of oil in soil treated with BSG and Jatropha plants may bedue to the presence of appreciable nitrogen (1%) and phosphorus(20.6 mg kg−1) contents in BSG (Table 1), this was recorded alsoin our previous works, where soil amended with BSG recorded(93–95%) loss of used lubricating oil in soil [15,16]. It was alsonoticed that Jatropha plant amended with BSG grows better andtaller (about 20% than other treatments) with lots of fibrous rootsthan other treatments in the experimental set up. The result is inagreement with that of Palmroth et al. [6], who recorded 60% loss ofdiesel fuel in 30 days in diesel-contaminated soil planted with pinetree and amended with NPK fertilizer. One-way ANOVA showedthat there is no significant difference between the soil treated withBS, BSG and SMC at (P < 0.05), whereas significant difference wasobserved between the soil treated with different organic wastes,soil with only Jatropha plants and soil without Jatropha plants.These results indicated that addition of organic wastes into thecontaminated soil planted with Jatropha increased the loss of oilin the soil by at almost 30%; this is in line with the findings ofVouillamoz and Milke [14], who observed that compost additioncombined with phytoremediation, increases the rate of removal ofdiesel fuel in soil.

3.4. Uptake of oil by Jatropha

Jatropha roots of different treatment were Soxhlet extractedto determine if there was phytoaccumulation of hydrocarbons inthe plant root. GC/MS analysis of the extract did not show pres-ence of hydrocarbons in all the treatments. This is in sharp contrastwith the results of Palmroth et al. [6], who observed an uptake ofdiesel oil by grass root, but agrees with the findings of Chaineauet al. [17] who did not observe uptake of hydrocarbons by maizeroot. However, the result is similar to that of Santosh et al. [11],

who observed that application of organic amendments stabilizesthe As, Cr and Zn in heavy metals-contaminated soil and reducedtheir uptake by plant tissues. The result suggests that the mecha-nism of hydrocarbons removal by the Jatropha plants may be via

894 P. Agamuthu et al. / Journal of Hazardou

Fig. 3. Counts of hydrocarbon utilizing bacteria in soil contaminated with 2.5% oil.Bars indicate standard error (n = 3).

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ig. 4. Counts of hydrocarbon utilizing bacteria in soil contaminated with 1% oil.ars indicate standard error (n = 3).

hizodegradation or phytovolatilization which has been well doc-mented [1,18]. Also, the removal of the oil may be as a result ofoot exudates produced by the Jatropha plant which enhance thectivities of soil microorganisms in mineralizing the oil in the soil.

.5. Bacterial counts

The counts of hydrocarbon utilizing bacteria in soil contam-nated with 2.5% and 1% used lubricating oil are shown inigs. 3 and 4. Contaminated soil treated with BSG and Jatrophaemediation shows high counts of HUB (240 × 105 CFU/g and93 × 105 CFU/g) in both soil contaminated with 2.5% and 1% oil,espectively. This is similar to the findings of Ijah and Antai [19],hereas the treatment with only Jatropha plant without organicastes amendments recorded low counts of HUB (48 × 105 and

5 × 105 CFU/g) in 2.5% and 1% pollution, respectively. The reasonor the increase in counts of HUB in contaminated soil amendedith organic wastes might be due to the presence of nutrients in the

rganic wastes especially nitrogen and phosphorus that enhancedhe multiplication of bacteria in the soil. The HUB isolated from theontaminated soil were identified as species of Pseudomonas, Bacil-us megaterium, Micrococcus and Corynebacterium. These bacterialpecies has been implicated in hydrocarbon degradation by dif-erent authors [20–22]. These bacterial species together with rootxudates of Jatropha plants possibly help in the removal of usedubricating oil from the soil.

. Conclusion

J. curcas shows a potential to withstand minimum concentra-ion (1% and 2.5%, w/w) of used lubricating oil in the contaminated

[

s Materials 179 (2010) 891–894

soil. However, no accumulation of hydrocarbon was detected in theplant tissue, but the rhizosphere of Jatropha harbours metabolicallydiverse bacteria measured as hydrocarbon utilizing bacteria. Thus,suggesting that oil loss from the soil might be through rhizodegra-dation mechanism. Addition of organic waste, especially BSG to thecontaminated soil further enhances the growth of Jatropha and pro-liferation of bacteria in the soil, thus accounting for the additionalremoval of oil by 33% and 29.3% in soil contaminated with 2.5%and 1% oil, respectively compared to the treatment with Jatrophaalone. The study therefore proves the viability of using J. curcas withBSG amendment in remediating hydrocarbon-contaminated soil.This affords an alternative method in removing oil contaminantsfrom soil while promoting growth of economically viable plant likeJatropha whose seed can be used for production of biodiesel.

Acknowledgements

The authors would like to acknowledge the support of Univer-sity of Malaya IPPP grant PS 244/2008C and FS269/2008C. Also,we would like to thank Dr. John of Nilai University College, Nilai,Malaysia who provided the Jatropha seedlings used for this study.

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