Journal of Hazardous Materials 179 (2010) 891–894
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Phytoremediation of soil contaminated with used lubricating oil using Jatropha curcas P. Agamuthu a , O.P. Abioye a,∗ , A. Abdul Aziz b a b
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Department of Chemical Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e
i n f o
Article history: Received 8 February 2010 Received in revised form 19 March 2010 Accepted 19 March 2010 Available online 25 March 2010 Keywords: Jatropha curcas Organic wastes Used lubricating oil Contaminated soil Phytoremediation
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 plant like Jatropha curcas offers an environmental friendly and cost-effective method for remediating the polluted soil. In this study, phytoremediation of soil contaminated with 2.5 and 1% (w/w) waste lubricating oil using J. curcas and enhancement with organic wastes [Banana skin (BS), brewery spent grain (BSG) and spent mushroom compost (SMC)] was undertaken for a period of 180 days under room condition. 56.6% and 67.3% loss of waste lubricating oil was recorded in Jatropha remediated soil without organic amendment for 2.5% and 1% contamination, respectively. However addition of organic waste (BSG) to Jatropha remediation rapidly increases the removal of waste lubricating oil to 89.6% and 96.6% in soil contaminated with 2.5% and 1% oil, respectively. Jatropha root did not accumulate hydrocarbons from the soil, but the number of hydrocarbon utilizing bacteria was high in the rhizosphere of the Jatropha plant, thus suggesting that the mechanism of the oil degradation was via rhizodegradation. These studies have proven that J. curcas with organic amendment has a potential in reclaiming hydrocarbon-contaminated soil. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Contamination of soil by organic chemicals (mostly hydrocarbons) is prevalent in oil producing and industrialized countries of the world, but pollution of soil by used lubricating oil is a common phenomenon in every major city across the globe. This may pose a great threat to the environment and human being at large. Different treatment methods have been employed to reclaim contaminated soil. Phytoremediation, a strategy that uses plant to degrade, stabilize, and/or remove soil contaminants [1] can be an alternative green technology method for remediation of hydrocarbon-contaminated soil. It offers an environmentally friendly, cost-effective and carbon neutral approach for the remediation of toxic pollutant in the environment [2]. Phytoremediation has now emerged as a promising strategy for in situ removal of many contaminants [3–5]. Microbe-assisted phytoremediation, including rhizoremediation appears to be particularly effective for the removal and/or degradation of organic contaminants from contaminated soil [1]. Furthermore, accord-
∗ Corresponding author. Tel.: +60 379674631. E-mail address:
[email protected] (O.P. Abioye). 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.03.088
ing to Palmroth et al. [6], root exudates from plants do help to degrade toxic organic chemicals and acts as substrates for soil microorganisms in the soil which directly results in increased rate of biodegradation of the organic contaminants. Different types of plants have been found useful for phytotreatment of soil contaminated by hydrocarbons. Alfalfa and horse radish was found to reduce concentration of kerosene-based jet fuel by 57–90% in 5 months [7]. Peng et al. [8] observed 41.61–63.2% total petroleum hydrocarbons (TPH) removal by Mirabilis jalapa L. in 127 days. Euliss et al. [9] found out that Carex stricta, Pannicum virgatum and Tripsacum dactyloides significantly reduced TPH by 70% after 1 year of growth. Studies by various authors show Jatropha curcas as a potential plant for remediation of heavy metals-contaminated soil. The plant (J. curcas) has been implicated 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 characteristics as non-edible plant which can grow in tropical areas and its commercial viability for the production of biodiesel, therefore the objective of this study is to determine the potential of J. curcas in removing hydrocarbons from soil and to investigate effects of different organic amendments on the ability of Jatropha in removing hydrocarbons. In addition, the mechanisms of removal of hydrocarbon will be determined.
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P. Agamuthu et al. / Journal of Hazardous Materials 179 (2010) 891–894 Table 1 Experimental design.
2. Experimental 2.1. Sample collection The soil samples used for the study were collected from a nursery section in Sungai Buloh, Kuala Lumpur, Malaysia. The J. curcas seedlings (3 weeks old) were provided by Dr John of the Nilai University College, Nilai, Malaysia. Used lubricating oil was collected from Perodua car service centre, Petaling Jaya, while the organic wastes were collected from different locations; banana skin (BS) was collected from IPS canteen, University of Malaya, brewery spent grains (BSG) was collected from Carlsberg brewery, Shah Alam, Selangor and spent mushroom compost (SMC) was collected from Gano mushroom farm, Tanjung Sepat, Selangor. 2.2. Physicochemical property determination of soil and organic wastes
Treatment
Details of treatment
A B C D E F G H I J K L
4 kg soil + 2.5% oil + 10% BS + Jatropha plant 4 kg soil + 2.5% oil + 10% BSG + Jatropha plant 4 kg soil + 2.5% oil + 10% SMC + Jatropha plant 4 kg soil + 2.5% oil + Jatropha plant 4 kg soil + 2.5% oil only 4 kg autoclaved soil + 2.5% oil + 0.5% NaN3 4 kg soil + 1% oil + 10% BS + Jatropha plant 4 kg soil + 1% oil + 10% BSG + Jatropha plant 4 kg soil + 1% oil + 10% SMC + Jatropha plant 4 kg soil + 1% oil + Jatropha plant 4 kg soil + 1% oil only 4 kg autoclaved soil + 1% oil + 0.5% NaN3
Four kilogram (4 kg) of sieved (2 mm) soil was contaminated with 2.5 and 1% (w/w) of used lubricating oil and thoroughly mixed, 5% (w/w) of different organic wastes (BS, BSG and SMC) were also mixed separately with the oil-contaminated soil. Plastic bags were filled with the soil–oil–organic waste mixture and allowed to stabilize for 4 days before transplanting the seedlings into the contaminated soil. Control treatment consisting of bags of the plant without used lubricating oil or organic wastes was also set up. Additional control treatment comprising of autoclaved soil containing 0.5% (w/w) NaN3 was also set up to determine non-biological loss of waste lubricating oil from the soil. All the treatments were set up in triplicate at room temperature (28 ± 2 ◦ C) with 24 h fluorescent lamps. The plants were moderately watered every 2 days with tap water to prevent leaching from the plastic bags. The appearance of the plants in response to the oil in soil was monitored to determine if there is phytotoxicity of the oil to the plants. The design of the experiment is as shown in Table 1.
the rhizosphere of each plant and analyzed for total bacterial and hydrocarbon utilizing bacterial counts. The root was rinsed thoroughly with tap water and the plant dry matter was determined after drying at 50 ◦ C for 48 h. The root tissue was extracted with dichloromethane in a Soxhlet extractor for 12 h to determine if the roots absorb the hydrocarbon from soil. The extracts were analyzed for hydrocarbons using gas chromatography with a mass-selective detector (GC/MSD) HP-6890 in scan mode. The GC was equipped with cross-linked 5% phenyl methyl siloxane capillary column; HP5MS. Helium was used as carrier gas. The temperature program was started at 40 ◦ C and raised by 10 ◦ C/min until 300 ◦ C, which was maintained for 8 min. HUB counts in the soil was determined by plating a serially diluted 1 g of the soil on oil agar (OA) [1.8 g K2 HPO4 , 4.0 g NH4 Cl, 0.2 g MgSO4 ·7H2 O, 1.2 g KH2 PO4, 0.01 g FeSO4 ·7H2 O, 0.1 g NaCl, 20 g agar, 1% (v/v) used lubricating oil in 1000 mL distilled water, pH 7.4], and incubated at 30 ◦ C for 72 h. The colonies on each plate were counted and recorded as colony forming unit per gram of soil (CFU/g). The pure culture of the bacterial isolates was identified by Gram staining technique and API 20NE for Gram negative bacteria and BBL Crystal rapid identification kit for Gram positive bacteria. The total extents of used lubricating oil biodegradation in soil were determined by suspending 10 g of soil in 20 mL of dichloromethane in a 250 mL capacity Erlenmeyer flask. After shaking for 1 h on an orbital shaker (Model N-Biotek-101), the solvent–oil mixture was filtered using Whatman number 4 filter paper into a beaker of known weight and the solvent was completely evaporated. The new weight of the beaker (now containing residual oil) was recorded. Percentage biodegradation of used oil was calculated using the formula of Ijah and Ukpe [13]:
2.4. Sampling
% biodegradation
Nitrogen content of soil used for phytoremediation and organic wastes was determined using the Kjeldahl method, while phosphorus, iron, aluminum and arsenic contents were determined using inductively coupled plasma-optical emission spectroscopy (ICPOES optima 4100 DV, PerkinElmer, USA) after acid digestion in a microwave oven. The pH was determined with pH meter (HANNA HI 8424) on 1:2.5 (w/v) soil/distilled water after 30 min equilibration. Triplicate determinations were made. 2.3. Soil preparation
Soil samples were taken within the rhizosphere zone of Jatropha from each plastic bag every 30 days for analysis of total petroleum hydrocarbon (TPH), pH, and hydrocarbon utilizing bacterial (HUB) counts. At the completion of the experiment (180 days), the plants were uprooted. The soil samples were collected from
=
weight of oil (control) − weight of oil (degraded) 100 × 1 Weight of oil (control)
Statistical analysis of the data was carried out using one-way ANOVA with SPSS Statistics version 17.0.
Table 2 Physicochemical properties of soil and organic wastes used for phytoremediation. Parameters
Soil
Nitrogen (%) Phosphorus (mg kg−1 ) Moisture content (%) pH Fe (mg kg−1 ) Al (mg kg−1 ) As (mg kg−1 )
0.6 32.1 10.1 6.8 76.3 193.6 0.4
BSG ± ± ± ± ± ± ±
0.02 2.5 1.3 0.5 6.2 9.1 0.01
BSG: Brewery spent grain, BS: Banana skin, SMC: Spent mushroom compost.
1.0 ± 20.6 ± 71.8 ± 6.7 ± – – –
BS 0.1 2.0 3.5 0.5
0.4 ± 21.2 ± 38.5 ± 7.0 ± – – –
SMC 0.01 1.4 2.8 0.3
0.5 ± 22.5 ± 62.3 ± 5.6 ± – – –
0.03 1.8 4.1 0.3
P. Agamuthu et al. / Journal of Hazardous Materials 179 (2010) 891–894
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Table 3 Dry mass of Jatropha plants used for phytoremediation. Treatment
Weight (g) Leaves
A B C D E F G H I
1.45 6.18 0.74 1.23 3.93 12.27 2.67 2.68 8.77
± ± ± ± ± ± ± ± ±
Stems 0.1 0.23 0.01 0.13 0.8 1.2 0.14 0.35 1.2
4.10 8.79 3.71 2.72 6.99 15.89 5.25 4.88 13.06
Roots ± ± ± ± ± ± ± ± ±
0.25 1.2 0.18 0.12 1.1 2.11 0.7 0.6 1.4
0.60 2.22 2.09 1.28 2.54 3.96 3.52 3.01 3.53
± ± ± ± ± ± ± ± ±
0.01 0.03 0.02 0.1 0.25 0.12 0.34 0.61 0.14
A, soil + 2.5% oil + BS; B, soil + 2.5% oil + BSG; C, soil + 2.5% oil + SMC; D, soil + 2.5% oil only; E, soil + 1% oil + BS; F, soil + 1% oil + BSG; G, soil + 1% oil + SMC; H, soil + 1% oil only; I, control soil i.e. without oil contamination.
3. Results and discussion 3.1. Physicochemical properties of soil and organic wastes used for phytoremediation The physicochemical properties of soil and organic wastes used for phytoremediation as shown in Table 2 revealed that the soil had low nitrogen content (0.6%), phosphorus content of the soil was 32.1 mg kg−1 . Of the organic wastes used, BSG had higher amount of nitrogen (1.02%) compared to BS (0.4%) and SMC (0.5%). The soil also contains certain heavy metals like Fe, Al and As. 3.2. Response of plants to the oil The appearance of the Jatropha plants in response to different concentration of oil was monitored throughout the 180 days of the experiment; no plant death was recorded in all the treatments of soil contaminated with 1% waste lubricating oil, however some of the plants showed signs of phytotoxicity such as yellowing of leaves, stunted growth compared with control, the signs are in line with the findings of Vouillamoz and Mike [14]. Plants in soil contaminated with 2.5% used lubricating oil showed high symptoms of phytotoxicity with death of at least one Jatropha plant recorded in each treatment (data not shown). This results shows that Jatropha plants can tolerate minimum degree of exposure to hydrocarbons. Dry mass of the Jatropha plants in each treatment was determined at the end of 180 days as shown in Table 3. 3.3. Loss of used lubricating oil in soil contaminated with 2.5% and 1% oil The percentage loss of used lubricating oil in soil treatment contaminated with 2.5% and 1% oil are shown in Figs. 1 and 2. The
Fig. 2. Percentage biodegradation of waste lubricating oil in soil contaminated with 1% oil. Bars indicate standard error (n = 3).
percentage loss of used lubricating oil at the end of 180 days in soil contaminated with 2.5% and 1% oil ranged from 11.6% to 89.6% and 14.8% to 96.6%, respectively in all the different treatments. Contaminated 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% and 90.1%) in 2.5% and 1% contaminated soil, respectively. The contaminated soil containing only Jatropha plant, without organic wastes treatment recorded 56.6% and 67.6% oil loss while control soil without 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% and 14.2% oil loss in soil contaminated with 2.5% and 1% oil may be due to non-biological factors like evaporation; this was recorded in autoclaved soil treated with sodium azide after 180 days. High loss of oil in soil treated with BSG and Jatropha plants may be due to the presence of appreciable nitrogen (1%) and phosphorus (20.6 mg kg−1 ) contents in BSG (Table 1), this was recorded also in our previous works, where soil amended with BSG recorded (93–95%) loss of used lubricating oil in soil [15,16]. It was also noticed that Jatropha plant amended with BSG grows better and taller (about 20% than other treatments) with lots of fibrous roots than other treatments in the experimental set up. The result is in agreement with that of Palmroth et al. [6], who recorded 60% loss of diesel fuel in 30 days in diesel-contaminated soil planted with pine tree and amended with NPK fertilizer. One-way ANOVA showed that there is no significant difference between the soil treated with BS, BSG and SMC at (P < 0.05), whereas significant difference was observed 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 the contaminated soil planted with Jatropha increased the loss of oil in the soil by at almost 30%; this is in line with the findings of Vouillamoz and Milke [14], who observed that compost addition combined with phytoremediation, increases the rate of removal of diesel fuel in soil. 3.4. Uptake of oil by Jatropha
Fig. 1. Percentage biodegradation of waste lubricating oil in soil contaminated with 2.5% oil. Bars indicates standard error (n = 3).
Jatropha roots of different treatment were Soxhlet extracted to determine if there was phytoaccumulation of hydrocarbons in the plant root. GC/MS analysis of the extract did not show presence of hydrocarbons in all the treatments. This is in sharp contrast with the results of Palmroth et al. [6], who observed an uptake of diesel oil by grass root, but agrees with the findings of Chaineau et al. [17] who did not observe uptake of hydrocarbons by maize root. However, the result is similar to that of Santosh et al. [11], who observed that application of organic amendments stabilizes the As, Cr and Zn in heavy metals-contaminated soil and reduced their uptake by plant tissues. The result suggests that the mechanism of hydrocarbons removal by the Jatropha plants may be via
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soil. However, no accumulation of hydrocarbon was detected in the plant tissue, but the rhizosphere of Jatropha harbours metabolically diverse bacteria measured as hydrocarbon utilizing bacteria. Thus, suggesting that oil loss from the soil might be through rhizodegradation mechanism. Addition of organic waste, especially BSG to the contaminated soil further enhances the growth of Jatropha and proliferation of bacteria in the soil, thus accounting for the additional removal of oil by 33% and 29.3% in soil contaminated with 2.5% and 1% oil, respectively compared to the treatment with Jatropha alone. The study therefore proves the viability of using J. curcas with BSG amendment in remediating hydrocarbon-contaminated soil. This affords an alternative method in removing oil contaminants from soil while promoting growth of economically viable plant like Jatropha whose seed can be used for production of biodiesel. Fig. 3. Counts of hydrocarbon utilizing bacteria in soil contaminated with 2.5% oil. Bars indicate standard error (n = 3).
Acknowledgements The authors would like to acknowledge the support of University 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. References
Fig. 4. Counts of hydrocarbon utilizing bacteria in soil contaminated with 1% oil. Bars indicate standard error (n = 3).
rhizodegradation or phytovolatilization which has been well documented [1,18]. Also, the removal of the oil may be as a result of root exudates produced by the Jatropha plant which enhance the activities of soil microorganisms in mineralizing the oil in the soil. 3.5. Bacterial counts The counts of hydrocarbon utilizing bacteria in soil contaminated with 2.5% and 1% used lubricating oil are shown in Figs. 3 and 4. Contaminated soil treated with BSG and Jatropha remediation shows high counts of HUB (240 × 105 CFU/g and 193 × 105 CFU/g) in both soil contaminated with 2.5% and 1% oil, respectively. This is similar to the findings of Ijah and Antai [19], whereas the treatment with only Jatropha plant without organic wastes amendments recorded low counts of HUB (48 × 105 and 45 × 105 CFU/g) in 2.5% and 1% pollution, respectively. The reason for the increase in counts of HUB in contaminated soil amended with organic wastes might be due to the presence of nutrients in the organic wastes especially nitrogen and phosphorus that enhanced the multiplication of bacteria in the soil. The HUB isolated from the contaminated soil were identified as species of Pseudomonas, Bacillus megaterium, Micrococcus and Corynebacterium. These bacterial species has been implicated in hydrocarbon degradation by different authors [20–22]. These bacterial species together with root exudates of Jatropha plants possibly help in the removal of used lubricating oil from the soil. 4. Conclusion J. curcas shows a potential to withstand minimum concentration (1% and 2.5%, w/w) of used lubricating oil in the contaminated
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