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Volume 3 • Issue 4 • 1000144J Bioremed Biodegrad ISSN: 2155-6199 JBRBD, an open access journal

Open Access

Adewole and Bulu, J Bioremed Biodegrad 2012, 3:4 DOI: 10.4172/2155-6199.1000144

Open Access

Research Article

Keywords: Calopogonium mucunoides; Phytoremediation; Crudeoil; Organic fertilizer; Soils

IntroductionThere is an increasing petroleum hydrocarbon pollution of the

soil ecosystem during oil exploration, exploitation, storage and transportation [1,2]. In Nigeria, tremendous increase in production and utilization of petroleum has led to a steady increase in the level of soil pollution by petroleum oil, especially in the Niger Delta region [3]. In addition to accidental discharge of petroleum oil [4], there exists the petroleum pollution of the soil ecosystem through the oil pipe vandalization and spillage [5]; and these have their attendant effects on the living soil organisms and vegetation [6].

Petroleum hydrocarbons are naturally occurring compounds that bind soil components thereby making the removal or degradation more difficult [7]. Also, their movement into the sub-soil leads to the expulsion of air thereby depleting oxygen reserves in the soil and impeding its diffusion to the deeper layers [8]. As the available soil oxygen diminishes, the soil microbial activities involved in the utilization of oxygen for biodegradation of the contaminants will reduce [9]. This will have adverse effect on the quality of soil and thereby reduce the growth performance of plants [10].

In the Niger Delta region of Nigeria, many of the petroleum hydrocarbon-polluted soils are found around the communities who practice subsistence fish and crop farming. Hence, the agri-business of many of the farmers in this region is in danger due to soil and freshwater pollution. Many of the soils that are good for agricultural purposes are being neglected because of their inability to support crops as a result of petroleum contamination. Phytoremediation is an emerging technology being used to clean-up polluted sites. Many of the established phytoplants such as Brassica juncea and Triticum aestivum [11] are temperate crops. However, tropical crops for example;Helianthus annuus [12] have been used at different times to remediate

soils polluted with heavy metals, but is rarely cultivated by Nigerian farmers [13].

Since, these established phytoplants are not readily available; there is therefore the need to look for phytoweeds that could be adaptable to tropical environment. During our preliminary survey of petroleum hydrocarbon-polluted soils, among many of the weeds with short duration life-span that survived on petroleum hydrocarbon-polluted soils is Calopogonium mucunoides. The specific objectives of the study therefore, were to (i) investigate the growth of C. mucunoides as a phytoremediating plant at different levels of crude oil soil contamination, (ii) assess the uptake of the total hydrocarbon, Pb and Cd by C. mucunoides in crude oil soil contamination, and (iii) evaluate the effectiveness of different organic-based fertilizers application on the remediating potential of C. mucunoides plant.

Materials and MethodsThe study was carried out in the greenhouse of the Faculty of

Agriculture, Obafemi Awolowo University, Ile-Ife, Nigeria. Bulk surface soil samples (0-15 cm) from an exhaustively cropped farm land within the University was collected, the soil was air-dried for 7 days, sieved using a 2 mm mesh and then analyzed to determine the physico-

*Corresponding author: MB Adewole, Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria, E-mail: [email protected]

Received February 16, 2012; Accepted April 12, 2012; Published April 14, 2012

Citation: Adewole MB, Bulu YI (2012) Influence of Different Organic-Based Fertilizers on the Phytoremediating Potential of Calopogonium mucunoides Desv. from Crude Oil Polluted Soils. J Bioremed Biodegrad 3:144. doi:10.4172/2155-6199.1000144

Copyright: © 2012 Adewole MB, et al. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Influence of Different Organic-Based Fertilizers on the Phytoremediating Potential of Calopogonium mucunoides Desv. from Crude Oil Polluted SoilsM. B. Adewole* and Y. I. BuluInstitute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria

AbstractA greenhouse experiment was conducted to investigate the growth of Calopogonium mucunoides in soils

contaminated by various concentrations of crude oil with a view of assessing its phytoremediating potential when different organic-based fertilizers were applied. The crude oil prepared at different concentrations of 0.0, 2.5, 5.0, 10.0 and 20.0% (v/v) acted as contaminants on 3 kg each of the air-dried soil collected from exhaustively cropped farm. Each treatment was replicated thrice in complete randomized design with four different fertility management levels, namely: 8t ha-1 compost organic fertilizer (CM); 8t ha-1 neem fortified organic fertilizer (NM); control1, without fertilizer application (C1) and Control2, where no fertilizer and no crop but crude oil was applied (C2). Significantly (p<0.05) highest total petroleum hydrocarbon (THC) uptake (10-2 mg kg-1) of 1.08, 0.52 and 0.21; 1.01, 0.51 and 0.11 in the roots and shoots for CM, NM and C1 treatments respectively were obtained at 2.5% contamination. Also, significantly (p<0.05) higher values of (10-2 mg kg-1) 2.57, 1.49 and 0.37; 3.02, 0.98 and 0.58 for Pb in the roots and shoots with CM, NM and C1 treatments respectively were phytoremediated at 5.0% contamination. Lower values of Cd were removed at different contamination levels and fertilizer treatments. With increased contamination, there was a reduction in the uptake of THC and Cd, while higher Pb bioaccumulated. The study concluded that C. mucunoides plant could be effectively used in the phytoremediation of crude oil contaminated soil when compost organic fertilizer is applied.

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chemical characteristics of the soil prior to sowing of the viable seeds of C. mucunoides. The soil properties obtained are presented in Table 1.

The air-dried and sieved soil was then filled into 60 buckets of radius 12.5 cm and height 17 cm leaving a space of 3 cm at the top end of the buckets to make allowance for watering. Each of the buckets contained 3 kg of the soil and each bucket was perforated at the base to avoid water logging and to increase the soil aeration. Crude oil obtained from Nigeria National Petroleum Corporation, Eleme, Rivers State, prepared to different concentration levels of 0.0%, 2.5%, 5.0%, 10.0% and 20.0% (v/v) were used as contaminants and the moistened buckets were left for one week to equilibrate. The crude oil was analyzed and has chemical composition [Total hydrocarbon (THC) 173.20 mg l-1, Pb 4.87 mg l-1, and Cd 0.85 mg l-1].

The seeds of C. mucunoides collected from the Teaching and Research Farm, the Faculty of Agriculture, Obafemi Awolowo University, Ile-Ife were scarified and their germination percentages were determined. Cotton wool was spread in a petri dish and moistened with clean water. Fifty seeds of C. mucunoides, randomly counted were put on the moistened cotton wool and another petri dish was used to cover it. This was replicated 3 times. The number of seeds that germinated was counted on the fifth day and a mean germination percentage of 75 were obtained. This assisted to decide the planting rate of four seeds of C. mucunoides per pot.

Each of the pollution concentration was replicated thrice in completely randomised design with four fertilizer application levels viz: 8 t ha-1 compost organic fertilizer (CM); 8 t ha-1 neem fortified organic fertilizer (NM); control 1, soil with no fertilizer application (C1) and control 2, soil with no fertilizer and no crop but with crude oil only (C2). The constituents of the organic-based fertilizers which were purchased at a local market are presented in Table 2. The four seeds of C. mucunoides planted in each of the pots were thinned to two seedlings per pots at 2 weeks after planting (WAP) and thereafter fertilized. The thinned seedlings were dropped into their respective pots. Throughout the duration of the experiment, distilled water was supplied to plants as often as soil dryness was observed to field moisture capacity and the pots were maintained weed-free. At 12 weeks of planting, the experiment was terminated to prevent the C. mucunoides weed seeds from spreading.

Plant growth measurement

The plant length, number of leaves and stem girth were determined weekly for a period of 12 weeks. Plant length was measured with a meter rule from soil level to the terminal bud. The stem girth was derived after measuring the diameter of the plant with a vernier calliper using the formula πd, where π = 22/7 and d = diameter of the plant. At harvest, plant roots and shoots were oven-dried at a temperature of 80°C for 48 hours, allowed to cool and their dry weights were determined.

Laboratory analysis

Soil and organic-based fertilizers analyses: The soil pH was potentiometrically determined in 1:1 soil-water ratio [14]. The particle size analysis was determined using hydrometer method in 5 % sodium hexametaphosphate as outlined by Bouyoucos [15]. Soil organic carbon was determined using Walkey-Black method [16] and micro Kjeldahl procedure was used for the determination of total nitrogen [17]. The available phosphorus was determined by Bray P1 method [18]. The exchangeable cations (Ca2+ + Mg2+ + K+ + Na+) were determined using 1M NH4OAc (Ammonium acetate) buffered at pH 7.0 as extractant [19]. The Na+ and K+ concentrations in the soil extracts were read on Gallenkamp flame photometer while Ca2+ and Mg2+ were read using a Buck Scientific Model 210 VGP (Norwalk, Connecticut, USA) Atomic Absorption Spectrophotometer (AAS).

Exchangeable acidity (H+ + Al3+) in the soil samples was extracted with 1M KCl [19]. Solution of the extract was titrated with 0.05M NaOH to a permanent pink endpoint using phenolphthalein as indicator. The amount of NaOH used is equivalent to the total amount of exchangeable acidity in the aliquot taken [20]. Lead and Cadmium were determined using 5 ml of the mixture (concentrated HNO3 and concentrated HClO4 in the ratio 2:1) with 5 ml of concentrated H2SO4 to digest 0.5 g of each soil sample for 2 hours at 150°C [21]. The digests were allowed to cool and each was made up to 25 ml with distilled water. Concentrations of Pb and Cd in the extract were read on using AAS.

Root and shoot analyses: Lead and Cd concentrations in the root and shoot samples were determined using 5 ml of the mixture (concentrated HNO3 and HClO4 in the ratio 2:1) with 5 ml of concentrated H2SO4 to digest 0.5 g of each sample for 2 hours at 150°C [21]. The digests were allowed to cool and each was made up to 25 ml with distilled water. The concentrations of Pb and Cd in the extracts were read on using AAS.

Determination of total hydrocarbon content

Greenberg et al. [22] approach was used for total hydrocarbon determination. Ten grams of each of the air-dried and sieved soil samples and 0.5 g each of the oven-dried and ground plant samples was weighed into a 250 ml conical flask each. 20 ml of xylene was added to each of the sample and then placed on a reciprocating shaker for 30 minutes. Each sample was later filtered using Whattman No. 1 filter paper of 11 cm into filtering bottle. The crude oil was used to prepare a set of standard: 0.00, 5.00, 10.00, 15.00, 20.00 and 25.00 ppm, with xylene as the solvent and thereafter used to calibrate the spectrophotometer before the filtrates of soil, root and shoot samples’ were read at 650 nm wavelength.

Translocation factor and bioaccumulation of the contaminants

The translocation factor (TF) was calculated as the concentration of Pb, Cd or THC in the shoots divided by the concentration in the

Property ValuepH (1:1 soil-water) 6.00Organic carbon (g kg-1) 29.70Total N (g kg-1) 2.56Available P (mg kg-1) 9.40Exchangeable cations (cmol kg-1) 2.75Exchangeable acidity (cmol kg-1) 0.15Total hydrocarbon content (mg kg-1) 0.17Pb (mg kg-1) 0.67Cd (mg kg-1) 0.11Clay (g kg-1) 82.00Silt (g kg-1) 60.00Sand (g kg-1) 858.00Textural class Loamy sand

Table 1: Physico-chemical properties of the soil used for the study.

Type of fertilizer N P K Ca MgCM 4.06 1.40 0.71 1.60 0.16NM 6.40 0.64 0.46 12.42 0.59

Table 2: Chemical compositions (%) of organic-based fertilizers used.



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roots. The bioaccumulation of the contaminants in the plant (shoots and roots) was calculated as the concentrations in the shoots and roots multiplied by their weights and thereafter, divided by the total weight of shoots and roots.

Statistical analysis

A statistical comparison of means was carried out with one-way analysis of variance (ANOVA) and treatment means were separated

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Figure 1: Effects of fertilizer treatments on the growth parameters after planting; Bars indicate level of significance at p < 0.05.



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using the Duncan range test available in SPSS 16 statistical package. Significance was set at p<0.05.

Results and DiscussionGrowth characteristics of Calopogonium mucunoides

The effects of fertilizer treatments and crude oil soil contaminations on the growth response of C. mucunoides are presented in Figure 1. The growth characteristics, namely, plant length, number of leaves and stem girth increased with fertilizer applications, but decreased with increase in crude oil contamination levels. From 8 WAP, the plant lengths were significantly (p<0.05) higher for 0% crude oil contamination among the fertilizer treatments; while significant difference was observed from 2 WAP for ≥ 2.5% soil contamination. Observations obtained in the plant lengths were similar to the number of leaves among the fertilizer treatments. However, significant (p<0.05) difference was observed in the stem girth of C. mucunoides from 6 WAP among different levels of crude oil contamination and fertilizer treatments. The order of increase in growth performance among the fertilizer treatments across the crude oil contamination levels was CM > NM > C1.

The higher the values of N and P in the fertilizers applied (Table 2), the higher was the growth performance of C. mucunoides. Makinde et al. [23] and Okokoh and Bisong [24] observed similar results of increased growth performance in a vegetable crop, Amaranthus hybridus with increase in organic N and P. Also, NM had higher organic N while CM had higher organic P in their available forms; and the possibility of good soil conditions created by organic manure in

the two fertilizers could account for good vegetative yield compared to CN plots. Whereas, in contaminated pots but without fertilizers, the physical properties of the crude oil may have imposed some stressful conditions which may interfere with water and gaseous exchange [9]. This may disrupts the normal growth of plant roots within the soil thereby reducing the normal physiological growth of the plant [25]. The non-germinability of C. mucunoides at 20% contamination may have resulted from the effect of toxicity of the crude oil to the embryo of the test crop. On the whole, the dry weights of C. mucunoides decreased with increase in soil contamination (Table 3).

Bioaccumulation of THC, Pb and Cd by Calopogonium mucunoides

The bioaccumulation of THC, Pb and Cd in the roots and shoots of C. mucunoides under different organic-based fertilizer applications are given in Table 4. The C. mucunoides remediated optimally, 1.04 × 10-2, 0.51 × 10-2, and 0.16 × 10-2 mg kg-1 of THC at 2.5%; 2.77 × 10-

2, 1.18 × 10-2, and 0.50 × 10-2 mg kg-1 of Pb at 5.0%; and 0.07 × 10-2, 0.04 × 10-2, and 0.02 × 10-2 mg kg-1 at 2.5% contamination with CM, NM, and C1 fertilizer applications, respectively. The increased organic manure in the soil brought about by the application of CM and NM fertilizers served as additional food for native micro-organisms to enhance their population and activities in the soil medium. Von Wedel et al. [26] earlier demonstrated that increased presence of mineral nutrients, either from organic or inorganic source enhanced the rate of microbial degradation of petroleum hydrocarbon, though in ground water. This also, agreed with the findings of Margesin et al. [27] that

CL (%)Root Shoot Root + Shoot

CM NM C1 CM NM C1 CM NM C10 6.3 ± 0.7a 4.7 ± 0.5a 3.0 ± 0.3a 8.7 ± 0.7a 7.2 ± 0.7a 3.6 ± 0.1a 15.0 ± 0.7a 11.9 ± 0.6a 6.6 ± 0.2a

2.5 3.2 ± 0.2b 2.0 ± 0.1b 1.4 ± 0.1b 5.5 ± 0.5b 4.6 ± 0.5b 1.4 ± 0.1b 8.7 ± 0.4b 6.6 ± 0.3b 2.8 ± 0.1b5.0 1.6 ± 0.2c 0.7 ± 0.1c 0.4 ± 0.1c 2.3 ± 0.2c 1.1 ± 0.2c 0.7 ± 0.1c 3.9 ± 0.2c 1.8 ± 0.1c 1.1 ± 0.1c

10.0 0.4 ± 0.1d 0.3 ± 0.0c 0.1 ± 0.0c 0.8 ± 0.2d 0.3 ± 0.1d 0.2 ± 0.0c 1.2 ± 0.2d 0.6 ± 0.1d 0.3 ± 0.0d20.0 - - - - - - - - -

Means followed by the same letter within a column are not significantly different at p < 0.05 by Duncan’s Multiple Range Test.CM = Compost organic fertilizer, NM = Neem fortified organic fertilizer, C1 = Control, THC = Total hydrocarbon, CL = Crude oil contamination levels.

Table 3: Mean (± standard error) dry weight (10-3 kg /plant) of roots and shoots of Calopogonium mucunoides.

CL (%) Root Shoot Root + ShootCM NM C1 CM NM C1 CM NM C1

THC0 0.15 ± 0.04 0.10 ± 0.03 0.07 ± 0.03 0.11 ± 0.04 0.13 ± 0.02 0.04 ± 0.01 0.13 ± 0.04a 0.12 ± 0.03a 0.05 ± 0.02b

2.5 1.08 ± 0.08 0.52 ± 0.08 0.21 ± 0.04 1.01 ± 0.05 0.51 ± 0.04 0.11 ± 0.04 1.04 ± 0.06a 0.51 ± 0.06b 0.16 ± 0.04c5.0 0.79 ± 0.10 0.40 ± 0.03 0.04 ± 0.01 0.65 ± 0.08 0.07 ± 0.02 0.02 ± 0.00 0.71 ± 0.09a 0.20 ± 0.02b 0.03 ± 0.00c

10.0 0.76 ± 0.08 0.29 ± 0.03 0.03 ± 0.00 0.41 ± 0.05 0.04 ± 0.01 0.00 ± 0.00 0.53 ± 0.07a 0.17 ± 0.02b 0.01 ± 0.00c20.0 - - - - - - - - -

Pb0 0.08 ± 0.02 0.06 ± 0.01 0.02 ± 0.00 0.05 ± 0.01 0.05 ± 0.01 0.02 ± 0.00 0.06 ± 0.01a 0.05 ± 0.01a 0.02 ± 0.00b

2.5 0.95 ± 0.06 0.61 ± 0.05 0.27 ± 0.02 0.87 ± 0.05 0.58 ± 0.02 0.11 ± 0.02 0.90 ± 0.06a 0.59 ± 0.04b 0.15 ± 0.02c5.0 2.57 ± 0.13 1.49 ± 0.11 0.37 ± 0.03 3.02 ± 0.18 0.98 ± 0.08 0.58 ± 0.05 2.77 ± 0.15a 1.18 ± 0.10b 0.50 ± 0.04c

10.0 2.26 ± 0.12 1.41 ± 0.11 0.35 ± 0.03 1.78 ± 0.10 0.94 ± 0.08 0.47 ± 0.03 2.11 ± 0.11a 1.18 ± 0.10b 0.43 ± 0.03c20.0 - - - - - - - - -

Cd0 0.02 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00b

2.5 0.09 ± 0.02 0.07 ± 0.01 0.03 ± 0.00 0.06 ± 0.01 0.02 ± 0.00 0.01 ± 0.00 0.07 ± 0.02a 0.04 ± 0.00a 0.02 ± 0.00b5.0 0.05 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.03 ± 0.00a 0.00 ± 0.00b 0.00 ± 0.00b

10.0 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a20.0 - - - - - - - - -

Table 4: Mean value (±standard error) of bioaccumulation of THC, Pb and Cd (10-2 mg kg-1) in the roots and shoots of Calopogonium mucunoides.



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carbon addition through organic nutrient supplement increased the ability of soil microbes to degrade crude oil. As more hydrocarbons are degraded, there will be sufficient carbon and energy to support large numbers of soil microbes.

The binding force that brought the soil components together to make its biodegradation difficult may have been found easy to break in this experiment, by these active native soil micro-organisms. This broken binding force positively enhanced the removal rates of THC, Pb, and Cd when CM and NM were applied. This was because the increased microbial activity in oil-contaminated soil due to organic manures application, increased the availability of soil nutrients, including heavy metals such as Pb and Cd. Pots without organic fertilizer application had significantly (p < 0.05) least values of THC (0.16 × 10-2 mg kg-1), Pb (0.50 × 10-2 mg kg-1), and Cd (0.02 × 10-2 mg kg-1) remediated. Parreira et al. [8] observed similar significant increase in the biodegradation and uptake of gasohol-contaminants due to increased microbial consortium brought about by organic manure application.

Translocation factor of THC, Pb and Cd in Calopogonium mucunoides

The translocation factors (TF) of THC, Pb and CD in the roots and shoots of C. mucunoides under different organic-based fertilizer applications are given in Table 5. The TF for THC, Pb and Cd increased when the soil contamination went up to 2.5% from zero and decreased thereafter from ≥ 5.0% in all the fertilizer applications. When the TF of THC, Pb and Cd were separately compared with the applied fertilizers, compost organic fertilizer was most effective, while control was the least at different levels of crude oil soil contamination.

ConclusionThe organic-based fertilizers enhanced the growth performance

of C. mucunoides in crude oil polluted soils. The biodegradation and bioaccumulation of petroleum hydrocarbon are also enhanced with organic-based fertilizer applications. However, compost organic fertilizer performed better in the bioaccumulation of the total petroleum hydrocarbon, Pb and Cd than either neem fortified organic fertilizer or zero fertilization when C. mucunoides was the test phytoplant.

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Fertilizer CL (%) THC CM Pb Cd THC NM Pb Cd THC C1 Pb Cd0 0.690 0.256 2.000 0.271 0.123 0 0.571 0.294 0

2.5 0.990 0.916 0.667 0.981 0.951 0.286 0.524 0.407 0.3335.0 0.855 1.444 0.333 0.175 0.658 0 0.500 1.568 0

10.0 0.519 1.336 0 0.138 0.667 0 0 1.343 020.0 - - - - - - - - -

CM = Compost organic fertilizer, NM = Neem fortified organic fertilizer, C1 = Control, THC = Total hydrocarbon, CL = Crude oil contamination levels.

Table 5: Translocation factor of THC, Pb and Cd in Calopogonium mucunoides.

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