biostimulation and phytoremediation treatment strategies of gasoline-nickel co-contaminated soil

Soil and Sediment Contamination, 23:227–244, 2014Copyright © Taylor & Francis Group, LLCISSN: 1532-0383 print / 1549-7887 onlineDOI: 10.1080/15320383.2014.812612

Biostimulation and Phytoremediation TreatmentStrategies of Gasoline-Nickel Co-Contaminated Soil

S. E. AGARRY, M. O. AREMU, AND O. A. AWORANTI

Biochemical Engineering and Biotechnology Laboratory, Department ofChemical Engineering, Ladoke Akintola University of Technology,Ogbomoso, Nigeria

This study investigated the potential effect of poultry dung (biostimulation) and stub-born grass (Sporobolus pyramidalis) (phytoremediation) on microbial biodegradationof gasoline and nickel uptake in gasoline-nickel-impacted soil. In addition, the potentialstimulatory effects of nickel on hydrocarbon utilization were investigated over a smallrange of nickel concentrations (2.5–12.5 mg/kg). The results showed that an increase innickel concentration increased hydrocarbon degraders in soil by a range of 8.4–17.2%and resulted in a relative increase in gasoline biodegradation (57.5–62.4%). Also, un-der aerobic conditions, total petroleum hydrocarbons’ (TPH) removal was 62.4% inthe natural gasoline-nickel microcosm (natural attenuation), and a maximum of 78.5%,85.7%, and 95.8% TPH removal was obtained in phytoremediation, biostimulation,and a combination of biostimulation- and phytoremediation-treated microcosms, re-spectively. First-order kinetics described the biodegradation of gasoline and nickeluptake very well. Half-life times obtained were 28.88, 18.24, 14.44, and 8.56 days forgasoline degradation under natural attenuation, phytoremediation, biostimulation, andcombined biostimulation and phytoremediation treatment methods, respectively. Theresults indicate that these remediation methods have promising potential for effectiveremediation of soils co-contaminated with petroleum hydrocarbons and heavy metals.

Keywords Biostimulation, gasoline, natural attenuation, phytoremediation, kinetics

Introduction

Environmental pollution has become a very important worldwide problem in recent years,influencing health, economic, and political issues (Myriam et al., 2005), and has assumedan unprecedented proportion (Bank et al., 2003), especially in Nigeria. The Niger Deltaregion of Nigeria has been associated with frequent oil spills resulting from petroleumexploration, exploitation, and production activities, oil pipeline vandalization, tanker ac-cidents, and accidental rupture of oil pipelines (Tanee and Kinako, 2008; Efe and Ok-pali, 2012). Sites co-contaminated with organic and metal pollutants are common andconsidered to be a more complex problem as the two components often cause a syner-gistic effect on cytotoxicity. Nickel is a metal-contaminating soil, especially in the vicin-ity of industrial sites where concentration can be as high as 40–4600 ug/g soil (Paton

Address correspondence to S.E. Agarry, Biochemical Engineering and Biotechnology Labora-tory, Department of Chemical Engineering, Ladoke Akintola University of Technology, Ogbomoso,Nigeria. E-mail: sam [email protected] or [email protected].

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228 S. E. Agarry et al.

et al., 2006; Robinson et al., 2008). Nickel has toxic effects on humans (Riley et al.,2005), plants, soil microflora, and fauna (Yuangen et al., 2006; Gikas, 2008). In addition,nickel (II) ions can replace nutrient cations (Ca2+, mg2+, and mn2+) from the cation ex-change places of forest soil and thus decrease the amount of available nutrients (Deromeand Lindroos, 1998). Therefore, for possible elimination of these effects, it is impera-tive to clean up these pollutants from the environment by applying remedial measures(Ellis et al., 1990).

Bioremediation has emerged as a highly promising secondary treatment option for oilremoval since its first application after the 1989 Exxon Valdez spill (Bragg et al., 1994).According to Sylvia et al. (2005), the term “bioremediation” can be used for any pro-cess that uses microorganisms, green plants, or their enzymes to return the environmentaltered by contaminants to its original condition. Several methods are used for petroleumoil bioremediation. These include biostimulation, bioaugmentation, and phytoremedia-tion. Phytoremediation is a broad term expressing the use of plants to remove, contain,or transform contaminants. Over the last few years, phytoremediation has been shownto be a promising approach due to its low cost and environmentally benign nature foraddressing the decontamination of both organic and inorganic contaminants in the soil(McClutchen and Schnoor, 2003; Sylvia et al., 2005; Kaimi et al., 2007; Shirdam et al.,2008; Zhang et al., 2009). Most studies on phytoremediation of contaminated soil havebeen on a single-pollutant system (Aprill and Sims, 1990; Schwab et al., 2006; Kechavarziet al., 2007; Basumatary et al., 2012). Reports in the literature on phytoremediation of soilco-contaminated with petroleum hydrocarbons and heavy metals are very limited. In thephytoremediation of organic pollutants based on microbial degradation in the rhizosphere,fertilization is essential for success to reduce competition between plants and microor-ganisms for limited nutrients in polluted soil. Only a very few workers have made use ofinorganic chemical fertilizer as an amendment agent to improve phytoremediation in theenhancement of petroleum hydrocarbon degradation (Hutchinson et al., 2001; Shtangeeva,2004; Efe and Okpali, 2012).

In developing countries like Nigeria, the uses of inorganic chemical fertilizers are notheavily subsidized as compared to Western Europe, and also are not sufficient for agri-culture, let alone for cleaning oil spills. Therefore, there is the need to search for cheaperand environmentally friendly options of enhancing soil fertility for petroleum hydrocarbondegradation. One such option is the use of livestock organic wastes (animal dung). Fewworkers have investigated the potential use of these livestock wastes like cow dung, pigdung, poultry manure, and goat dung (Okolo et al., 2005; Adesodun and Mbagwu, 2008;Singh and Fulekar, 2009; Agarry et al., 2010a) as biostimulating agents in the clean-up ofsoil contaminated alone with petroleum hydrocarbons. These were found to show positiveinfluence on petroleum hydrocarbon biodegradation in a polluted environment. Further-more, only a very few workers have reported the potential use of these livestock wastesas biostimulating agents for the bioremediation of soil co-contaminated with petroleumhydrocarbons and heavy metals (Agarry and Ogunleye, 2012). Thus, to the best of ourknowledge there is a dearth of information on the use of livestock manure as an amendmentagent to support or improve phytoremediation in enhancing the bioremediation of soilsco-contaminated with petroleum hydrocarbons and heavy metals. Also, the evaluation andcomparison of livestock-derived organic waste (e.g., poultry dung) and the use of plantsto remediate soil co-contaminated with organic pollutant and heavy metal have not beenreported in the literature.

The overall goal of this study is to investigate the concurrent removal of gasolineand nickel in co-contaminated soil using poultry dung and stubborn grass (Sporobolus

Gasoline-Nickel Co-Contaminated Soil 229

pyramidalis) alone and/or in combination as agents of biostimulation and phytoremediation,respectively, in the mitigation of gasoline-nickel toxicity in soil. This study also attemptsto examine the effect of low concentration of nickel on gasoline degradation in soil and toevaluate and compare the use of biostimulation and phytoremediation through determiningthe kinetics of gasoline biodegradation as well as nickel uptake.

Materials and Methods

Collection of Samples

The soil sample used for the study was collected from the top surface soil (0–15 cm) ofLadoke Akintola University of Technology (LAUTECH) agricultural farm land, Ogbomoso,Nigeria. The soil samples were air-dried, homogenized, passed through a 2-mm (pore size)sieve, stored in a polyethylene bag, and kept in the laboratory prior to use. The gasolineoil was obtained from a commercial gasoline station in Ogbomoso, Nigeria. The poultrydung (PD) used as soil amendment was obtained from the poultry farm of LAUTECH,Ogbomoso, Nigeria. The poultry dung was sun-dried for two weeks, ground, and sievedto obtain uniform-size particles (0.0625 mm) that were the same size as the sample soil,which was then stored in a polyethylene bag and kept prior to use. The stubborn grass(Sporobolus pyramidalis) was obtained from the vicinity of LAUTECH farm. This wasselected based on certain features thought to enhance phytoremediation. These include theability of the plant to survive adverse environmental conditions, fibrous root system, andhigh rate of evapo-transpiration to enhance phytostabilization. Sporobolus pyramidalis (S.pyramidalis) is a coarse, tufted, perennial stubborn grass with a strong root-system andunbranched culms that can attain a height of 0.6 to 0.7 m, and can grow well in sunny orsomewhat shaded places.

Characterization of Soil Sample and Amendment Agents

The soil samples were characterized for total carbon (TOC), total nitrogen (N), total phos-phorus, moisture content, and pH according to standard methods. Soil pH was determinedaccording to the modified method of McLean (1982); total organic carbon was determinedby the modified wet combustion method (Nelson and Sommers, 1982) and total nitrogenwas determined by the semi-micro-Kjeldahl method (Bremner and Mulvaney, 1982). Avail-able phosphorus was determined by Brays No. 1 method (Olsen and Sommers, 1982) andmoisture content was determined by the dry weight method. The total hydrocarbon de-grading bacteria (THDB) populations were determined by the vapor phase transfer method(Amanchukwu et al., 1989). The physicochemical and microbiological characterized pa-rameters are presented in Table 1.

Identification of Soil Microorganisms

The soil samples were subjected to serial dilution and plated on oxoid nutrient-agar (NA)using spread plate technology and incubated at room temperature for 24 h, as described byCruickshank et al. (1980). Plates that yielded 30 to 300 colonies of bacterial isolates wereaccepted. After incubation, isolated bacterial species were subjected to morphological,cultural, and biochemical tests, as reported by Cruickshank et al. (1980) and Gerhardt et al.


Table 1Soil sample and poultry dung physicochemical and microbiological analysis

Parameter Soil Poultry Dung

Organic carbon (%) 10.01 ± 0.03 40.94 ± 0.03Total nitrogen (%) 0.37 ± 0.05 4.33 ± 0.01Phosphorus (%) 0.11 ± 0.02 1.28 ± 0.04pH 7 ± 0.1 6.7. ± 0.1Moisture (%) 10.2 ± 0.04 8.8 ± 0.01Total hydrocarbon-degrading bacteria

(THDB) (cfu/g)0.02 ± 1.88 × 106 0.03 ± 5.82 × 106

Note: Each value is a mean of three replicates and ± indicates standard deviation among them.

(1981), to identify them. The microorganisms present in the LAUTECH agricultural soilwere identified to be made up of mainly Bacillus and Pseudomonas species.

Experimental Soil Treatment Design

Soil samples (4 kg) was put into seven different plastic bins (microcosm) with a volume ofabout 3 L and labeled T1 to T8, respectively. The soil in plastic bin T8 was sterilized threetimes by autoclaving at 121◦C for 15 minutes. The soil in each microcosm was spiked with10% (w/w) gasoline and was thoroughly mixed using a short iron rod to achieve completeartificial contamination. A total of 10% spiking was adopted in order to achieve severecontamination because, above 3% concentration, oil has been reported to be increasinglydeleterious to soil biota and crop growth (Osuji et al., 2005). After pollution, the gasoline-contaminated soil was left for one week to allow for weathering before the differentremediation treatments were applied. A weight of the nickel salt that gave a corresponding,2.5, 7.5, and 12.5 mg/kg of nickel heavy metal was weighed and dissolved in 100 mlof deionized water. These were left to stand for 30 min to obtain complete dissolution.Thereafter, contents in each microcosm T2, T3, and T4 were further spiked with 2.5, 7.5,and 12.5 mg/kg of nickel, respectively, and thoroughly mixed together using a short ironrod. To the contents in microcosms T5, T6, T7 and T8 was added 12.5 mg/kg nickel each,while stubborn grass was transplanted into microcosm T5 (phytoremediation treatment),20 g of poultry dung was added to microcosm T6 (biostimulation treatment), and 20 g ofpoultry dung and stubborn grass (Sporobolus pyramidalis) was added to microcosm T7(combined biostimulation and phytoremediation treatment), respectively. The contents inmicrocosms T1 and T8 had no poultry dung amendment and no stubborn grass planted,both of which served as control A and control B, respectively. The experimental procedureis presented in Table 2. The microcosms were covered with aluminum foil. The moisturecontent was adjusted to 50% water-holding capacity by the addition of sterile distilled waterand incubated at room temperature (28 ± 2◦C). The content of each microcosm was tilledtwice a week for aeration, and the moisture content was maintained at 50% water-holdingcapacity. The experiment was set up in triplicate. In total, 24 microcosms were settled andincubated for six weeks (42 days). Periodic sampling from each microcosm was carried outat seven-day intervals for 42 days to determine the residual total petroleum hydrocarbon(TPH) and residual nickel heavy metal, respectively.


Table 2Remediation treatment of gasoline and nickel co-contaminated soil

Microcosm Code Remediation Treatment and Description

T1 (Control A) 4 kg Soil + 400 g GasolineT2 4 kg Soil + 400 g Gasoline + 2.5 mg/kg NickelT3 4 kg Soil + 400 g Gasoline + 7.5 mg/kg NickelT4 4 kg Soil + 400 g Gasoline + 12.5 mg/kg NickelT5 4 kg Soil + 400 g Gasoline + 12.5 mg/kg Nickel + Stubborn

grassT6 4 kg Soil + 400 g Gasoline + 12.5 mg/kg Nickel + 20 g

Poultry dungT7 4 kg Soil + 400 g Gasoline + 12.5 mg/kg Nickel + Poultry

dung + Stubborn grassT8 (Control B) 4 kg Autoclaved Soil + 400 g Gasoline + 12.5 mg/kg Nickel

Total Petroleum Hydrocarbon Determination

The total petroleum hydrocarbon content of the soil samples was determined gravimetricallyby a modified form of the Adesodun and Mbagwu (2008) solvent extraction method. Soilsamples (approximately 10 g) were taken from each microcosm and put into a 50-ml flaskand 20 ml of n-hexane was added. The mixture was shaken vigorously on a magnetic stirrerfor 30 min to allow the hexane to extract the oil from the soil sample. The solution was thenfiltered using a Whatman filter paper and the liquid phase extract (filtrate) diluted by taking1 ml of the extract into 50 ml of hexane. The absorbance of this solution was measuredspectrophotometrically at a wavelength of 400 nm HACH DR/2010 spectrophotometerusing n-hexane as blank. The total petroleum hydrocarbon in soil was estimated withreference to a standard curve derived from fresh lubricating oil of different concentrationsdiluted with n-hexane. The TPH concentrations were expressed as mg of hydrocarbons perkg of dry soil.

Determination of Nickel Concentration

The soil-contaminated samples were digested using the wet oxidation method and the nickelmetal present was determined using an atomic absorption spectrophotometer (UNICAM929 AA Spectrometer). A total of 5 g of air-dried ground contaminated soil sample wastransferred to a 25-ml conical flask and 5 ml of concentrated H2SO4 was added, followedby 25 ml of concentrated HNO3, and 5 ml of concentrated HCl, respectively. The content ofthe flask was heated at 200◦C for 1 h in a fuming hood and then cooled to room temperature.After cooling, 20 ml of deionized water was added and the mixture was filtered to completethe digestion. Finally, the mixture was transferred to a 50 ml volumetric flask, filled to themark, and allowed to settle for at least 15 h. The filtrate was analyzed for available nickel(Ni2+) by atomic absorption spectrophotometer

Biodegradation Kinetics and Estimation of Half-life Time

Kinetic analysis is a key factor for understanding biodegradation process, bioremediationspeed measurement, and development of efficient clean-up for an organic chemical


contaminated environment. Biodegradability of hydrocarbon can be explained byfirst-order kinetics (Agarry et al., 2010a; Zahed et al., 2011) and this is given as in Eq. (1)

Ct = Coe−kt (1)

Similarly, the uptake of heavy metal from the soil can also be explained by first-orderkinetics, where Co is the initial gasoline or nickel content in soil (mg/kg), Ct is the residualgasoline or nickel content in soil at time t (mg/kg), k is the biodegradation rate constant ormetal uptake rate constant (day−1) and t is time (day).

The biodegradation half-life is the time taken for a substance to lose half of its amount.Biodegradation half-life times (t1/2) are calculated using Eq. (2) (Zahed et al., 2011)

t1/2 = ln 2

k(2)

where k is the biodegradation rate constant (day−1). The half-life model is based on theassumption that the biodegradation rate of hydrocarbons positively correlated with thehydrocarbon pool size in soil (Yeung et al., 1997).

Data Analysis

The data were subjected to one-way analysis of variance (ANOVA) at 5% probability.Means of the different treatments were tested for level of significant differences at P = 0.05by Tukey (Honestly Significant Difference) test. The data analysis was performed using astatistical package for social sciences, version 16.0 (SPSS Inc., Chicago, IL, USA).

Results and Discussion

Soil Properties

Trindade et al. (2005) suggested that the optimum ratio of carbon to nitrogen to phos-phorous (C:N:P) was 100:1.25:1 for bioremediation of hydrocarbon-contaminated soils.Table 1 shows that both total nitrogen and available phosphorus were seriously deficientin the contaminated soil before bioremediation, hence the need to amend the soil with nu-trient. When the population of indigenous microorganisms capable of degrading the targetcontaminant is less than 105 colony-forming units (cfu) g−1 of soil, bioremediation willnot occur at a significant rate (Hinchee et al., 1995; Liu et al., 2010). It can be seen fromTable 1 that the population of hydrocarbon-degrading bacteria in the soil is sufficient forbioremediation to occur at a significant rate.

Effect of Nickel on Gasoline Biodegradation

The biodegradation of gasoline increased with increase in remediation time, as depicted inFigures 1 and 2, respectively. This indicates that the complete biodegradation or mineraliza-tion of any organic pollutant is time-dependent. Figure 1a shows the effect of nickel on thebiodegradation of gasoline in soil. It is seen that the concentration of TPH in gasoline-nickelsoil microcosms T2, T3, and T4 co-contaminated with 2.5, 7.5, and 12.5 mg/kg nickel con-centration was reduced from 99,699, 99,760, and 99,765 mg/kg to 42,272, 39,405, and37,512 mg/kg of dry soil, respectively, which corresponded to 57.6%, 60.5%, and 62.4%gasoline degradation, respectively. The TPH concentration was reduced from 99,897 to


Figure 1. Effect of nickel on gasoline biodegradation: (a) time course of gasoline biodegradation;(b) time course of nickel uptake. Bars indicate the average of triplicate samples while the error barsshow the standard deviation. (Color figure available online).

47,251 mg/kg of dry soil in gasoline-soil microcosm T1 (Control A), and this correspondedto 52.7% gasoline degradation. Thus, the TPH percentage degradations in T2, T3, and T4were significantly higher (P = 0.05) than that in T1 (control A) at the end of the remedi-ation time (42 days), indicating that the presence of nickel had increased and stimulatedTPH degraders in the soil. It can therefore be seen that gasoline biodegradation increasedwith increase in nickel concentration used in this study. Nevertheless, this observation isin contrast with the observation reported by Al-Saleh and Obuekwe (2009). They reportedan inhibitory effect of nickel on the mineralization of petroleum hydrocarbons in Kuwait


Figure 2. Effect of biostimulation and phytoremediation on gasoline-nickel removal: (a) time courseof gasoline biodegradation; (b) time course of nickel uptake. Bars indicate the average of triplicatesamples while the error bars show the standard deviation. (Color figure available online).

soil. Previous studies have shown that in heavy metal-organic pollutant combined systems,biodegradation of organic contaminants is often severely inhibited by toxic metals (Maslinand Maier, 2000; Said and Lewis, 1991). However, in some cases, the addition of metals hasbeen observed to stimulate microbial activity (Zhang et al., 2009) and thereby stimulatedbiodegradation even at low concentrations (Kuo and Genthner, 1996).

Furthermore, the TPH concentration in gasoline-nickel autoclaved soil microcosm T8(control B) marginally attained a percentage degradation of 2.6%. The marginal reductionof TPH concentration observed in T8 (control B) may be due to abiotic degradation ofgasoline and evaporation losses or volatilization. A similar observation has been reported


for kerosene biodegradation (Shabir et al., 2008; Agarry et al., 2010b) and for degradationof lubricating oil (Agarry et al., 2013). This observation indicates the role and importanceof intrinsic or autochthonous microorganisms in the biodegradation process of organiccontaminants present in the environment. Thus, the efficiency of bioremediation is a functionof the microbial viability in the natural environment (Joo et al., 2008; Agarry et al., 2013).

On the other hand, Figure 1b shows that the nickel concentration in the unamendedgasoline–nickel-contaminated microcosms T2, T3, and T4, reduced from 2.5, 7.5, and12.5 mg/kg (day 0) to corresponding values of 0.35, 1.45, and 3.0 mg/kg (day 42) equiva-lent to 86%, 80.7%, and 76% nickel uptake, respectively. This indicated that the intrinsicmicrobial species in the soil has great potential in bioaccumulating the nickel metal asso-ciated with the gasoline. However, no reduction in nickel concentration was observed inthe autoclaved gasoline-nickel soil microcosm T8 (control B). This observation revealedthe role and importance of intrinsic microorganisms in the uptake of heavy metals such asnickel from heavy-metal-contaminated soil.

Effect of Biostimulation and Phytoremediation on Gasoline Biodegradationand Nickel Uptake

Figure 2 shows the effect of biostimulation and phytoremediation on the biodegradation ofgasoline and nickel uptake in soil microcosms co-contaminated with gasoline and nickel. Itcan be seen that the TPH concentration decreased with time. Initially, TPH concentrationwas 99, 765, 99,762, 99,689, and 99,764 mg/kg dry soil in gasoline-nickel soil microcosmsT4 (natural attenuation), T5 (phytoremediation), T6 (biostimulation), and T7 (combinedbiostimulation and phytoremediation), respectively. After 42 days of bioremediation, itwas reduced to 37,512, 21,444, 14,256, and 4,190 mg/kg dry soil, which is equivalent to aTPH percentage degradation of 62.4%, 78.5%, 85.7%, and 95.8% in T4, T5, T6, and T7,respectively (Figure 2a).

The higher TPH percentage degradation observed in stubborn grass vegetated gasoline-nickel soil microcosms T5 (phytoremediation) over the unvegetated gasoline-nickel soilmicrocosms T4 (natural attenuation) is in agreement with the findings of Aprill and Sims(1990), Ayotamuno et al. (2006), Muratova et al. (2008), Peng et al. (2009), and Basumataryet al. (2012), who all observed higher petroleum hydrocarbon reduction in planted (vege-tated) petroleum hydrocarbon-impacted soil than in unplanted soil. This observation showedthat stubborn grass was able to stimulate the intrinsic microorganisms in the soil by in-creasing the microbial activities as a result of increased microbial population. Thus, thepromotion of hydrocarbon degradation by stubborn grass (Sporobolus pyramidalis) maybe due to the complexity of plant-microorganisms interactions, which is similar to thefindings of Liste and Prutz (2006), Muratova et al. (2008), and Basumatary et al. (2012),respectively. The enhanced degradation or removal of persistent organic pollutants in soilsby plants has been widely researched and could be attributed to the following phenomena,such as: (1) increased microbial activity in the rhizosphere soil; (2) plant uptake, thoughplant uptake might be minimal, as reported by Nakamura et al. (2004) and He et al. (2005);(3) degradation mediated by the enzymes secreted by plants in the root zone. Kaimi et al.(2006) found that the number of aerobic bacteria and the amount of soil dehydrogenaseactivity in planted soil were higher than that in unplanted soil and also showed a correlationwith the growth of roots. The degradation rate of diesel oil showed a correlation with soildehydrogenase activity in both planted and unplanted soil.

Also, the higher TPH percentage biodegradation observed in poultry dung amended-gasoline-nickel soil microcosms T6 (biostimulation) over that obtained in the unamended


Table 3aAnalysis of variance (ANOVA) for the different treatments based on TPH

Source Sum of squares Degree of freedom Mean of squares F-value P-value

Treatment 1.64×1010 4 4.10×109 7.07×108 3.31×10−42

Error 58 10 5.8Total 1.64×1010 14

gasoline-nickel soil microcosms T4 (natural attenuation), as depicted in Figure 2, indicatesthat the biodegradation rate of gasoline was enhanced by the poultry dung amendment.Similar observations have been reported for the use of animal manure, such as cow dung,pig dung, goat dung, and poultry dung, in the biodegradation of organic pollutants (Okoloet al., 2005; Adesodun and Mbagwu, 2008; Singh and Fulekar, 2009; Liu et al., 2010;Agarry et al., 2010a; Agarry and Owabor, 2011).

Furthermore, the higher TPH percentage degradation observed in stubborn grassvegetated/poultry-dung-amended gasoline-nickel microcosm T7 (combined biostimulationand phytoremediation) over the unamended and/or unvegetated gasoline-nickel soil micro-cosms T4 (natural attenuation) (Figure 2) revealed that the rate of gasoline biodegradationwas greatly increased as a result of combined provision of nutrients/microbial biomass bypoultry dung as well as the exudates secreted by the grass in the root zone. Generally, theobserved TPH percentage biodegradation was highest in the remediation treatment of soilmicrocosms amended with poultry dung and vegetated with stubborn grass T7 (combinedbiostimulation and phytoremediation). This was followed by remediation treatment in soilmicrocosms amended with poultry dung alone T6 and soil microcosm phytoremediatedalone with stubborn grass T5.

A one-way ANOVA analysis was conducted to compare the bioremediation efficiencyof the natural attenuation, biostimulation, and phytoremediation methods and the resultsare presented in Table 3a. The results in Table 3a suggest that natural attenuation (T4),phytoremediation (T5), biostimulation (T6), and combined biostimulation and phytoreme-diation (T7) had a statistically significant effect on the biodegradation of gasoline in soilat the 5% probability level (P = 0.05). Post-hoc comparisons using Tukey’s (HSD) testat 5% probability level were carried out to actually determine the significant differencein bioremediation efficiency between the treatment methods, and the results are shown inTable 4a. The grouping of TPH mean using the Tukey’s test for the different treatmentmethods revealed a much significant differences between the treatment methods.

Similarly, Figure 2b shows that the nickel concentration reduced from 12.5 mg/kg (day0) to 0.6, 1.6, and 0 mg/kg (day 42) and this corresponded to 95.2%, 87.2%, and 100%nickel uptake in the gasoline-nickel contaminated microcosms T5 (phytoremediation), T6

Table 3bAnalysis of variance (ANOVA) for the different treatments based on nickel uptake

Source Sum of squares Degree of freedom Mean of squares F-value P-value

Treatment 316.52 4 79.13 3596 9.70×10−16

Error 0.22 10 0.022Total 316.74 14


Table 4aGrouping of TPH mean for the different treatments computed by Tukey’s method

Microcosm Treatments TPH Mean (mg/kg) Standard Deviation

T8 Control B 97448A 2T4 Natural attenuation 37512B 2T5 Phytoremediation 21449C 1T6 Biostimulation 14256D 2T7 Combined Biostimulation and

Phytoremediation4190E 4

Means that do not share the same letter are significantly different.

(biostimulation), and T7 (combined biostimulation and phytoremediation), respectively.The level of nickel uptake was 76% in the unamended gasoline-nickel contaminated mi-crocosm T4 (natural attenuation). These observations indicated that phytoremediation,biostimulation, and combined biostimulation and phytoremediation enhanced the uptakeof nickel from the gasoline-nickel-contaminated soil. Enhancement of the nickel uptakewas as a result of increase in the microbial activities supported by nutrients and biomassprovided by the poultry dung as well as the exudates provided by the stubborn grass andits metal-accumulating ability. Agarry and Ogunleye (2012) have reported a similar ob-servation for the uptake of hexavalent chromium in a spent-engine-oil-contaminated soilusing pig dung and NPK fertilizer as biostimulating agents. Also, the use of NPK fertil-izer and urea as biostimulating agents to enhance heavy metal uptake by microorganismsin a heavy-metal/petroleum-hydrocarbon-contaminated soil has been reported (Odokumaand Akpona, 2010; Agarry et al., 2013). However, no decrease in nickel concentrationwas observed in the autoclaved gasoline-nickel soil microcosm (control B). A similar ob-servation has been reported for the uptake of lead in a lubricating-oil/lead-contaminatedsoil microcosm (Agarry et al., 2013). This observation indicates the potential of intrinsicmicroorganisms (such as Bacillus and Pseudomonas species present in the LAUTECHfarm soil) in the uptake or accumulation of heavy metals such as nickel from heavy-metal-contaminated soil. Bacillus and Pseudomonas species have been identified as bacteria thathave the ability to bioaccumulate or bioconcentrate heavy metals (Odokuma and Akponah,2010; Agarry and Ogunleye, 2012; Agarry et al., 2013).

A one-way ANOVA statistical analysis performed to compare the nickel uptake effi-ciency in natural attenuation (T4), biostimulation and phytoremediation methods (T5, T6,and T7) suggests that natural attenuation (T4), biostimulation (T6), phytoremediation (T5),and combined biostimulation and phytoremediation (T7) had a statistically significant ef-fect on the nickel uptake from gasoline-nickel-contaminated soil at the 5% probability level(P = 0.05) (Table 3b). In addition, post-hoc comparisons using Tukey’s (HSD) test at the5% probability level that were carried out to actually determine the significant difference innickel uptake efficiency between the treatment methods showed there are many significantdifferences between the treatment methods (Table 4b).

Effect of Nickel, Biostimulation, and Phytoremediation on Microbial Growth

The growth profile of intrinsic microorganisms in unamended gasoline and gasoline-nickelsoil microcosms is shown in Figure 3a. Figure 3a revealed that THDB count in the soil


Figure 3. Growth profile of total hydrocarbon degrading bacteria (THDB) in (a) unamended gasolineand gasoline-nickel soil microcosms; (b) unamended and amended gasoline-nickel microcosms. Barsindicate the average of triplicate samples while the error bars show the standard deviation. (Colorfigure available online).

increased with the presence of nickel as compared with soil microcosm T1 (control A)not contaminated with nickel and that the THDB growth increased with an increase inthe nickel concentration (2.5 to 12.5 mg/kg). The THDB exhibited a 60.8%, 64.2%, and69.3% growth increase in gasoline-soil microcosms T2, T3, and T4 co-contaminated with2.5, 7.5, and 12.5 mg/kg nickel concentration, respectively. The gasoline-contaminated soil(control A) had a 53.5% THDB growth increase. Nevertheless, no growth was observed inautoclaved gasoline-nickel soil microcosm (control B). Generally, the presence of heavymetals in the microbial environment has a negative effect on microbial growth rate (Madoniet al., 1996; Leduc et al., 1997). However, trace amounts of selected heavy metals are oftenbeneficial to the microbial growth (Wood and Tchobanoglous, 1975; Gokcay and Yetis,1991). Congeevaram et al. (2007) observed that Aspergillus species isolated from nickel (II)


Table 4bGrouping of nickel uptake mean for the different treatments computed by Tukey’s method

Microcosm Treatments TPH Mean (mg/kg) Standard Deviation

T8 Control B 12.5A 0T4 Natural attenuation 3.0B 0.3T5 Phytoremediation 0.6D 0.1T6 Biostimulation 1.6C 0.1T7 Combined Biostimulation and

Phytoremediation0.0E 0

Means that do not share the same letter are significantly different.

ions-contaminated soil was able to grow in the presence of 50–500 mg/l nickel concentrationin liquid culture but the growth decreased above 100 mg/l nickel concentration; and it wasobserved by Lankinen et al. (2011) that basidiomycetous litter-degrading fungi were ableto grow in a 20 mg/kg nickel-contaminated soil.

Figure 3b shows the growth profiles of microorganisms in the gasoline-nickel micro-cosms subjected to natural attenuation, biostimulation, and phytoremediation treatments.It can be seen that in each of the gasoline-nickel microcosm treatments, the microbialpopulation (as depicted by the THDB count) increased with time. The THDB showeda growth increase of 79.8%, 87.5%, and 97.1% for stubborn grass vegetated gasoline-nickel microcosm (T5), poultry-dung-amended gasoline-nickel microcosm (T6) and stub-born grass vegetated/poultry-dung-amended gasoline-nickel microcosm (T7), respectively.Meanwhile, in the unamended and/or unvegetated gasoline-nickel microcosm (T4), theTHDB revealed a 69.3% growth increase. These observations showed that the populationsof hydrocarbon degraders in the treatment soil microcosms (T5, T6, and T7) were signifi-cantly higher (P = 0.05) than in the gasoline-nickel soil microcosm T4 (natural attenuation)at the end of the remediation trial (42 days), indicating that amendment with poultry dungand vegetation with stubborn grass had increased and stimulated TPH degraders in the soil.A similar observation has been reported by Wang et al. (2008) and Basumatary et al. (2012),respectively, that microbial numbers in the planted soil petroleum hydrocarbon impactedsoil were significantly higher than the unplanted petroleum hydrocarbon-contaminated soil.Similarly, it has also been reported that petroleum-contaminated soil amended with poultry

Table 5aBiodegradation rate constant (k1), correlation coefficient (R2), and half-life time (t1/2)

obtained for gasoline biodegradation in gasoline-nickel soil bioremediation

Microcosm Remediation Treatmentk1

(day−1) R2 t1/2(days)

T4 Natural attenuation 0.024 0.990 28.88T5 Phytoremediation 0.038 0.911 18.24T6 Biostimulation 0.048 0.942 14.44T7 Combined

Biostimulation andPhytoremediation

0.081 0.950 8.56


Table 5bNickel uptake rate constant (k2), correlation coefficient (R2), and half-life time (t1/2) ob-

tained for nickel uptake in gasoline-nickel soil bioremediation

Microcosm Remediation Treatment k2(day−1) R2 t1/2 (days)

T4 Natural attenuation 0.035 0.974 19.80T5 Phytoremediation 0.078 0.983 8.89T6 Biostimulation 0.052 0.985 13.33T7 Combined

Biostimulation andPhytoremediation

0.121 0.986 5.73

manure promotes the growth of indigenous degraders in it (Rahman et al., 2002; Liu et al.,2010).

Kinetics and Half-life of Gasoline Biodegradation and Nickel Metal Uptake

The first-order kinetics model equation (Eq. (1)) fitted to the gasoline biodegradation andnickel uptake data was used to determine the rate of gasoline biodegradation and nickeluptake in the various remediation treatments. The biodegradation rate constants (k1) andnickel uptake rate constant (k2) for the different remediation treatments are presented in Ta-bles 5a and b. It can be seen from Table 5a that the soil microcosms vegetated with stubborngrass and amended with poultry dung T7 (combined biostimulation and phytoremediation)had a higher gasoline biodegradation rate constant than others, closely followed by that ofsoil microcosm amended with poultry dung T6 (biostimulation), stubborn grass T5 (phy-toremediation), and unamended gasoline-nickel soil microcosms T4 (natural attenuation),respectively. Also, from Table 5b, gasoline-nickel soil microcosms amended with the com-bination of poultry dung and vegetated with stubborn grass T7 (combined biostimulationand phytoremediation) showed a higher nickel uptake rate constant than other forms of soiltreatments. This was relatively followed by gasoline-nickel soil microcosm vegetated withstubborn grass T5, poultry dung T6, and unamended gasoline-nickel soil microcosms T4,respectively.

The biodegradation half-life times for gasoline biodegradation and the half-life fornickel uptake were calculated using Eq. (2) and the values are presented in Tables 5a and b.The highest half-life of 28.88 days for gasoline biodegradation (Table 5a) and 19.80 days fornickel uptake (Table 5b) was observed for unamended and/or unvegetated gasoline-nickel-contaminated soil T4 (natural attenuation). This was reduced to a lesser number of days forgasoline-nickel-contaminated soil amended with poultry dung (biostimulation) and vege-tated with stubborn grass (phytoremediation), respectively (either alone or in combination).Nevertheless, gasoline-nickel-contaminated soil amended with the combination of poultryand vegetated with stubborn grass T7 (combined biostimulation and phytoremediation) hadthe lowest half-life of 8.56 days for gasoline degradation (Table 3a) and 5.73 days for nickeluptake or reduction (Table 5b).

Conclusions

The effects of nickel metal, poultry dung (biostimulation), and stubborn grass (Sporoboluspyramidalis) (phytoremediation) on gasoline biodegradation and growth rate of intrinsic


soil microbial species as indicated by THDB have been assessed for an ex-situ bioreme-diation system. The nickel metal has been found to stimulate gasoline biodegradation andmicrobial growth at concentrations of 2.5, 7.5, and 12.5 mg/kg, respectively. Also, thepoultry dung (organic nutrient) and stubborn grass (Sporobolus pyramidalis) have beenobserved to enhance biodegradation of gasoline, nickel uptake, and growth rate of intrinsicsoil microorganisms. Thus, biostimulation with poultry dung and phytoremediation withstubborn grass (Sporobolus pyramidalis) had the tendencies to enhance the gasoline-nickelbiodegradation and uptake process, indicating that they are good remediation treatmentoptions in petroleum hydrocarbon/heavy-metal-polluted soil. The fact that soil microcosmsT7 amended with the combination of biostimulation and phytoremediation have the highestTPH loss, nickel uptake, and microbial growth, as indicated by a higher gasoline biodegra-dation rate constant (0.081 day−1) and higher nickel uptake rate constant (0.121 day−1), thanthe other treatment options indicated that they are a preferred remedial option. Therefore,this study has shown that poultry dung and stubborn grass (Sporobolus pyramidalis) canrespectively be a suitable organic nutrient and a potential plant species for biostimulationand phytoremediation of gasoline-nickel-contaminated soil.

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