Journal of
Soil Science and Environmental Management

  • Abbreviation: J. Soil Sci. Environ. Manage.
  • Language: English
  • ISSN: 2141-2391
  • DOI: 10.5897/JSSEM
  • Start Year: 2010
  • Published Articles: 286

Full Length Research Paper

Biostimulation of hydrocarbon utilizing bacteria in soil contaminated with spent engine oil using banana and plantain agro-wastes

Victor Taghoghor Omoni
  • Victor Taghoghor Omoni
  • Department of Biological Sciences, University of Agriculture, P. M. B. 2373, Makurdi, Benue State, Nigeria
  • Google Scholar
Celestine Uzoma Aguoru
  • Celestine Uzoma Aguoru
  • Department of Biological Sciences, University of Agriculture, P. M. B. 2373, Makurdi, Benue State, Nigeria
  • Google Scholar
Emmanuel Odogbo Edoh
  • Emmanuel Odogbo Edoh
  • Department of Microbiology, Adekunle Ajasin University, P. M. B. 001, Akungba-Akoko, Ondo State, Nigeria
  • Google Scholar
Oluwatayo Makinde
  • Oluwatayo Makinde
  • Department of Microbiology, Adekunle Ajasin University, P. M. B. 001, Akungba-Akoko, Ondo State, Nigeria
  • Google Scholar


  •  Received: 22 May 2015
  •  Accepted: 21 July 2015
  •  Published: 01 August 2015

 ABSTRACT

hydrocarbon utilizing bacteria in soil contaminated with spent engine oil, as an alternative to inorganic fertilizers was investigated. Two types of agro-wastes were used for biostimulation of hydrocarbon degraders in soil contaminated with 10% w/w spent engine oil. 100g w/w each of plaintain and banana agro-wastes were supplemented and mixed with spiked soil. The rates of biodegradation of the oil were studied for a period of 70 days under laboratory conditions. The physiochemical property of agro-waste, total heterotrophic bacteria count, hydrocarbon-utilizing bacterial counts, percentage net loss and biostimulant efficiency were observed. The results of the total heterotrophic bacterial count (THBC) and hydrocarbon utilizing bacterial (HUB) counts were observed to be higher in plantain peels treatment option (38×107 and 41×106 cfu/g) than banana peels treatment options (29×107 and 29×106 cfu/g), and when compared with control (2.9×107 and 2.2×106 cfu/g) throughout the 70 days of bioremediation study. The study showed higher THBC and HUB counts at day 28 and day 42. Plantain peels treatment showed higher percentage of degradation (93.5%) and biostimulant efficiency (32.4%) than banana peels treatment with 84.11 and 24.9% respectively. Half-life for plantain peels treatment, banana peels treatment and unamended soil were 21.521, 24.146 and 43.043 day-1 respectively. These organic materials are widely available as wastes in the environment; hence, they can serve as “waste-to-environmental cleanup”.
 
Key words: Biostimulation, agro- wastes, spent oil, petroleum hydrocarbons, bio-stimulant efficiency, bioremediation, half-life.
 


 INTRODUCTION

Global increase in the usage of petroleum products has resulted in the rapid increasing contamination of soil by spent engine oil (SEO) (Mandri and Lin, 2007). Spillage of used motor oils such as engine  oil,  diesel  or  jet  fuel contaminates the natural environment with hydrocarbon (Husaini et al., 2008). Hydrocarbon contamination of the air, soil, and fresh water especially by polycyclic aromatic hydrocarbon (PAHs) attracts  public  attention   because
 
majority of PAHs are toxic, mutagenic, and carcinogenic (Bumpus,1989; Clemente et al., 2001; Cerniglia and Sutherland, 2001) and SEO contain metals such as arsenic, zinc, cadmium and potentially toxic substances, such PAHs  (Hagwell et al., 1992; Boonchan et al., 2000), which can seep through different layers of aquifers into the soil and contaminate ground water.
 
The illegal spread of spent engine oil is an environmental hazard with global concerns (Blodgett, 2001). Prolong exposure to high concentration of hydrocarbon containing compounds may cause the development of liver or kidney disease, possible damage to the bone marrow, and an increased risk of cancer (Mishra et al., 2001).
 
Lack of essential nutrient such as nitrogen and phosphorus is one of the major factors affecting biodegradation of hydrocarbon by microorganisms in soil and water environment (Abioye et al., 2012). Therefore, the addition of inorganic or organic nitrogen-rich nutrients (biostimulation) is an effective means to enhance the bioremediation process (Hollender et al., 2003; Semple.et al., 2006; Walworth et al., 2007). Concentration of petroleum hydrocarbon in soil impacted environment determines to a greater extent the rate of breakdown of hydrocarbons by either authochthonous or allochthonous microorganisms. Hence, high concentration of hydrocarbon can be inhibitory to microorganisms, and concentration at which inhibition occurs depends on the compound (Abioye et al., 2012).
 
Banana and plantain peels are thick ropey-textured which are green to yellow colored skin. In western society, banana and plantain peels are discarded while the flesh is eaten; however, many animals in the primate family consume bananas or plantains peel, flesh and whole. Banana and plantain peels have a high nutritional content to boost the soil, in a cheap, natural and effective way.
 
In Nigeria, as in other part of the developing world, oil spills at auto-mechanic workshops have been left uncared for over the years and its continuous accumulation may cause serious environmental problems because of its hazardous nature.
 
Crude oil pollution adversely affects the soil ecosystem through adsorption to soil particles, provision of an excess carbon that might be unavailable for microbial use and an induction of a limitation in soil nitrogen and phosphorus (Atlas, 1981; Baker and Herson, 1994). The present study evaluate the potential of agro wastes (banana peels and plantain peels) as biostimulation and bulking agents for bacterial biomass suppliers. The addition of organic waste materials such as plantain peels and banana peels to the soil facilitates aeration through small pores and increases the water-holding capacity of the soil, thus enhancing bioremediation (Jobson et al., 1974; Amadi et al., 1992).

 


 MATERIALS AND METHODS

Collection and processing of samples
 
The soil sample used was collected randomly with a metal soil auger at a depth of 15 to 30 cm from the agricultural garden of University of Agriculture, Makurdi. They were bulked to form a composite sample and transported in a black polythene bag to the laboratory, air dried and sieved through a 2 mm mesh. Fresh spent engine oil was collected from Auto-Mechanic workshop, Mechanic Villa, Makurdi. The organic wastes (ripe plantain and banana peels) were collected from different canteens at Wurukum market, Makurdi. The organic wastes were sun-dried for 14 days and ground into fine powder to obtain a uniform particle size and stored in a sterilized airtight plastic container.
 
Soil preparation (or experimental set up)
 
1 kg of soil (sieved with 2 mm mesh size) was placed in rubber bowl with a volume of about 3000cm3 and then polluted with 10% w/w (100 ml) of spent engine (SEO) and thoroughly mixed. The concentration was chosen to simulate slightly heavy contamination in the field. 10% w/w (100 g) of different organic wastes (sun-dry) plantain peels (PP) and banana peels (BP) were individually introduced into each oil-polluted soil and thoroughly mixed. Also, rubber bowl container containing 1 kg soil was spiked with 10% w/w (100 ml) of spent engine oil as control.
 
The moisture content of the amended soils and control were adjusted by adding distilled water and incubation was at room temperature (26°C). The content of each vessel were tilled thrice a week for proper aeration, and moisture content was maintained at 60% water holding capacity by addition of distilled water. The experiment was set up in triplicate. Periodic sampling from each vessel was carried out at 14-day interval for 70 days. The design of the experiment is as shown in Table 1.
 
 
Physicochemical analyses
 
The pH of soil sample was measured by electrode  pH  meter  using the method described by Mclean (1982); electrical conductivity was determined using the aqueous extraction method according to Mathieu   and   Pieltain   (2003).   Particle    size    distribution    was determined by hydrometer method as described by Sheldrick and Wang (1993). Organic carbon determination was by Anne method (modified Walkley-Black) (Mathieu and Pieltain, 2003). Total nitrogen was determined by the microkjeldahl digestion method (Bremner and Mulvancy, 1982). Available phosphorus was measured using alkaline oxidation method as described by Dick and Tabatabai (1977). Moisture content was determined by gravimetric method as described by Black (1965).
 
Biodegradation of total hydrocarbon content in soil contaminated with spent engine oil
 
The residual petroleum hydrocarbon content of the soil samples (amended and unamended) during study period was determined gravimetrically by solvent extraction method including the unamended as control (Adesodun and Mbagwu, 2008). 10 g of soil samples (triplicates) was taken from each microcosm and transferred into a 50-ml flask and the hydrocarbon content in oil polluted soil was extracted using 20 ml of n-hexane. The mixture was shaken vigorously on a magnetic stirrer for 30 min and allowed to stand for 10 min until the hexane extract completely separate the oil from the soil sample. The solution was then filtered using a Whatman filter paper and the liquid phase extract (filtrate) diluted by taking 1 ml of the extract into 50 ml of hexane. The absorbance of this solution was measured spectrophotometrically at a wavelength of 420 nm spectrophotometer (Spectronic 721 Model) using n-hexane as blank. The total hydrocarbon in soil sample was estimated with reference to a standard curve derived from fresh spent engine oil of different concentration diluted with n-hexane. Biodegradation percentage was calculated using the following formula:
 
 
Net loss of total hydrocarbon content and biostimulant efficiency in soil contaminated with spent engine oil
 
The evaluation of the efficiency of each organic wastes applied to the oil contaminated soil was estimated by determining the net percentage loss and biostimulant efficiency based on each soil amendment microcosm. The % biostimulant efficiency was calculated using the equation stated by Agarry (2013) using the formula:
 
% Biostimulant efficiency (BE) = percentage loss in THC of oil polluted soil amended with organic wastes - % loss in THC of unamended polluted soil (control).                                                 (2)
 
First order kinetics and half-life for biodegradation of total petroleum hydrocarbons in spent oil contaminated soil
 
Bioremediation kinetics is very important in biodegradation studies. It gives information on the kinetics of soil bioremediation and characterizes the concentration of the contaminant remaining at any time and allows predicting the level likely to be present at any time (Agarry, 2013; Agarry et al., 2013). Several researchers have used the first order kinetics to effectively explain the biodegradability of motor engine oil (Yeung et al., 1997;  Abioye  et al., 2009; Agarry et al., 2013; Onuoha, 2013). This model is given as:
 
 
Statistical analysis
 
The data in this study were subjected to one-way analysis of variance (ANOVA) at p < 0.05.  Relationship between variables and comparison of means of the different treatments were tested for level of significant differences at p<0.05 using Least significant Difference (LSD) test. The data analysis was performed using software (Statistical package for social sciences (SPSS), version 17.0.
 
 
 

 


 CONFLICT OF INTEREST

The authors have not declared any conflict of interest.

 


 RESULTS AND DISCUSSION

The baseline physicochemical properties of the soil before spiking and nutrients amendment in the study revealed low amount of nitrogen, phosphorus and organic matter (Table 2).
 
The hydrocarbon degrading bacteria isolated and identified during the bioremediation process were Micrococcus sp., Pseudomonas sp., Bacillus sp., Corynebacterium sp., Nocardia sp., Achromobacter and Klebsiella sp. (Table 4). These bacteria species have been reported in hydrocarbon degradation by different authors (Ijah, 1998; Van et al., 2003; Bento et al., 2005; Abioye et al., 2009; Onuoha et al., 2011; Onuoha, 2013). In similar findings by Onuoha (2013), the populations of hydrocarbon degraders from the treatment vessels in this study showed that majority of the bacteria isolated were Gram positive belonging to the Actinobacteria group. Meanwhile, the study also observed the presence of Gram negative bacteria as hydrocarbon utilizers. Some studies have shown that oil polluted soils are dominated by Gram-negative bacteria (Macnaughton et al., 1999; Kaplan and Kitts, 2004; Chikere et al., 2012).The results obtained in this study showed different degree of hydrocarbon utilization in the treatment options by the bacterial isolates with the spent engine oil serving as the sole source of carbon and energy (Figure 2). Also, there was gradual reduction  in  hydrocarbon  utilizers  in   the control as the number of days increases in the study period.
 
The current study showed high utilization of hydrocarbon by the soil autochthonous organisms (Figure 3). However, high oil pollution has a reduced effect on the rate of microbial reduction of total hydrocarbon in soil. The ineffectiveness of some organic wastes in high spent engine oil contaminated environment could be attributed to reduction in the activity of the soil microbes at that level of oil pollution. Bossert and Bartha (1984) stated that sensitivity of soil microflora to petroleum hydrocarbon is a factor of quantity and quality of oil spilled and previous exposure of the native soil microbe to oil. Schaefer and Juliane (2007) stated that bioremediation is useful method of soil remediation if pollutant concentrations are moderate.
 
The total heterotrophic bacteria count and Hydrocarbon utilizing bacteria count in all the soil amended with various organic wastes were higher compared to unamended control soil as shown in Figures 1 and 2; the counts of hydrocarbon degrader in oil-polluted soil in this study was ×107 for THB and ×106 for HUB. Similar results were reported by several researchers who observed counts of hydrocarbon utilizers in oil-polluted soil to be x106 cfu/g (Ijah and Antai, 2003; Onuoha, 2013), but lower than those obtained by Antai and Mgbomo (1989) whose counts of HUB in hydrocarbon-contaminated soil was ×108 CFU/g. However, this may be due to differences in microbial ecology of the soil or characteristics of the experimental soil. The reason for the higher counts of the bacteria in amended soil may be the result of the presence of appreciable quantities of nitrogen and phosphorus in the agro-wastes (Table 3).
 
In the amended samples, there was an increase in total heterotrophic bacteria for plantain peels from day 0 to 28 (3.3×107 to 38×107 cfu/g) and then decreased at day 42 (34×107 cfu/g) while banana peels increased from day 0 to 42 (2.3×107 to 29×107cfu/g) and decreased at day 56 (26×107cfu/g). It was also observed that the hydrocarbon utilizing bacteria count for soil amended with plantain and banana peels showed a marked increase from day 0 crude oil degradation after  biostimulation  and  found  out that nutrient enhancement increased bacteria counts which correlated significantly with hydrocarbon attenuation. This same observation was made by several workers (Okpokwasili et al., 1986; Okpokwasili and Amanchukwu, 1988; Margesin et al., 2007; Ruberto et al., 2006; Quatrini et al., 2008).
 
 
 
 
The percentage loss of total petroleum hydrocarbon (TPH) is shown in Figure 1. There was a remarkable reduction of total petroleum hydrocarbon during the studied period in soil  supplemented  with  agro  wastes (banana and plantain peels). At the end of 14 days, soil polluted with 100,000 mg/kg (10%) spent engine oil showed a significant reduction in TPH of 65.76, 35.67 and 11.33% in soil amended with plantain peels, banana peels and unamended soil, respectively, while 93.47% (6522 mg/kg), 84.11% (15888 mg/kg) and 63.15% (36850 mg/kg) of TPH reduction were observed at day 70 in soil amended with plantain peels, banana peels and control respectively. The results clearly showed that natural attenuation occurs in the control at day 70 due to 50% removal as a result of abiotic factors such as sorption and volatilization based on the nature of the soil. Ijah and Antai (2003) reported   high   degradation   of hydrocarbons in soil contaminated with 10 and 20% crude oil compared to those contaminated with 30 and 40% crude oil which experienced partial degradation of hydrocarbons within a period of 12 months. Rahman et al. (2002) reported also the percentage of degradation by mixed bacterial consortium decreased from 78 to 52%, as
 the concentration of crude oil increased from 1 to 10%.
 
The results of statistical analysis showed that there was significance difference in all the treatment options. It can be stated that the high loss of petroleum hydrocarbon in unamended soil could be as a result of natural attenuation such as physical and chemical processes, such as dispersion, dilution, sorption, volatilization, and abiotic transformations (USEPA, 1999).
 
The highest net percentage loss of TPH was observed at day 14 in soil supplemented with plantain peels (54.43%) and banana peels (35.67%). At day 56, there was an increase in net percentage loss of TPH in soil amended with plantain peels (33%), (Table 5). Similar results was observed and reported by Abioye et al. (2009). It is noteworthy that the two agro-wastes were effective in biostimulating the hydrocarbons utilizers that subsequently led to a reduction in petroleum hydrocarbon in the soil polluted with spent engine oil. Similar study had been carried by several researchers and reported the effectiveness of plantain and banana peels in stimulating hydrocarbon degraders in oil polluted  (Ekpo  et  al., 2012; Abioye et al., 2009).
 
 
The result from the study revealed higher biostimulation efficiency (BE) in plantain peels (32.44%) than that of banana peels (24.92%). However, the observed reduction in petroleum hydrocarbons in spent oil may not only be due to the biodegradation process induced by nutrient   additions, but other processes such as volatilization, adsorption to organic compounds, other abiotic factors are equally implicated in the reduction (Onuoha, 2013).
 
The results obtained from the study fitted with the first order kinetic model used to determine the rate of biodegradation. Table 6 shows the biodegradation rate constant (K) and half-life (t½) for the different treatments within the period of study. The kinetic parameters observed in this study showed that the rate of degradation of spent oil in soil amended with Plantain and Banana Peels was high at the level of concentration of oil pollution.
 
It was observed that soil amended with plantain peels showed a higher biodegradation constant of 0.0322 and lower half-life of 21.52 days  than  banana  peels with a lower biodegradation rate constant of 0.0287 and higher half- life of 24.52 days (Table 7). The biodegradation rate constant of unamended soil (control) was least with a 0.016 day-1 and a half-life of 43.04 days. Since oil degradation is a natural process limited by temperature, pH, and scarcity of nutrients such as nitrogen and phosphorus (Ladousse and Tramier, 1991; Leahy and Colwell, 1990), the higher rate of hydrocarbon reduction reported in this study with the addition of plantain and banana  peels  could  be  due   to   bioavailability of the nutrients in these organic wastes to  bacterial  species  in the oil polluted soil (Chukwudozie, 2013).
 
In the present study, the THB and HUB counts obtained from the nutrient-amended sample when compared with those from the oil-contaminated-unamended were statistically significant at p < 0.05. Further research and development will require focus on the cheap, environmental friendly and widely available nutrients that can be used to enhance the microbial and plant activities in mineralizing hydrocarbons in soil. It therefore necessitates the research for cheaper and environmentally friendly options for enhancing petroleum hydrocarbon degradation. These organic materials are widely available as wastes in the environment; hence, they can serve as “waste-to-environmental cleanup”.
 
Also, further studies also need to be carried out in order to study in details the genetics of the hydrocarbon degrading bacteria in spent oil contaminated soil to ascertain the degradative genes/enzyme involved in the degradation.
 
 
 
 
 

 


 CONCLUSION

The results obtained from the study indicated that bioremediation of spent engine oil-contaminated soil with the use of organic fertilizer (obtained from plantain and banana peels) as biostimulating agents resulted in the accelerated removal of petroleum hydrocarbon. Hence, the agro wastes greatly and significantly enhance the degradation of petroleum hydrocarbon in soil contaminated with motor engine oil. However, the presence of heavy metals in spent engine oil could reduce the degradation rate (percentage degradation) of its petroleum hydrocarbon components. Bioremediation of hydrocarbon using agro-wastes in spent engine oil contaminated soil offers a better and more environmentally friendly technique that if properly and thoroughly explored in agricultural lands will lead to a safer environment for both plant and animal.



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