Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 1 MYU Tokyo
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1 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 1 MYU Tokyo ES577 Bioremediation of Crude Oil-Polluted Soil Effect of Poultry Droppings and Natural Rubber Processing Sludge Application on Biodegradation of Petroleum Hydrocarbons C.O. Okieimen* and F.E. Okieimen 1 Department of Chemical Engineering, University of Benin, Benin City, Nigeria 1 Department of Chemistry, University of Benin, Benin City, Nigeria (Received September 8, 2003; accepted September 3, 2004) Key words: bioremediation, crude oil-contaminated soil, poultry droppings, natural rubber processing sludge, soil phase, slurry phase Laboratory bioremediation experiments were carried out on crude oil-polluted soil samples by applying various amounts of poultry droppings and natural rubber processing sludge as nutrient supplements at 29 C and using slurry-phase and solid-phase biodegradation techniques. Changes in the total hydrocarbon content of the soil were determined using a spectrophotometric technique as a function of time. It was found that the extent of crude oil degradation in untreated soil samples was markedly lower (by up to 100%) than in the soil samples treated with nutrient supplements. Hydrocarbon degradation efficiency was higher in the slurry-phase than in the soil-phase technique. 1. Introduction Soil pollution, a very serious environmental problem, has been attracting considerable attention in recent years. It is now generally recognized that land as a component of the environment deserves the same attention and protection as water and air. (1,2) This recognition has arisen perhaps because of increased incidents of land pollution, the scarcity of land, and increased awareness and concern about long-term effects of land pollution on terrestrial and aquatic ecosystems and on groundwater quality. Contamination of land results from a wide range of human activities, including industrial discharge processes, disposal of wastewater and agriculture. The environmental impact of exploration, production and refining of crude 1
2 2 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen oil is a major concern in both developed and developing countries. Because of the known adverse effects of petroleum hydrocarbons on soil ecology and fertility (3 6) there has been growing interest in the development of efficient technologies for the remediation of contaminated land, with a view toward making it available for further use. Crude oil is a natural product and as such is susceptible to degradation by naturally occurring microflora. However, crude oil contamination is accompanied by a depletion in both nutrient and oxygen levels of soil (6 9) and this retards the ability of the natural microflora to degrade crude oil. The recognition of crude oil as a complex but biodegradable mixture of hydrocarbons and the knowledge that microbial hydrocarbon-degraders can be enriched in many environments have contributed to the development of bioremediation techniques. Bioremediation involves the use of microorganisms to degrade environmental contaminants. It has numerous applications, including the clean up of groundwater, soil, lagoons, sludges and process waste streams. (10) Conditions for bioremediation are optimized by soil water conditioning, aeration, ph, temperature and nutrient addition. Many workers have reported the use of inorganic fertilizers to stimulate indigenous microbial population/activity in soil contaminated with petroleum hydrocarbons. (11 15) In a previous report, we examined the effect of the application of cow dung and poultry droppings on petroleum hydrocarbon degradation in soil. (17) In the present report we compare the effects of poultry droppings and natural rubber processing sludge on the degradation of petroleum hydrocarbon in soil using the solid-phase and slurry-phase treatment systems. Natural rubber latex obtained from the Hevea brasiliensis tree is a dispersion of rubber particles in an essentially aqueous medium. The latex is usually processed into intermediate products such as crumb rubber, rubber sheets and others. This process generates large volumes of wastewater with high pollution potential and large deposits of sludge (natural rubber processing sludge) in the wastepit. 2. Materials and Methods 2.1 Materials Crude oil (Atan blend) was obtained from the Petroleum Training Institute, Warri. It was weathered by exposure to atmospheric conditions for two weeks with occasional stirring and then stored for use. A surface soil sample (0 10 cm) was collected from a fallow piece of land in the Ugbowo Campus of the University of Benin, Benin City. It was air dried and sieved through a 2-mm sieve. The physico-chemical characteristics of the soil sample (18) are given in Table 1. Natural rubber processing sludge was obtained from the Osse Rubber Estate Factory (Michelin Group) near Benin City. Poultry droppings were collected from the university poultry, air-dried and powdered. The nitrogen and phosphorus contents of poultry droppings and natural rubber processing sludge are given in Table Solid-phase treatment technique The air-dried soil sample was treated with the weathered crude oil (1 g oil kg 1 soil) over a period of one week at a time in order to acclimate microorganisms in the soil to the oil. During this period, the oil-contaminated soil was aerated by turning the mixture over at regular intervals. At the end of the second week an additional amount of the weathered crude
3 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 3 Table 1 Some characteristics of the soil nutrient supplement. Nutrient supplement* Parameter Soil Sludge a Poultry droppings b Moisture content (%) 3.7 ± ± ± 0.04 Water retention capacity (%) ± 0.21 ND ND Total organic carbon (%) 2.09 ± 0.03 ND ND Total nitrogen (mg/kg) ± ± ± 0.09 Phosphorus (mg/kg) 2.46 ± ± ± 0.11 ph 6.82 ± ± 0.06 ND a = Okikhomonuan (1997) (18), b = Okurumeh and Okieimen (1992) (17) oil (3 g oil kg 1 ) was applied to the soil and thoroughly mixed. The total crude oil applied to the soil was 5 parts per 1000 parts of soil, which is equivalent to 5000 mg oil kg 1 soil. Approximately 1 kg of the oil-contaminated soil was placed in each of five beakers. Into four of the beakers, various amounts of sludge were added: flask A contained oilcontaminated soil without sludge, and flasks B, C, D and E contained oil-contaminated soil with 20, 50, 100 and 200 mg sludge, respectively (representing 10 wt% of the total sludge loading). The amount of sludge applied was based on the crude oil content of the soil. After each weekly sampling during the first three weeks, an additional 20, 30, and 40% of the total sludge load were added, respectively. Thus, at the end of this period, flasks B, C, D and E contained oil-contaminated soil and a total sludge application of 250, 500, 1000 and 2000 mg, respectively, which is equivalent to 5, 10, 20 and 40 wt% of crude oil in the soil. Application of poultry droppings to the oil-contaminated soil was carried out as described for the sludge at 2, 5 and 10 wt% of crude oil in the soil. 2.3 Slurry-phase treatment technique The weathered crude oil was applied to the soil at a rate of 5g kg 1 soil per week for five weeks. At the end of the fifth week an additional amount of the weathered crude oil (25 g kg 1 soil) was applied to the soil. The total crude oil applied to the soil was 5 parts of oil per 100 parts of soil which is equivalent to 50,000 mg oil kg 1 soil. Approximately 1 kg of the oil-contaminated soil was placed in four conical flasks each containing 300 ml of deionized water. Into three of the flasks, various known amounts of sludge were added. Flask A contained oil-contaminated soil without sludge, and flasks B, C, and D contained oilcontaminated soil with 100, 500 and 1000 mg of sludge, respectively. The reactors were continually stirred and were fitted with whisper aerators. The initial level of water in the reactor was maintained throughout the period of the experiment. A solid-phase treatment experiment was set-up consisting of oil-contaminated soil (1 kg) and sludge (100 mg) which was applied as an aqueous suspension in 100 ml of deionised water and aerated by turning the mixture over.
4 4 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen The experiments were carried out at 29 C and monitored at weekly intervals for total petroleum hydrocarbon content. After each weekly sampling (for the first five weeks), sludge was applied as described above. Thus, at the end of this period, flasks B, C, and D had a total sludge application of 500, 250 and 500 mg, respectively, representing a total sludge loading of 1, 5 and 10 wt% based on the crude oil content of the contaminated soil. The total sludge loading for the solid-phase treatment was 10 wt% based on the crude oil content of the contaminated soil. 2.4 Determination of total petroleum hydrocarbon The total petroleum hydrocarbon content of the soil samples was determined spectrophotometrically. Each soil sample (10 g) was extracted with two 50-ml portions of xylene and the combined extract was centrifuged for 10 min. The absorbance of the supernatant was read at 400 nm in a HACH DR A calibration curve was prepared by mixing calculated amounts of crude oil (Atan blend) in xylene. 3. Results and Discussion 3.1 Solid-phase treatment The extent of biodegradation of petroleum hydrocarbons in the untreated and sludgetreated soil samples is given in Table 2. The initial rates of biodegradation are relatively high and may correspond to the degradation of the n-alkane fraction of the petroleum hydrocarbons. (17) Within the six-week period of this study, the results show that biodegradation of petroleum hydrocarbons is enhanced by the application of natural rubber processing sludge. The total petroleum hydrocarbons degraded (THD) in the soil samples treated with the sludge were markedly higher (by more than two-fold) than in the untreated soil (Table 3). The results in Table 3 show that sludge application at more than 20 wt% of the crude oil did not produce a corresponding increase in the extent of degradation of petroleum hydrocarbons. In fact, at a relatively high sludge loading, the extent of petroleum hydrocarbon degradation decreased. Reductions in the extent of petroleum hydrocarbon biodegradation at a relatively high nutrient supplement loading have been explained in terms of the utilization of the nutrient supplement as an energy source in preference to the hydrocarbon contaminant. (19) In addition, the change in the ph of the soil (ph 6.82) upon application of the sludge (ph 8.41) may induce additional strees on the indigenous microflora and could result in the observed reduced values of THD at high sludge loading. The effect of poultry droppings on the extent of petroleum hydrocarbon degradation is shown in Table 4. The results show that within the 6-week period of study, the total hydrocarbon content of the soil samples was markedly reduced. The maximum levels of reduction in the petroleum hydrocarbon contents in the soil (Table 4) were lower in the soil treated with poultry droppings than for the sludge-treated oil-contaminated soil samples at comparable levels of nutrient supplement application. The difference in the N and P contents of the nutrient supplements is thought to account for the observed efficiency of biodegradation in the soil treated with poultry droppings.
5 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 5 Table 2 Effect of natural rubber processing sludge application on the degradation of petroleum hydrocarbons in soil at 29 C. Initial level of petroleum hydrocarbons in soil = 5000 mg/kg. Residual petroleum hydrocarbons (%) Time (weeks) A B C D E ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.02 Table 3 Total petroleum hydrocarbons degraded in soil samples treated with natural rubber processing sludge. Contaminated Total petroleum hydrocarbons THD/THD soil sample degraded THD (%) A ± 1.01 B ± C ± D ± E ± THD, THD = Total petroleum hydrocarbons degraded in the presence and absence of sludge application Table 4 Effect of poultry droppings on the petroleum hydrocarbon degradation of soil at 29 C. Initial concentration of petroleum hydrocarbons in soil = 500 mg/kg. Nutrient supplement loading a (%) Reduction in petroleum hydrocarbon levels* (%) ± ± ± 1.05 a) basis: amount of weathered crude oil added to soil
6 6 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 3.2 Slurry-phase treatment technique The extent of biodegradation in the oil-polluted soil samples as a function of time is shown in Table 5. The results show that within the 12-week period of the study, the total hydrocarbon content of the soil samples was markedly reduced. The maximum levels of reduction in the petroleum hydrocarbon content of the soil samples are shown in Table 6. It can be seen from the results in Table 6 that the extent of petroleum hydrocarbon degradation was markedly higher (by up to 107%) in the sludge-treated soil than in the untreated soil. The results in Table 6 show that at the same levels of nutrient loading, degradation off petroleum hydrocarbons in the slurry-phase was markedly higher than in the solid-phase process. Effective mixing and the role of oxygen as an electron acceptor in the degradation process may explain the observed enhanced degradation of petroleum hydrocarbons in the slurryphase process. The results in Table 6 show that the extents of crude oil degradation using natural rubber processing sludge as a nutrient supplement are higher than those reported for cow dung (17) and poultry droppings. The results show that the slurry-phase treatment technique is more effective than the solid-phase technique in reducing the levels of petroleum hydrocarbon in contaminated soil. The slurry-phase treatment is intrinsically an ex-situ technique and would be suitable for bioremediation of polluted soil excavated from localized hot-spots in oil spills on land. Natural rubber processing sludge and poultry droppings are biological waste materials with little or no potential for causing secondary deleterious effects on soil ecology. They are available at minimal cost and require minimal processing. Table 5 Biodegradation of petroleum hydrocarbon-contaminated soil in the presence of natural rubber processing sludge in a slurry-phase reactor at 29 C. Initial concentration level of petroleum hydrocarbon = 50,000 mg/kg. Petroleum hydrocarbon degraded (%)* Time (weeks) A B C D E ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.40 A = Slurry phase with no sludge added; C = Slurry phase with 5 wt% sludge; E = Slurry phase with 10 wt% sludge B = Slurry phase with 1 wt% sludge D = Solid phase with 10 wt% sludge
7 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 7 Table 6 Effect of natural processing sludge application on the extent of petroleum hydrocarbon degradation in slurry-phase treatment technique. Nutrient supplement Total nutrient Maximum reduction of supplement loading (%) petroleum hydrocarbon levels (%) Nutrient rubber Processing sludge ± 0.40 Processing sludge ± 0.37 Processing sludge ± 0.41 Processing sludge ± 0.04 (35.6 ± 0.39) Value obtained for solid-phase biodegradation technique in parenthesis. * Basis: amount of weathered crude oil applied to soil. 4. Conclusion Biodegradation of petroleum hydrocarbons in soil is enhanced by the application of poultry droppings and natural processing sludge as nutrient supplements in both soil-phase and slurry-phase treatment techniques. A potential for the application of these biological wastes in the remediation of contaminated soil and in the amelioration of marginal land for sustainable agricultural development is indicated. References 1 Smith, M.A. (1989): Report to NATO-CMS pilot study on contaminated land. In: Smith M.A. Ed.: Contaminated land, Reclamation and Treatment. Plenum Press, New York, pp Tadesse, B., Donaldson, J.D. and Grimes, S.M. (1994): Contaminated and polluted land. A general review of decontamination and control. Bioresour. Technol. 60: Ellis, A. and Adams, R.M. (1961): Contamination of soil by petroleum hydrocarbons. Adv. Agron. 13: Odu, C.T.I. (1972): Microbiology of soil contaminated with petroleum hydrocarbons I. Extent of contamination and some soil microbial properties after contamination. J. Inst. Petrol. 58: Udo, E.J. and Fayemi, A.A (1975): The effect of pollution on germination and nutrient uptake of corn. J. Environ. Qual. 4: Amadi, A. (1990): Nitrification and nitrogen mineralisation in chemical demulsifier contaminated soil. Oil Pollut. 7: Atlas, R.M. (1991): Petroleum biodegradation and oil spill bioremediation. Marine Pollut. Bull. 31: Atlas R.M. and Bartha, R. (1993): Stimulated biodegradation of oil slicks using oleophilic fertilizer. Environ. Sci. Technol. 7: Bossert, R. and Bartha, R. (1994): In: Altas, R.M. Ed.: The fate of petroleum hydrocarbon in soil environment. Petroleum Microbiology. McMillian, New York.
8 8 Environmental Sciences, 12, 1 (2005) C. Okieimen and F. Okieimen 10 Boopathy, R. and Manning, J. (1999): Surfactant-enhanced bioremediation of soil contaminated with 2,4,6-trinitrotoluene in soil slurry reactors. Water Environ. Res. 71: Lehtomake, M. and Niemela, S. (1975): Improved microbial degradation of oil in soil. Ambio. 4: Gudin, C. and Syratti, W.T. (1975): Biological aspects of land rehabilitation following hydrocarbon contamination. Environ. Pollut. 13: Oliveri, R., Bacculum, P., Robetiolo, A., Oddo, N., Deggen, L. and Tonola, A. (1976): Microbial degradation of oil spills enhanced by slow-release fertilizer. Appl. Environ. Microbiol. 31: Lode, M.A. (1986): Changes in bacterial community after application of oily sludge in soil. Appl. Microbiol. Biotechnol. 25: Amadi, A., Dickson, A.A. and Maate, G.O. (1993): Remediation of oil polluted soil. Effect of organic and inorganic supplement on the performance of maize (Zea mays). Water, Air, Soil Pollut. 66: Odu, C.T.I. (1978): The effect of nutrient application and aeration on oil degradation in soil. Environ. Pollut. 15: Okurumeh, O.A. and Okieimen, F.E. (1992): Effect of cow dung and poultry dropping application on petroleum hydrocarbon degradation in soil. Niger. J. Appl. Sci. 10: Ohikhomouan, C.O. (1997): Physico-chemical characterization of natural rubber processing wastewater and sludge. MSc. Thesis, University of Benin, Benin City. 19 Raymond, R.L., Hudson, J.O. and Jamison, V.W. (1976): Oil degradation in soil. Appl. Environ. Microbiol. 31:
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