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Bioremediation of oil sludge-contaminated soil


Environment International 26 (2001) 409 ± 411

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Bioremediation of oil sludge-contaminated soil
N. Vasudevan*, P. Rajaram
Centre for Environmental Studies, Anna University, Chennai 600 025, India

Abstract Bioremediation has become an important method for the restoration of oil-polluted environments by the use of indigenous or selected microbial flora. Several factors such as aeration, use of inorganic nutrients or fertilizers and the type of microbial species play a major role in the remediation of oil-contaminated sites. Experiments were undertaken for bioremediation of oil sludge-contaminated soil in the presence of a bacterial consortium, inorganic nutrients, compost and a bulking agent (wheat bran). Experiments were conducted in glass troughs for the 90-day period. Bulked soil showed more rapid degradation of oil compared to all other amendments. During the experimental period, wheat bran-amended soil showed 76% hydrocarbon removal compared to 66% in the case of inorganic nutrients-amended soil. A corresponding increase in the number of bacterial populations was also noticed. Addition of the bacterial consortium in different amendments significantly enhanced the removal of oil from the petroleum sludge from different treatment units. D 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Bioremediation; Contaminated soil; Oil sludge; Degradation

1. Introduction Oil production and shipping operations result in accidental contamination of soil with petroleum hydrocarbons. Petroleum refining also results in the generation of large quantities of oil sludge consisting of hydrophobic substances and substances resistant to biodegradation. Clean-up technologies such as incineration and burial of sludge in secure landfills are expensive. Land treatment disposal of oil refinery sludge generally gives good results (Bartha, 1986). Controlled land treatment, i.e., land farming, is cheaper and also environmentally safe (Bonnier et al., 1980; El-Nawawy et al., 1987). Aerobic conditions and appropriate microorganisms are necessary for an optimal rate of bioremediation of soils contaminated with petroleum hydrocarbons. In soils, the oxygen content depends on microbial activity, soil texture, water content and depth. A low oxygen content in soils has been shown to limit bioremediation of soils contaminated with petroleum hydrocarbons (von Wedel et al., 1988) and in a laboratory experiment, mineralization of hydrocarbons from soil was severely limited when the oxygen content

was below 10% (Freijer, 1986). Tillage is a mechanical manipulation of soil to improve soil conditions (Hillel, 1980). It alters physical and chemical properties of soil in such a way that it stimulates microbial activity (Melope et al., 1987). Tillage redistributes carbon, nitrogen and water and reduces spatial distribution within the soil (Rhykerd et al., 1999). Bulking agents are materials of low density that lower soil bulk density, increase porosity and oxygen diffusion, and can help to form water-stable aggregates. These activities increase aeration and microbial activity (Hillel, 1980). The aim of this study was to enhance the remediation of soil contaminated with oil sludge by the use of a bacterial consortium, inorganic supplements, bulking agents and compost. 2. Materials and methods 2.1. Soil preparation The soil used in the study was collected from open fields near the petroleum refinery site in Chennai city. Surface litter was removed and soil was collected to a depth of 25 cm and sieved to remove large roots, macrofauna and stones. For physico-chemical properties, the soil was airdried and passed through a sieve (2 mm); soil pH was

* Corresponding author. Tel.: +91-44-2354-296; fax: +91-44-2354717. E-mail address: nvasu30@yahoo.com (N. Vasudevan).

0160-4120/01/$ ± see front matter D 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 1 ) 0 0 0 2 0 - 4

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N. Vasudevan, P. Rajaram / Environment International 26 (2001) 409±411

measured in water using a 1:5 soil/water ratio. Soil texture, pH and organic matter content were determined according to Parmer and Schmidt (1964). 2.2. Sludge Petroleum refinery sludge was analyzed gravimetrically as described by Dibble and Bartha (1994). The following results were obtained after Soxhlet extraction. It contained 24% ether-extractable hydrocarbons, 5% water and 71% ash. 2.3. Biodegradation experiments Experiments were carried out in glass troughs of 23 ? 12.5 cm containing 5 kg soil and 5% w/w oil sludge. Experiments were conducted with the following treatment combinations: Soil + oil Soil + oil Soil + oil Soil + oil Soil + oil tium Soil + oil sludge (abiotic control) sludge sludge + compost sludge + bacterial consortium sludge + inorganic nutrients + bacterial consorsludge + wheat bran + bacterial consortium

53% ether-extractable hydrocarbons. The troughs were covered with sterile aluminium foil and incubated at 30°C for 90 days. To achieve sufficient aeration, the contents of the troughs were mixed thoroughly every alternative day. After the start of the experiment and at intervals of 15 days, ether extractable hydrocarbons and bacterial population in soil were determined. The ether extractable hydrocarbons were determined by extracting 25 g of soil using diethyl ether. The bacterial counts in different treatment units were determined by plating on nutrient agar medium and the colony forming units were counted after 24 h of incubation at 30°C. All determinations were carried out in duplicate.

3. Results and discussion The soil from site near industrial area had a loamy texture (34% sand, 40% silt and 26% clay). The soil organic matter content was 2.5%. Gas chromatographic analysis revealed that the oil sludge contained 53% saturated hydrocarbons, 24% aromatic hydrocarbons and 12% asphaltic hydrocarbons. 3.1. Biodegradation of oil sludge In order to investigate the optimum conditions for the biodegradation of oil sludge, soil was supplemented with nutrients, and inorganic amendments, bulking agent and a bacterial consortium as inoculum. Results showed a corresponding influence due to the different amendments in the remediation of oil sludge-contaminated soil (Table 1). Addition of inorganic nutrients produced little effect on oil removal compared to the soil amendment without inorganic nutrients. The soil microbial population played a major role in the treatment of hydrocarbons. Addition of organic compost instead of the inorganic nutrients did not enhance the removal of petroleum hydrocarbon compared to the treatment unit containing inorganic nutrients, indi-

The abiotic control containing soil, oil sludge and 0.3% w/w AgNO3 was used to monitor the abiotic loss of hydrocarbons. The treatment unit with inorganic nutrients received 24 g of ammonium nitrate and 4.37 g of dipotassium hydrogen phosphate as an additional supplement. The experimental units containing bacterial consortium received approximately 106 colony forming units (cfu)/g of soil as inoculum. Homogenizations of soil, oily sludge, compost, bacteria and wheat bran in different reactors were carried out in a stainless steel blending machine. The moisture level of the different soil mixtures was maintained at 20%. The bacterial population in the unsterilized soil was 103 cfu/g. The oil sludge contained 8% moisture, 26% ash and
Table 1 Relative percent biodegradation of oily sludge and bacterial count in soil

Time after sludge application (days) 15 Sample no. 1 2 3 4 5 6 7
a

30 ± 4 (2 ? 104) 6 (3 ? 105) 7 (3 ? 106) 8 (1 ? 108) 13 (2 ? 109) 15 (3 ? 1010)

45 ± 12 16 16 19 27

60 ± 18 (2 ? 105) 3 (3 ? 106) 24 (1 ? 107) 28 (1 ? 109) 44 (5 ? 1011) 45 (3 ? 1012)

75 ± 24 27 27 34 54

90 ± 25 28 29 40 65

Treatment Soil + oil sludge (abiotic control) Soil + oil sludge Soil + oil sludge + compost Soil + oil sludge + activated sludge Soil + oil sludge + bacterial consortium Soil + oil sludge + inorganic nutrients + bacterial consortium Soil + oil sludge + wheat bran + bacterial consortium

(Percent degradation and cell number)a ± 1 (6 ? 103) 2 (8 ? 104) 2 (3 ? 105) 2 (1 ? 107) 4 (6 ? 107) 5 (1 ? 109) (6 ? 104) (1 ? 106) (8 ? 106) (2 ? 108) (6 ? 1010) (3 ? 105) (6 ? 106) (2 ? 107) (4 ? 109) (2 ? 1012) (5 ? 105) (9 ? 106) (3 ? 107) (2 ? 1010) (3 ? 1012)

28 (3 ? 1011)

56 (2 ? 1013)

72 (6 ? 1013)

Cell number (cfu/g) in parentheses.

N. Vasudevan, P. Rajaram / Environment International 26 (2001) 409±411

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cating the lack of suitable hydrocarbon-degrading strains in the compost. In the case of the treatment unit containing inorganic nutrients, nearly 66% oil degradation was recorded. The initial bacterial count of 5 ? 106 cfu/g had increased to 3 ? 1012 cfu/g in 90 days (Table 1). During the period of 90 days, up to 76% of petroleum hydrocarbons were degraded in bulked soil compared to other amendments. An increase of 32% over the control unit without wheat bran addition was noticed and a corresponding increase in the bacterial population from 6 ? 106 to 6 ? 1013 cfu/g of soil. There was a 120-fold increase in the oil-degrading bacterial population in bioaugumented soil with wheat bran whereas in the nutrients-amended soil, about a 100-fold increase in bacterial population was observed. Earlier reports by ElNawawy et al. (1992) indicated up to 71% oil removal from the 5.8% oil sludge-amended with fertilizer in 112 days; the rates decreased with the increase in the amount of oil sludge in soil, whereas Sandvik et al. (1986) showed 45% oil removal from the oily sludge during bioremediation for 9 months. Tillage and bulking with wheat bran seemed to influence the disappearance of the hydrocarbons (Rhykerd et al., 1999). The addition of bulking agents tend to have a priming effect on microbial populations. It has also been noted previously that addition of organic material to soil enhances oil degradation (Chang and Weaver, 1998). Tillage of soil might have enhanced biodegradation by increasing bioavailability of the oil. Small clumps were noted during the early stages of the experiment, but disappeared during tillage and this probably enhanced redistribution of the oil, making it more available for microbial degradation. Bulking agent might have also played a role in reducing soil bulk density as well as serving as an additional organic material during the bioremediation process. In the present study, the increased oil degradation could be attributed to the selected bacterial consortium comprising of strains of Acinetobacter, Pseudomonas, Bacillus, Flavobacterium, Corynebacterium and Aeromonas. Earlier studies in our laboratory confirmed the emulsification capacity of Pseudomonas on different hydrocarbon sub-

strates (Rahman et al., 1999). The results of the present study indicated that the use of bulking agent played an important role in the bioremediation of oil-contaminated soil. In general, tillage of soil might enhance the contact between oil and bacterial populations thereby enhancing the bioremediation process. References
Bartha R. Biotechnology of petroleum pollutant biodegradation. Microb Ecol 1986;12:155 ± 72. Bonnier PD, Akoun GL, Cadron EC, Edwards ED, Hockness W. A technique for the disposal of oily refinery wastes. Report No.3/10, The Hague: Concawe, 1980. Chang ZZ, Weaver RW. Organic bulking agents for enhancing oil bioremediation in soil. J Soil Contam 1998;1:173 ± 80. Dibble JT, Bartha R. Effect of environmental parameters on the biodegradation of oil sludge. Appl Environ Microbiol 1994;37:729 ± 39. El-Nawawy AS, Ghobrial F, Elimam A. Feasibility study of disposal of oily sludge in Kuwait. Proceedings of the 2nd International Conference on New Frontiers for Hazardous Waste Management, Pittsburgh, PA, 27 to 30 September. 1987;162 ± 8. El-Nawawy AS, El-Bagouri IH, Abdal M, Khalafai MS. Bio-degradation of oily sludge in Kuwait soil. World J Microbiol Biotechnol 1992;8:618 ± 20. Freijer JI. Mineralization of hydrocarbons in soil under decreasing oxygen availability. J Environ Qual 1986;25:296 ± 304. Hillel D. Soil structure and aggregation. In: Introduction to soil physics, London: Academic Press, 1980. pp. 40 ± 52, 200 ± 204. Melope MB, Griwe IC, Pege ER. Contributions by fungi and bacteria to aggregate stability of cultivated soils. J Soil Sci 1987;38:71 ± 7. Parmer D, Schmidt EL. Experimental soil microbiology. Minneapolis: Burgess Publishing, 1964. pp. 6 ± 13. Rahman KSM, Vasudevan N, Lakshmanaperumalsamy P. Enhancement of biosurfactant production to emulsify different hydrocarbons. J Environ Pollut 1999;6:85 ± 93. Rhykerd RL, Crews B, McInnes KJ, Weaver RW. Impact of bulking agents, forced aeration and tillage on remediation of oil-contaminated soil. Biores Technol 1999;67:279 ± 85. Sandvik S, Lode A, Pedersen TA. Biodegradation of oily sludge in Norwegian soils. Appl Microbiol 1986;23:297 ± 301. von Wedel RT, Mosquera SF, Goldsmith CD, Hater GR, Wong A, Fox TA, Hunt WT, Paules MS, Quiros JM, Wiegand JW. Bacterial biodegradation of petroleum hydrocarbons in ground water: in situ augumented bioreclamation with enrichment isolates in California. Water Sci Technol 1988;20:501 ± 3.


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