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 acciden-

tal contamination of soil with petroleum hydrocarbons.

Petroleum refining also results in the generation of large

quantities of oil sludge consisting of hydrophobic substan-

ces 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 remedia-

tion 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 air-

dried and passed through a sieve (2 mm); soil pH was

0160-4120/01/$ ± see front matter D 2001 Elsevier Science Ltd. All rights reserved.

PII: S0 1 6 0 - 4 1 2 0 ( 0 1 ) 0 0 0 20 - 4

* Corresponding author. Tel.: +91-44-2354-296; fax: +91-44-2354-

717.

E-mail address: nvasu30@yahoo.com (N. Vasudevan).

www.elsevier.com/locate/envint

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 con-

tained 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 sludge (abiotic control)

Soil + oil sludge

Soil + oil sludge + compost

Soil + oil sludge + bacterial consortium

Soil + oil sludge + inorganic nutrients + bacterial consor-

tium

Soil + oil sludge + wheat bran + bacterial consortium

The abiotic control containing soil, oil sludge and 0.3%

w/w AgNO3 was used to monitor the abiotic loss of hydro-

carbons. The treatment unit with inorganic nutrients

received 24 g of ammonium nitrate and 4.37 g of dipotas-

sium 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

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 determina-

tions 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% satu-

rated 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 corre-

sponding 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-

Table 1

Relative percent biodegradation of oily sludge and bacterial count in soil

Time after sludge application (days)

15 30 45 60 75 90

Sample no. Treatment (Percent degradation and cell number)a

1 Soil + oil sludge (abiotic control) ± ± ± ± ± ±

2 Soil + oil sludge 1 (6� 103) 4 (2� 104) 12 (6� 104) 18 (2� 105) 24 (3� 105) 25 (5� 105)

3 Soil + oil sludge + compost 2 (8� 104) 6 (3� 105) 16 (1�106) 3 (3� 106) 27 (6� 106) 28 (9� 106)

4 Soil + oil sludge + activated sludge 2 (3� 105) 7 (3� 106) 16 (8� 106) 24 (1�107) 27 (2� 107) 29 (3� 107)

5 Soil + oil sludge + bacterial consortium 2 (1�107) 8 (1�108) 19 (2� 108) 28 (1�109) 34 (4� 109) 40 (2� 1010)

6 Soil + oil sludge + inorganic nutrients

+ bacterial consortium

4 (6� 107) 13 (2� 109) 27 (6� 1010) 44 (5� 1011) 54 (2� 1012) 65 (3� 1012)

7 Soil + oil sludge + wheat bran

+ bacterial consortium

5 (1�109) 15 (3� 1010) 28 (3� 1011) 45 (3� 1012) 56 (2� 1013) 72 (6� 1013)

a Cell number (cfu/g) in parentheses.

N. Vasudevan, P. Rajaram / Environment International 26 (2001) 409±411410

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 El-

Nawawy 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 bioremedia-

tion 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 compris-

ing 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.

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