Soil and surface water contamination by used lubricating oil is a common occurrence in most developing countries. This has been shown to have harmful effects on the environment and human beings at large. Bioremediation can be an alternative green technology for remediation of such hydrocarbon-contaminated soil. Bioremediation of soil contaminated with 5% and 15% (w/w) used lubricating oil and amended with 10% brewery spent grain (BSG), banana skin (BS), and spent mushroom compost (SMC) was studied for a period of 84 days, under laboratory condition. At the end of 84 days, the highest percentage of oil biodegradation (92%) was recorded in soil contaminated with 5% used lubricating oil and amended with BSG, while only 55% of oil biodegradation was recorded in soil contaminated with 15% used lubricating oil and amended with BSG. Results of first-order kinetic model to determine the rate of biodegradation of used lubricating oil revealed that soil amended with BSG recorded the highest rate of oil biodegradation (0.4361 day−1) in 5% oil pollution, while BS amended soil recorded the highest rate of oil biodegradation (0.0556 day−1) in 15% oil pollution. The results of this study demonstrated the potential of BSG as a good substrate for enhanced remediation of hydrocarbon contaminated soil at low pollution concentration.
Contamination of soil by used lubricating oil is rapidly increasing due to global increase in the usage of petroleum products [
Prolonged exposure to high oil concentration may cause the development of liver or kidney disease, possible damage to the bone marrow, and an increased risk of cancer [
Lack of essential nutrients such as nitrogen and phosphorus is one of the major factors affecting biodegradation of hydrocarbon by microorganisms in soil and water environment. Therefore, the addition of inorganic or organic nitrogen-rich nutrients (biostimulation) is an effective approach to enhance the bioremediation process [
Concentration of petroleum hydrocarbon determines to a greater extent the rate of breakdown of the hydrocarbons from soil environment. High concentration of hydrocarbon can be inhibitory to microorganisms, and concentration at which inhibition occurs varied with the compound. Ijah and Antai [
The objectives of this study are to determine the potential of banana skin, brewery spent grain, and spent mushroom compost for enhanced biodegradation of used lubricating oil in soil, as an alternative to the use of inorganic fertilizers. These organic materials are widely available as wastes in our environment. The study also aimed to determine the effects of oil concentration on biodegradation of used lubricating oil.
The soil sample used was collected from the Nursery Section of the Asia-European Institute, University of Malaya, Kuala Lumpur, Malaysia, in a sack and transported to the laboratory for analysis. Used lubricating oil was collected from the Perodua Car Service Centre, Petaling Jaya, while the organic wastes were collected from different locations; banana skins (BS) were collected from the IPS Canteen, University of Malaya, brewery spent grains (BSG) were collected from Carlsberg Brewery, Shah Alam, Selangor, and spent mushroom compost (SMC) was the collected from Gano Mushroom Farm, Tanjung Sepat, Selangor.
1.5 kg of soil (sieved with 2 mm mesh size) was placed in plastic vessels with a volume of about 3000 cm3, and 5% and 15% (w/w) used lubricating oil was added separately, thoroughly mixed, and left undisturbed for 48 hours to allow the volatilization of toxic components of the oil. After two days, 10% of each organic waste (ground dry banana skin (BS), brewery spent grain (BSG), and spent mushroom compost (SMC)) were individually introduced into each oil-polluted soil and thoroughly mixed. The moisture was adjusted to 60% water holding capacity and incubated at room temperature (28 ± 2°C). Treatment with only soil and used lubricating oil served as control. Additional control was also set up which contained autoclaved soil poisoned with 0.5% (w/w) sodium azide to monitor nonbiological loss of oil in the oil-contaminated soil. The content of each vessel was tilled twice a week for aeration and the moisture maintained at 60% water holding capacity by the addition of sterile distilled water. The experiment was set up in triplicate. Periodic sampling from each vessel was carried out at 14-day intervals for 84 days. Composite samples were obtained by mixing 5 g of soil collected from four different areas of the plastic vessels for isolation and enumeration of hydrocarbon utilizing bacteria and determination of total petroleum hydrocarbon.
Nitrogen contents of soil used for bioremediation and organic wastes were determined using the Kjeldahl method, while phosphorus and carbon contents were determined using ICP-QES and furnace method, respectively. pH was determined with pH meter (HANNA HI 8424) on 1 : 2.5 (w/v) soil/distilled water after 30minute equilibration. Triplicate determinations were made.
Residual hydrocarbon contents of the soil samples were determined by toluene cold extraction method of Adesodun and Mbagwu [
Three replicate samples from each oil-polluted soil were withdrawn every 14 days for the enumeration of hydrocarbon utilizing bacteria (HUB). 0.1 mL of serially diluted samples were plated on oil agar prepared from mineral salt medium of Zajic and Supplisson [
Gram-negative bacterial isolates were identified using API 20 NE. Pure culture colonies of bacterial sample were transferred into an ampoule of API NaCl 0.85% medium (2 mL) with the aid of inoculating wire loop to prepare a suspension with a turbidity equivalent to 0.5 McFarland standard. Tests of NO3 to PNPG in the API panel were inoculated by distributing the saline suspension into the tubes using sterile pipette. 200
Toxicity of the remediated soils was assessed using germination test. Lettuce was used in this study owing to its sensitivity to hydrocarbon in soil [
Germination index of lettuce seed on the remediated soil was calculated using the formula of Millioli et al. [
Statistical analysis of data was carried out using Analysis of Variance (ANOVA).
The physicochemical properties of soil and organic wastes used for the bioremediation studies are shown in Table
Physicochemical properties of soil and organic wastes used for bioremediation.
Parameter | Soil | Organic wastes | ||
---|---|---|---|---|
BSG | BS | SMC | ||
pH |
|
|
|
|
Nitrogen (%) |
|
|
|
|
Phosphorus (mg/kg) |
|
|
|
|
Organic C (%) |
|
|
|
|
Moisture (%) |
|
|
|
|
Sand (%) |
|
— | — | — |
Silt (%) |
|
— | — | — |
Clay (%) |
|
— | — | — |
HUB (CFU/g) |
|
|
|
|
Texture | Clayey | — | — | — |
BSG: Brewery spent grain, BS: banana skin, SMC: spent mushroom compost, HUB: hydrocarbon utilizing bacteria.
The percentage of oil biodegradation in the soil contaminated with 5% and 15% used lubricating oil is shown in Figures
Biodegradation of petroleum hydrocarbon in soil contaminated with 5% used lubricating oil and amended with 10% organic wastes.
Biodegradation of petroleum hydrocarbon in soil contaminated with 15% used lubricating oil and amended with 10% organic wastes.
At the end of 28 days in soil contaminated with 15% oil, there were 17%, 24%, and 5% total petroleum hydrocarbon (TPH) degradation in soil amended with BSG, BS, and SMC, respectively. The reason for the low percentage of oil degradation within the first 28 days might be attributed to the toxicity of the oil on the microbial flora of the soil, due to high concentration of oil which might likely had negative effects on the biodegradative activities of the microbial population in the contaminated soil. This initial trend of low biodegradation due to high oil concentration has been reported by different authors [
BSG-amended soil recorded highest percentage biodegradation (92% and 55%) throughout the 84 days period in 5% and 15% oil-contaminated soil, respectively. This might be due to high N and P contents present in BSG. N and P are known as the most important nutrients needed by hydrocarbon-utilizing bacteria to carry out effective and efficient biodegradative activities of xenobiotics in the soil environment [
First-order kinetics was used to determine the rate of biodegradation of used lubricating oil in the various treatments as shown in Table
Biodegradation rates of hydrocarbon in used lubricating-oil-contaminated soil.
Treatment | Biodegradation constant ( |
---|---|
Soil + 5% oil + BS | 0.4010b |
Soil + 5% oil + BSG | 0.4361b |
Soil + 5% oil + SMC | 0.3100b |
Soil + 5% oil | 0.1886a |
Autoclaved soil + 5% oil | 0.0079a |
Soil + 15% oil + BS | 0.0556b |
Soil + 15% oil + BSG | 0.0479a |
Soil + 15% oil + SMC | 0.0216b |
Soil + 15% oil | 0.0092a |
Autoclaved soil + 15% oil | 0.0033a |
Values followed by letter b indicate significant difference at
The results show significant relationships between the rate of biodegradation and concentration of oil in the contaminated soil. From the results, higher biodegradation rates were recorded in soil contaminated with 5% oil; this high biodegradation rate could be attributed to increase in the activity of soil microbes in this oil pollution level [
Count of hydrocarbon utilizing bacteria (HUB) in soil contaminated with 5% used lubricating oil and amended with organic wastes is shown in Figure
Hydrocarbon-utilizing bacteria (HUB) in soil contaminated with 5% used lubricating oil and amended with organic wastes.
Hydrocarbon-utilizing bacteria (HUB) in soil contaminated with 15% used lubricating oil and amended with organic wastes.
The counts of hydrocarbon utilizing bacteria (HUB) in all the soil amended with organic wastes were appreciably higher compared to those of unamended and poisoned control soil. The reason for higher counts of bacteria in amended soil might be as a result of presence of appreciable quantities of nitrogen and phosphorus in the organic wastes, especially high nitrogen content in BSG, which are necessary nutrients for bacterial biodegradative activities [
The HUB isolated from the used lubricating-oil-contaminated soil were identified as species of
Lettuce (
Toxicity test based on seed germination (%).
Percentage of oil pollution | Treatments | |||||
---|---|---|---|---|---|---|
A | B | C | D | E | F | |
5 | 80 ± 6.0 | 100 | 80 ± 6.0 | 40 ± 6.0 | 20 ± 0 | 100 |
15 | 40 ± 5.8 | 40 ± 6.0 | 20 ± 0 | 10 ± 0 | 0 | 100 |
A = Soil + Oil + BS, B = Soil + Oil + BSG, C = Soil + Oil + SMC, D = Soil + Oil, E = Autoclaved soil + Oil + NaN3, F = Uncontaminated soil.
Germination index of lettuce seed on the remediated soil was calculated using the formula of Millioli et al. [
Seed germination toxicity index (%).
Percentage of oil pollution | Germination toxicity index (%) | ||||
---|---|---|---|---|---|
A | B | C | D | E | |
5 | 40.00 | 83.33 | 33.34 | 13.33 | 3.27 |
15 | 6.53 | 13.33 | 5.00 | 1.65 | 0.00 |
A = Soil + Oil + BS, B = Soil + Oil + BSG, C = Soil + Oil + SMC, D = Soil + Oil, E = Autoclaved soil + Oil + NaN3.
Amendment of soil contaminated with used lubricating oil with organic wastes positively enhanced the rate of biodegradation of used lubricating oil in soil within the period of 84 days. The results of the studies in soil contaminated with 5% and 15% used lubricating oil amended with organic wastes (BS, BSG, and SMC) show low (55%) oil biodegradation in soil contaminated with 15% oil compared with 92% oil biodegradation recorded in 5% oil pollution, thus, showing that level of oil contamination influenced the rate of oil biodegradation in soil environment. Contaminated soil amended with BSG recorded highest rate of oil biodegradation and counts of hydrocarbon utilizing bacteria compared to soil amended with BS and SMC in both 5% and 15% oil pollution. Results of germination toxicity test carried out on the remediated soil showed less toxicity to lettuce in 5% oil-contaminated soil compared to those of 15% oil-contaminated soil. Therefore, brewery spent grain, which is a waste from brewery, can be utilized effectively to reclaim soil contaminated with used lubricating oil.
The authors would like to acknowledge the support of University of Malaya, IPPP grant PS 244/2008C, and FRGS Grant FP014/2010A. Also, they would like to thank the management of the Carlsberg Brewery for providing brewery spent grain and the Ganofarm Sdn. Bhd for the provision of spent mushroom compost.