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ISSN: 2155-6199
Journal of Bioremediation & Biodegradation
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Bioremediation of Soil Contaminated With Used Motor Oil in a Closed System

Abdulsalam S1*, Adefila SS2, Bugaje IM3 and Ibrahim S4
1Chemical Engineering Programme, Abubakar Tafawa Balewa University, Nigeria
2Department of Chemical Engineering, Ahmadu Bello University, Zaria, Nigeria
3Department of Chemical Engineering, University of Maiduguri, Borno state, Nigeria
4Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria
Corresponding Author : Abdulsalam S
Chemical Engineering Programme
Abubakar Tafawa Balewa University
Bauchi, Nigeria, PMB 0248
Bauchi State, Nigeria
Tel: +2348032839412
E-mail: [email protected]
Received May 02, 2012; Accepted November 21, 2012; Published November 23, 2012
Citation: Abdulsalam S, Adefila SS, Bugaje IM, Ibrahim S (2013) Bioremediation of Soil Contaminated With Used Motor Oil in a Closed System. J Bioremed Biodeg 4:172. doi: 10.4172/2155-6199.1000172
Copyright: © 2013 Abdulsalam S, et al. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

A study was carried out on biodegradation of soil contaminated with used motor oil in aerobic fixed bed bioreactors. Six treatments, labeled TR1 to TR6, were investigated in an experimental rig for 70 days continuously. Bioremediation indices such as Total Petroleum Hydrocarbon (TPH), Oil and Grease content (O&G), Total Heterotrophic Bacteria Count (THBC), Hydrocarbon Degrading Bacteria Count (HDBC) and CO2 respiration rates were monitored. In addition, environmental factors such as temperature, pH and moisture content were also monitored. Results revealed that 50, 63, 66, 57, 68 and 75% biodegradation were achieved for TR1, TR2, TR3, TR4, TR5 and TR6, respectively, in terms of the O&G content removal. Furthermore, CO2 respiration showed cumulative generations of 4 472, 5 226, 5 493, 5 279, 5 667 and 6 242 mg/kg CS for TR1, TR2, TR3, TR4, TR5 and TR6, respectively. From the results obtained, the biostimulation option (TR6) gave the best result and thus, can be used to develop a safe, robust and economical treatment technology for SCUMO.

Keywords
Biodegradation; Biostimulation; Used motor oil; Contamination; Soil; Bioreactor
Abbreviations
AB: Absorber; AC: Air Compressor; AFM: Air Flow Meter; B: Bioreactor; BV: Ball Valve; CFU: Colony Forming Unit; CS: Contaminated Soil; DF: Dilution Factor; EB: Exogenous Bacteria; GV: Gate Valve; HD: Humidifier; HDBC: Hydrocarbon Degrading Bacteria Count; HS: Heat Sterilization; N: Nitrogen; OC: Organic Carbon; O&G: Oil and Grease content; P: Phosphorus; PG: Pressure Gauge; RV: Relief Valve; SCUMO: Soil Contaminated with Used Motor Oil; SP: Sampling Point; THBC: Total Heterotrophic Bacteria Count; TR: Treatment; WAC: Water Absorption Capacity; WC: Water Content
Introduction
Used motor oil is the brown-to-black oily liquid removed from a motor vehicle, when the oil is changed. Used motor oil is similar to unused oil, except that it contains additional chemicals that are produced or build up in the oil, when it is used as an engine lubricant at high temperatures and pressures, inside an engine as it runs [1]. Used motor oil also contains metals such as aluminium, chromium, copper, iron, lead, manganese, nickel, silicon and tin that comes from engine parts, as they wear down [2].
Some of these metals in used motor oil can dissolve in water and move through the soil easily, and may be found in surface water and ground water. Thus, metals from used oils can build up in plants, animals, soil, sediments and non-flowing surface water. Heavy metals and chemicals in used motor oil are absorbed and distributed into various tissues of human, plants and animals by their movement in the environment, which can result in serious health problem, such as anemia, tremor and consequently, resulting in death [2]. Other health hazards which can result from used motor oil include mutagenicity and carcinogenicity [3-5].
In most countries of the world, oil spills at auto-mechanic workshops have been left uncared for over the years, and its continuous accumulation is of serious environmental concern because of the hazard associated with it, as discussed above. The physicochemical treatment technologies currently in use are expensive and not environmentally friendly. In addition, some of these technologies only transfer the contamination from one place to another.
In recent times, a lot of efforts have been made towards reducing environmental pollution, by using natural processes to treat environmental pollution. These techniques include: bioremediation (use of microorganisms to degrade pollutants) and phytoremediation (use of plants to clean pollutants by bioaccumulation into the plant’s tissues).
Bioremediation is the naturally occurring process by which microorganisms transform environmental contaminants into harmless endproducts, in order to obtain the sources of carbon and energy. During the process of bioremediation, which involves the activity of microorganisms to remove pollutants, environmental parameters such as temperature, pH, oxygen and moisture content, are optimized to achieve accelerated biodegradation.
Basically, there are two different approaches to bioremediation technologies, depending on the pollution situation and type of micro-organisms being used. The first is the one which involves the activation of the indigenous microflora in the polluted area by addition of nutrients and forming the best conditions of other chemical, physical and biological factor, or known as biostimulation. The second (bioaugumentation) is the one which involves the addition of oiloxidizing micro-organisms isolated from other sites, or addition of genetically engineered micro-organisms [6].
A lot of works have been reported on bioremediation of hydrocarbon pollutants, but just a few had reported on bioremediation of soil contaminated with used motor oil. Most work reported in the literature on the biological treatment of soil contaminated with used motor oil had been focused on the identification of microorganisms, which can be used to degrade used motor oil [7], and the use of plants for the degradation of used motor oil [8]. In addition, other works prior to this investigation were based on biological treatments of soil contaminated with spent motor oil in open systems [9,10].
The objectives of this study were to examine the effectiveness of bioremediation of soil contaminated with used motor oil, using aerobic fixed bed bioreactor to determine the best treatment option, which can be used to develop a safe, robust and economical treatment technology for soil contaminated with used motor oil, and to identify the types of indigenous bacteria in the contaminated soil. To our knowledge, this is the first experimental study which investigated biodegradation of soil contaminated with used motor oil in a closed aerobic fixed bed bioreactor.
Materials and Methods
Materials
Sample collection: Prior to this investigation, a preliminary investigation was carried out, in which five sites contaminated with used motor oil were investigated. The preliminary study was carried out in order to identify similarities and major differences in physicochemical and microbiological characteristics of the polluted soils, details of investigation are contained in [11]. From the preliminary investigation, sample collected from Baban Kaduna Auto-Mechanics Workshop was chosen as a representative sample due to its age of contamination.
Top soil (0-16.4 cm) contaminated with spent motor oil was collected from Baban Kaduna Auto-Mechanics Workshop located at Tudun Wada in Zaria, Kaduna State-Nigeria, and was stored at 4°C until it was required for the experiment. In addition, the bacteria (Pseudomonas aeruginosa and Bacillus subtilis) were obtained from the National Veterinary Research Institute Vom, Plateau State-Nigeria, and the Fertilizer (NPK 20:10:10) used for this study was obtained from Bauchi Fertilizer Company Limited, Bauchi-Nigeria.
Methods
Experimental design: Bioremediation of SCUMO was investigated in an aerobic fixed bed bioreactor. Six treatments were investigated as described in table 1.
Experimental apparatus: The experimental apparatus consisted of air compressor, pressure gauge, air pretreatment unit, humidifying unit, Air flow meter, Bioreactors, VOC Filters or Adsorbers, CO2 Absorbers and a stand; the process flow diagram is presented in figure 1.
Process description: These biodegradation investigations were carried out in six aerobic fixed bed bioreactors (TR1, TR2, TR3, TR4, TR5 and TR6), connected in parallel with 1.5 kg of contaminated soil; this included where appropriate, the various additives at room temperatures, such as inoculation of 2133 cfu/g CS of consortium of Pseudomonas aeruginosa and Bacillus subtilis, or heat sterilization at 12°C or 30.42 g of NPK 20:10:10 and 5.6 g of KH2PO4 to give carbon, nitrogen and phosphorus molar ratio of 100:10:1. The bioreactors were completely closed in order to avoid CO2 leakage to the environment, before passing into the CO2 traps. The process flow diagram is presented in figure 1.
The moisture content in all the six treatments was set at 20% (w/w) at the initiation of bioremediation. The airflow rate was maintained in all cases at an average rate of 10 L/hr, using a flow meter for fourteen (14) hours daily for the period of investigation.
Physicochemical and microbiological characteristics of contaminated soil: The following physicochemical and microbiological tests were carried out on all the treatments on weekly basis: Oil & Grease content (O&G), moisture content, pH, temperature, Total Heterotrophic Bacteria Count (THBC) and Hydrocarbon Degrading Bacteria Count (HDBC). In addition, carbon dioxide (CO2) respiration rate was monitored every 48 hr throughout the duration of this study. In addition, the isolation and identification of bacteria in SCUMO were carried out. Furthermore, the Total Petroleum Hydrocarbon (TPH) was measured before and after bioremediation processes.
Physicochemical and microbiological properties of each treatment were determined by the following standard methods.
• Particle size distributions were determined based on the unified soil classification [12].
• Bulk and particle densities were determined using the gravimetric analysis, and soil porosities calculated from bulk and particle densities values [13].
• Soil pH was determined using the method of Bates [14] and moisture contents by the ASTM-D2216 [15].
• Available phosphorus in soil samples was determined using the spectrophotometer, and total nitrogen content by the Kjedahl method.
• Total Petroleum Hydrocarbon (TPH) was carried out, based on the Modified U.S EPA 418.1 Method [16].
•The temperature was measured using a digital thermometer.
• Indigenous bacteria identification was carried out by morphological and biochemical characterization of petroleum hydrocarbon utilizers, following the methods of Buchanan and Gibbons [17].
• Total Heterotrophic Bacteria Counts (THBC) were carried out by employing the standard plate counting technique and Hydrocarbon Bacteria Degrading Counts (HDBC) were carried out by employing the Most Probable Number (MPN) analysis with 5 tubes, using the Bushnell-Haas medium [18].
• The CO2 respiration rates were determined using the titrimetric analysis.
Determination of O&G content (spectrophotometry method): Moist samples were collected aseptically from each bioreactor and airdried for 48 h. 5 g of each air-dried sample was extracted by vigorous hand shaking for 3 minutes, with 20 mL of toluene in a separation funnel. The mixtures were allowed to settle and the extracts were decanted into a volumetric flask and plug. The above procedure was repeated two times using 20 mL of toluene each time [19]. The total extracts were combined and diluted in the ratio 1:3 (extract: toluene), and then the absorbance of each sample was quantified using a CE 1020 (1000 Series) UV Spectrophotometer at 400 nm. Oil and grease contents were extrapolated from a standard curve of absorbance (A400 nm) against concentration. Values of concentration obtained were multiplied by the Dilution Factor (DF), to give the actual concentration.
Organic carbon in soil samples (colorimetric method): An amount of 0.5 g of contaminated soil sample sieved though 2 mm sieve was weighed into a glass beaker, 10 mL of 1N K2Cr2O7 was added to the glass. In addition, 10 mL of concentrated H2SO4 was carefully added. The solution was shaken gently and allowed to cool at room temperature. The solution was washed into 100 mL volumetric flask and made to the mark, with distilled water. In addition, a blank solution was prepared by adding 10 mL of 1N K2Cr2O7 and 10 mL of concentrated H2SO4. 0, 2, 4, 6, 8 and 10 mL of 2000 ppm sucrose solutions were pipetted into different flasks, 10 mL of 1N K2Cr2O7 and 10 mL of concentrated H2SO4 were added to each flask. Both the blank and standard solutions were washed into 100 mL flask and made to mark with distilled water. The standards now contain 40, 80, 120, 160, 200 ppm. The blank, standards and samples were read on a spectrophotometer at 600 nm. Samples concentrations were calculated using the following relation.
(1)
Results and Discussion
Environmental conditions
Bioreactors temperature: Variations in temperatures with time during the 70 days of bioremediation study in aerobic fixed bed bioreactors are presented in figure 2. The temperature range was between 26 and 32°C; this temperature range fell within the optimum range of 10 to 40°C, required for effective bioremediation to occur [20]. Therefore, temperature was not a limiting factor in this study.
Moisture content in bioreactors: Variations in moisture content with time for the ten weeks of bioremediation study in fixed bed bioreactors are presented in figure 3. The moisture content profiles in all the bioreactors (TR1 to TR6) were erratic, and their values deviated significantly from the optimum range of 10 to 20% (w/w) [21].
Low moisture contents observed can be attributed to the presence of oil in the soil, which resulted in the blockage of the pores, hence low water retention. Deviations of TR5 and TR6 were less compared to those of other treatments. This can be attributed to the high generations of water in TR5 and TR6 during the oxidation process, as evident by high CO2 generations for these treatments (Figure 4). These observations show that moisture content was a limiting factor to effective bioremediation in this study. The unusual moisture content of TR2 in week 6 requires further investigation to ascertain, if it is a phenomenon or due to experimental error.
pH in bioreactors: Figure 5 depicts the variations in pH with time in all the bioreactors. The pH range for TR1, TR2 and TR4 fell within the range of 5.5-8.5, required for effective bioremediation [21,22]. pH for TR5 and TR6 were above the upper limit of 8.5 in weeks 1 and 2 for TR5, and weeks 2 and 3 for TR6. These variations could be attributed to the addition of NPK (20:10:10) and KH2PO4 to TR5 and TR6, resulting in increase in alkalinity, hence, high pH values noticed in the first three weeks of the bioremediation processes.
In addition, in week 6, the pH value for TR5 fell below the lower limit, and in week 7, the pH value for TR3 was below the lower limit. TR3 and TR5 were the two treatment options, which were bioaugmented using consortium of Bacillus subtilis and Pseudomonas aeruginosa. The fall in pH could be attributed to some complexity within the medium, during the stated periods.
Total Petroleum Hydrocarbon (TPH) removal: The Total Petroleum Hydrocarbon for all the treatment options (TR1 to TR6) was determined before and after the bioremediation process, as shown in figure 6. At time zero (at the initiation of bioremediation process), the TPH concentrations of the contaminated soils in the bioreactors, TR1, TR2, TR3, TR4, TR5, and TR6 were 215, 215, 192, 326, 169 and 192 mg/kg dry weight, respectively. After 70 days of bioremediation, TPH concentrations reduced to 111, 94, 54, 129, 66 and 54 mg/kg dry weight, which are equivalent to 49, 56, 72, 60, 61 and 72% removals for TR1, TR2, TR3, TR4, TR5 and TR6, respectively. The extent of biodegradation obtained in the entire treatments investigated, fell within the range of 30-75%, reported for lubricating oils at optimum conditions [23].
Of the six treatment options investigated, treatment option 3 (TR3) and treatment option 6 (TR6) resulted in maximum TPH removal (72%). Treatment option 1 (TR1), in which the indigenous microorganisms were reduced by heat sterilization gave the least TPH removal (49%). The appreciable degradation obtained for TR1 could be linked to the residual bacteria that resisted heat sterilization action. In addition, since all the treatments were humidified and aerated at equal rates, this residual bacterium would proliferate in the vessel and hence, degrade the pollutant.
Oil and grease biodegradation: Although, the TPH removal can be used to monitor the extent of biodegradation of SCUMO as described above, the magnitude of TPH in used motor oil is low, which could be attributed to reduction in the C-H bonds in lubricating oils as it is being used, that leads to loss in viscosity. Because of the low magnitude of TPH in used motor oil, the TPH alone cannot be used to assess the extent of contamination of SCUMO. The oil and grease content is a better index for monitoring SCUMO, since the method is not limited to the C-H stretching.
The levels of reduction in the oil and grease content at different periods during the course of the study are shown in figure 7. From this figure, the percentage O&G content removal increased with time, which is typical of any degradation process. The process was characterized by a period of fast decrease in hydrocarbon concentrations during the first five weeks (40, 45, 51, 40, 59 and 63% for TR1, TR2, TR3, TR4, TR5 and TR6, respectively), followed by a period of slower activity (past week 5). The degradation pattern followed shifting order (1-0) degradation [24].
At the initiation of bioremediation (at time zero), the concentrations of O&G contents in bioreactors TR1, TR2, TR3, TR4, TR5 and TR6 were 29 010, 37 966, 35 519, 33 746, 32 027 and 38 592 mg/kg dry weight, respectively. After 70 days, their concentrations reduced to 14 439, 14 088, 12 085, 14 438, 10 115 and 9 830 mg/kg dry weight, which are equivalent to 50, 63, 66, 57, 68 and 75% losses in O&G contents.
Of the six treatments employed in this study, TR6 resulted in the maximum bioremediation response (75% reduction in O&G). This observation is in line with the literature that biostimulation strives well at sites that have been contaminated for sometimes, or aged contaminated sites [25].
CO2 generation in bioreactors: CO2 evolution was also used as indicator of bacteria respiration (a product of bioremediation process). Monitoring of CO2 generated in all the bioreactors (TR1 to TR6) during the 70-day experimental study of soil contaminated with used motor oil is presented in figure 8 and 4. The pattern of 48-hourly CO2 generation rates depicted in figure 4, shows an overall trend with random irregularities, typical of microbial respiration in soil [26].
Initially, the CO2 generation in the bioreactors was small (0 to 10 days), then increased, thus erratic and got to maximum on the 26th day from beginning (this corresponds to period of high pollutant removals) and then started declining (past 30 days), which correspond to period of slow pollutant removal. The decline in the CO2 generation can be attributed to exhaustion in the available nutrients or production of toxic metabolic products, which probably inhibits the activity of microorganisms. Finally, CO2 generations between the 66th and 70th days were almost constant, which was an indication of end of bioremediation, as evidenced by the 0, 1, 0, 1, 0 and 1% pollutant removals for TR1, TR2, TR3, TR4, TR5 and TR6, respectively, between weeks 9 and 10.
In addition, the 48-hourly measurements of CO2 generated by bioremediation process allowed the estimation of cumulative CO2 generation over the treatment period (Figure 4). All the treatment options (TR1 to TR6) appears to show a trend of adaptation period (1 to 10 days), maximum oil degrading period (25 to 55 days), and a decaying rate of oil degradation period (past 60 days).
The cumulative CO2 generated in each bioreactor increased with pollutant or oil degradation. The cumulative CO2 generated for TR1, TR2, TR3, TR4, TR5 and TR6 were 4 276, 5 226, 5 493, 5 279, 5 667 and 6 249 mg/kg, respectively. Treatment option 6 (TR6) gave the best CO2 generation, which corresponds to the best TPH (72%) and O&G content (75%) removals. The control (TR1) showed the least CO2 generation, which also corresponds to least TPH (49%) and O&G (50%) contents removals. In addition, it could be observed that profiles for all treatments had almost reached a plateau (Figure 4).
Bacteria counts in bioreactors
Total Heterotrophic Bacteria Count (THBC) in bioreactors: Profiles for the Total Heterotrophic Bacteria Count for all the treatments (TR1 to TR6) are presented in figure 9. These profiles follow typical microbial growth pattern for a batch culture of microorganisms with adaptation period, phase of exponential growth, stationary phase and the death phase [27].
At the initiation of the bioremediation process, THBC in TR1, TR2, TR3, TR4, TR5 and TR6 were 4.70E+06, 3.75E+08, 5.40E+06, 6.50E+06, 1.70E+07 and 2.69E+08 cfu/g of dry soil, respectively. In week 1, the THBC for TR2, TR5 and TR6 decreased to 1.40E+07, 1.50E+07 and 1.50E+07 cfu/g of dry soil, respectively. Decrease in TR2 and TR6 were of high magnitude, compared to that of TR5. Decrease in TR5 and TR6 could be attributed to the addition of NPK (20:10:10) and KH2PO4, which could cause upsets in carbon and inorganic nutrient balance for the indigenous population, caused by the presence of hydrocarbon pollutant. The low magnitude of decrease experienced in TR5 could be because of the consortium of Pseudomonas aeruginosa and Bacillus subtilis, used to augment the indigenous bacteria. At the end of the 70-day experiment, the cumulative THBC were 1.89E+09, 3.92E+12, 6.72E+12, 6.13E+12, 4.86E+12 and 5.28E+12 cfu/g of dry soil for TR1, TR2, TR3, TR4, TR5 and TR6, respectively.
All the treatments, except TR1, appear to show a similar trend of adaptation period, which lasted for 7 days. This suggests that indigenous and added bacteria could be of the same genus. Another striking feature is that all the treatments, except TR1, showed almost identical exponential growth phase (between weeks 1 and 4); this is the period in which microorganisms utilized the oil pollutant as their sole carbon and energy sources, thereby degrading the pollutant at faster rates (period of maximum oil degradation).
Bacteria populations in all bioreactors, except TR1, increased and reached maximum value of ≥ 1013 cfu/g of dry soil within four weeks, then remained stable between weeks 4 and 9, before declining. This observation is unusual since the treatments were not the same. This could be attributed to certain inhibitors or activators, or complexity of medium. On the other hand, similar observation was noticed for TR1, but it has low magnitude of bacteria population (≥ 1010 cfu/g of dry soil). In addition, TR1 showed a prolong adaptation period of 3 weeks, followed by 1 week of exponential growth, then like other treatments, the bacteria growth attained a maximum value in week 4 and remained constant up till week 9, before declining.
An increase in oil degradation between weeks 1 and 4 in all bioreactors was corresponding to an increase in the total heterotrophic bacteria number during the degradation processes, demonstrating the ability of utilizing used motor oil as the energy source. In addition, decline in bacterial populations between weeks 9 and 10 resulted in the termination of the bioremediation processes for all treatments, as could be attested to by inactivity of oil degradation during this period.
Furthermore, bacterial species identified in the test soil were Bacillus subtilis and Micrococcus leteus. The identification of these species was in line with previous investigation on spent motor oil contaminated soil [7].
Total hydrocarbon degrading bacteria counts: Figure 10 depicts the variation in HDBC with time for the ten weeks of experimental study of SCUMO in aerobic fixed bed bioreactors. From the profiles obtained, HDBC showed a general increase in bacteria count for all the treatments. At initiation of bioremediation processes, HDBC for TR1, TR2, TR3, TR4, TR5 and TR6 were 4.50E+02, 2.38E+03, 5.63E+03, 1.25E+03, 2.13E+03 and 1.63E+03 MPN/g dry weight, respectively. After 70 days, their concentrations were 7.57E+04, 4.07E+04, 4.25E+04, 6.61E+04, 6.17E+04 and 6.28E+04 MPN/g dry weights.
Treatment options TR4, TR5 and TR6 got to a maximum (≥ 10E+06 MPN/g dry weight) within the first 35 days, which corresponds to period of fast increase in hydrocarbon degradation (40, 59 and 63% removal in O&G content for TR4, TR5, and TR6, respectively). On the other hand, the period of decrease in HDBC correspond with time of slow rate of removal of pollutant. In addition, the HDBC for TR3 got to a maximum after 8 weeks, then decreased and leveled off. This corresponds to the high TPH (72%) and O&G content (66%) removals in TR3.
Selection of treatment technology for SCUMO: Based on the results obtained for TPH, O&G content and CO2 generation, Treatment option 6 (TR6), the sample stimulated with NPK (20:10:10) and KH2PO4 gave the best result. Therefore, TR6 could be used to develop a full-scale treatment technology for soils contaminated with used motor oil.
The 72 and 75% removals of TPH and O&G contents, respectively, could be improved by intermittent addition of nutrients as the biodegradation process proceeds. In addition, optimizing the moisture content level that was a limiting factor in this work, could further improve the hydrocarbon removal rate.
Conclusions
Based on the results obtained in this investigation, the following conclusions can be drawn.
1. Biodegradation of soil contaminated with used motor oil is successful to an appreciable extent in a closed bioreactor (solid-phase) system.
2. Petroleum removal efficiencies, in terms of oil and grease content removals, can reach 75% over a period of 70 days in aerobic fixed bed bioreactor, within the range of experimental conditions in this study.
3. The biostimulation option was selected as the treatment technology for soil contaminated with used motor oil, as it produced the highest contaminants removal (75% in 70 days).
4. Two types of indigenous bacteria were identified from the test soil: they were Bacillus subtilis and Micrococcus luteus.
Recommendations
1. Results obtained in this work should be used to develop a pilotplant rehabilitation technology for soils contaminated with used motor oil.
2. The influence of fertilizer types should be investigated on oil and grease removal rates, in order to ascertain the type(s) of fertilizer that would lead to the most efficient hydrocarbon removal rates. For instance, there are three classes of fertilizers: agricultural, slow-release and oleophilic formulations.
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