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Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil
International Biodeterioration & Biodegradation 63 (2009) 1054–1060
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Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil Ragheb A. Tahhan*, Rouba Youssef Abu-Ateih Department of Natural Resources and Environment, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 March 2009 Received in revised form 4 September 2009 Accepted 4 September 2009 Available online 9 October 2009
The main objective of this study was to evaluate the effect of oily sludge concentration on its biodegradability in soil. Oily sludge was collected and applied to microcosms at full-, half-, or quarterstrength concentrations equivalent to 44.2, 22.2, and 11.1 g kg1 soil, respectively, of total petroleum hydrocarbons (TPH) contained in oily sludge. The biodegradability of oily sludge was evaluated by measuring CO2 evolution and by measuring removal of TPH as well as its main composing fractions; namely; alkanes, aromatics, NSO-compounds, and asphaltenes. The collected soil contained 3.63 106 cfu g1 soil of hydrocarbon-degrading bacteria, which is satisfactory to drive successful biodegradation of hydrocarbons in soil. These numbers increased significantly with oily sludge addition at a rate proportional to the added TPH reaching 3.35 107 cfu g1 soil in the half-strength treatment. TPH mineralization rate followed the same pattern. However, TPH-mineralization efficiency was the greatest in quarter-strength treatment at 18.3%. TPH-removal efficiency was also highest in quarterstrength treatment at 30.9%. Nutrients addition caused mineralization inhibition. Since nutrients were added as a ratio of the added carbon, inhibition was the greatest with the highest TPH treatment. While alkanes were degraded, aromatics and asphaltenes were not, and NSO-compounds were enriched. Although SDS was completely biodegradable in soil, its addition promoted mineralization and removal of TPH from soil. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Oily sludge Biodegradation TPH TPH-extraction TPH-fractionation
1. Introduction Petroleum refining unavoidably generates considerable volumes of oily sludge during oil production and processing activities. Total petroleum hydrocarbons (TPH) are the most deleterious components in oily sludge, because of their potential hazard to human health and the environment. TPH are primarily composed of alkanes, aromatics, nitrogen-, sulfur-, and oxygen- (NSO)-containing compounds, and asphaltene fractions (Admon et al., 2001; Ali and Hassan, 2002; Mishra et al., 2001a). Bioremediation techniques are sound alternative methods for the treatment of TPH-contaminated soil. Such techniques are economically and politically attractive and have shown promising results in the treatment of soil contaminated with organic compounds, particularly with petroleum hydrocarbons (Chaineau et al., 2005; Garcia-Blanco et al., 2007; Yerushalmi et al., 2003).
* Corresponding author. Tel.: þ962 2 7201000x22050; fax: þ962 2 7201078. E-mail address: [email protected] (R.A. Tahhan). 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.09.001
However, there are still several technical aspects regarding bioremediation of oily sludge that are yet to be resolved. The addition of oily sludge to soil (5–30%; equivalent to 9–61 mg TPH/g soil) resulted in 70–90% degradation of TPH during two months regardless of their initial concentrations (Admon et al., 2001). However, Lazar et al. (1999) found that oily sludge concentration has an effect on the percentage of its biodegradation; the best biodegradation occurred for the treatment that received 10% oily sludge (equivalent to 3.2% initial TPH). Nutrient deficiency might be a limiting factor in the biodegradation process. Admon et al. (2001) found that hydrocarbon loss was observed only after nutrients were amended to oily sludgecontaminated soil at C:N:P ratio equivalent to 50:10:1. Jordanian oil refinery operations end up with storage of the produced oily sludge in concrete lined pools. This process will eventually lead to accumulation of large quantities of this material beyond their storage capacity or safe disposal processes. In addition, the operations involved in production, transport, and disposal of oily sludge result in spills of oily sludge on large soil areas. This study was designed to evaluate the effect of TPH concentration on its biodegradation in soil. The study also proposes a possible approach for remediating TPH-contaminated soil.
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2. Materials and methods 2.1. Soil collection and preparation The soil used in this study was collected from an orchard area inside the oil refinery campus in Zarka. This soil is regularly irrigated with treated oily wastewater, which is characterized with high salinity (6 dS m1) and an inhibitory effect on soil carbon mineralization activity (data not shown). Briefly, surface soil layer (15 cm deep) was collected in plastic bags. A small portion of the soil was packed on ice for immediate analysis of microbiological characteristics. The soil was brought to the laboratory, mixed thoroughly, and passed through a 6.3 mm sieve in order to remove large roots and plant residues. This preparation was intended for use in biodegradation studies. For physical, chemical, and biological analysis, however, the soil was passed through a 2 mm sieve (Vasudevan and Rajaram, 2001). 2.2. Soil analyses Organic matter was determined by measuring organic carbon, using Walkley Black method (Nelson and Sommers, 1996). pH was determined in 1:1 soil:water mixture by standard soil analytical methods (Thomas, 1996). Soil texture was determined by hydrometer method (Gee and Bauder, 1986). Total heterotrophic bacterial counts (THBC) and hydrocarbon-degrading bacterial counts (HCDBC) in soil were determined by spread plating method (Zubber, 1994). R2A agar plates (Margesin and Schinner, 2001) were used to enumerate heterotrophic microorganisms. Plates were incubated for 48 h at 28 C. Hydrocarbon utilizing bacteria were quantified on MSM agar plates. MSM had the following composition, in g/L DD H2O:KH2PO4, 1.0; Na2HPO4, 1.0; NH4NO3, 0.5; (NH4)2SO4, 0.5; MgSO4$7H2O, 0.2; CaCl2$2H2O, 0.02; FeCl3, 0.002; MnSO4$2H2O, 0.002 and agar, 16 (Herman et al., 1997). MSM plates were spread with the appropriate dilution and then sprayed with diethyl ether-dissolved oily sludge as a carbon source. Plates were incubated for two weeks at 28 C. All measurements were done in triplicate. 2.3. Oily sludge collection and analyses Oily sludge samples were collected from oily sludge storage basins inside the refinery campus. Moisture content was determined by oven drying overnight at 105 C (Nikitina et al., 2003). To determine TPH content in oily sludge, a triplicate of 1 g of air dried oily sludge was extracted with 100 ml of each hexanes, dichloromethane, and chloroform, successively. The extracts were pooled and air dried to evaporate the solvents. TPH was then determined gravimetrically (Mishra et al., 2001b). We collected several samples of oily sludge over 2 years. Considerable variations in content were observed between these samples. However, only one sample was used in the biodegradability experiments for this study. The composition of this sample was as follows: 64.91 0.16% moisture; 24.45 1.46% TPH; and 10.76 1.05% residue. This analysis was one in triplicate.
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(100 ml each). After evaporation of the solvents, each fraction (including asphaltenes) was determined gravimetrically (Mishra et al., 2001b). TPH composition for the sample used in this study was as follows: 66.65 2.93% alkanes, 14.71 1.72% aromatics, 7.77 5.33% NSO-compounds, and 11.99 1.83% asphaltenes. These analyses were done in triplicate. 2.5. Oily sludge biodegradability study Oily sludge biodegradability was evaluated via C-mineralization experiments (Zibilski, 1994). These experiments (treatability studies) were conducted in order to determine the biodegradability of various concentrations of oily sludge in soil. Four rates of oily sludge were tested. TPH concentrations with the applied oily sludge were; control (no addition), 11.1 (quarter-strength), 22.2 (halfstrength), and 44.2 g kg1 soil (full-strength). In addition, nutrients (NH4NO3 and KH2PO4) were added to achieve C:N:P ratio equivalent to 50:10:1 (Admon et al., 2001; Yerushalmi et al., 2003). Fifty grams of air-dried soil were mixed with oily sludge, and then incubated in sealed jars. Carbon dioxide evolution was periodically collected in alkali and evaluated by titration with HCl. Moisture content was monitored gravimetrically during the experimental period and maintained at 60% of the water holding capacity. At the end of the incubation period, all treatments were subjected to TPH extraction as mentioned below, TPH fractionation, total heterotrophic bacterial counts and hydrocarbon-degrading bacterial counts. This experiment was replicated four times. 2.6. Synthetic surfactants aided biodegradation Two different synthetic surfactants; Triton X-100 (non-ionic surfactant) and Sodium dodecyl sulfate, SDS (anionic surfactant) were used in this study as they were recommended previously in the biodegradation of hydrocarbons. The Critical Micelle Concentration (CMC) was considered to be 0.24 mM for Triton X-100 and 8.1 mM for SDS (Jones and Chapman, 1995). Oily sludge rates were similar to the oily sludge biodegradability study described above. Surfactants were added at two rates to achieve the following concentrations in soil water; below the critical micelle concentration (sub-CMC ¼ ½ CMC) and above the critical micelle concentration (supra-CMC ¼ 2 CMC). In addition, separate surfactants biodegradability experiments were conducted. All these experiments were done in four replicates. 2.7. TPH extraction from soil TPH was extracted from 25 g soil with two portions of 100 ml of each hexane, dichloromethane, and chloroform, successively, as described above for TPH extraction from oily sludge. Solvents were evaporated to a constant weight before TPH was determined gravimetrically. 3. Results and discussion 3.1. Soil properties
2.4. TPH fractionation TPH was extracted as described by Mishra et al. (2001b). Extracted TPH (0.2–0.5 g) were dissolved in n-pentane and separated into soluble and insoluble fractions (asphaltenes). The soluble fraction was loaded onto a silica gel column. Alkane fraction was eluted with 100 ml of hexanes, aromatic fraction was eluted with 100 ml benzene, and finally, NSO-containing compounds (resins) fraction was eluted with a mixture of chloroform and methanol
Soil properties are presented in Table 1. Organic carbon level (1.3%) is typical for an area such as Zarka, which is characterized by a hot dry climate. pH of the collected soil is slightly alkaline, which is also typical for this area. Such pH values are not reported to be inhibitory for biodegradative abilities of microorganisms to hydrocarbons. Electrical conductivity of the soil is fairly high. This is mostly a result of irrigation with the saline oily wastewater. High salinity in the soil probably imposes stress on microorganisms.
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Table 1 Physical, chemical, and microbiological properties of soil collected from the oil refinery campus.
Cumulative C-CO2 (mg kg-1 soil)
3500
Property Organic carbon SD (%) pH SD EC (dS m1) Soil texture Bulk density (g/cm3) Porosity (%) THBC SD (cfu 106) HCDBC SD (cfu 106)
1.31 7.44 12.12 Clay 1.124 57.58 6.07 3.63
0.095 0.021
1.29 1.39
EC: Electrical Conductivity. THBC: Total Heterotrophic Bacterial Count. HCDBC: Hydrocarbon-Degrading Bacterial Count. cfu: colony forming unit. SD: Standard Deviation.
3000
2500
2000
1500
1000
500
0
However, it seems that soil bacteria are adapted to this salinity level because they have shown high numbers (6.07 106 cfu g1 soil). Total heterotrophic bacterial counts in this soil are high enough, and above the recommended range for successful hydrocarbon degradation (U.S. EPA, 2004). Moreover, the soil contains high counts of hydrocarbon-degrading bacteria (3.63 106 cfu g1 soil). This is probably a result of adaptation to the continuous addition of hydrocarbons contained in the treated oily wastewater applied to the soil. According to Mishra et al. (2001a), hydrocarbon-degrading bacteria that are indigenous to soil with populations exceeding 105 cfu g1 soil are usually enough to satisfactorily carry out the degradation process without the need for bioaugmentation.
0
20
40
60
80
100
120
Time (days) Fig. 1. Organic carbon mineralization from unamended refinery soil (7 zero-strength; control), and soil amended with different rates of oily sludge, measured by C–CO2 evolution. C full-strength, B half-strength, ; quarter-strength. Error bars represent the standard deviation.
pattern was common to all oily sludge loading rates studied. It was characterized by an initial period of 40 days, where C–CO2 evolution rate was low. This period was considered as an acclimation period. A second phase started after 40 days indicating an active TPH mineralization. A third phase that started after 90 days was roughly identified. It was characterized by lower rate of C-mineralization especially for half- and full-strength application rates. Bossert et al. (1984), noted that an active phase of CO2 evolution was followed by a late phase (closure phase) that showed ‘‘greatly decreased’’ CO2 evolution activity. TPH removal is defined as the amount of extracted TPH subtracted from the amount of added TPH. TPH-removal efficiency is the ratio of TPH removal to the amount of added TPH. TPH removal in the quarter-, half-, and full-strength treatments that received oily sludge alone also followed the same trend as that in TPH mineralization. TPH removal was 3.4, 4.4 and 8.8 g kg1 soil, for quarter-, half-, and full-strength treatments, respectively (Table 2). In addition, TPH-removal efficiency in quarter-strength treatment was the greatest (31%). TPH removal was about 20% in both half- and fullstrength treatments. The carbon used to build new cell mass was calculated based on documented cell yield value of 1.12 (Sukatsch and Johnson, 1972). If we consider that TPH in oily sludge is identical to TPH in petroleum oil, then, the amount of TPH removal should be equivalent or greater than the summation of mineralized-C and biomass-C. However, our results show exactly the opposite of that, where removal was smaller than the summation of mineralized-C
3.2. Effect of oily sludge loading rate on biodegradation As oily sludge loading rate increases, TPH concentration and any other potentially toxic compound increase as well. Thus, it is necessary to test the effect of diluting oily sludge on the rate and extent of biodegradation. TPH mineralization is represented by the amount of carbon evolved as carbon dioxide. TPH-mineralization efficiency is the ratio of carbon evolved as carbon dioxide to the amount of TPH-carbon added to the soil. The amount of mineralized TPH increased with increasing oily sludge loading rate. For instance, when soil was amended with oily sludge alone, the amounts of carbon dioxide evolved were 3.1, 2.4, and 1.7 g kg1 in full-, half-, and quarter-strength treatments, respectively (Table 2). However, mineralization efficiency was the greatest for quarterstrength treatment, and generally, inversely proportional to the amount of TPH added. This shows that the mineralized carbon is proportional to the amount of TPH added. Fig. 1 shows the cumulative C–CO2 evolved from refinery soil amended with different levels of oily sludge. Application of oily sludge increased the rate of C–CO2 evolution by 2.2, 3.1, and 3.9 fold for quarter-, half-, and full-strength treatments. A mineralization
Table 2 Mineralization from refinery soil (control), and mineralization, and TPH removal from refinery soil amended with different rates of oily sludge with or without nutrients. TPH removal (g kg1 soil) SD
TPH removal (%) SD
Mineralized C, C–CO2 (mg kg1 soil) SD
Mineralized C, C–CO2 (%)
C-Biomass (mg kg1 soil)
C–CO2 þ C-biomass (%)
Sludge alone Control (no sludge) Quarter-strength Half-strength Full-strength
NA 3.4 0.4 4.4 0.2 8.8 1.0
NA 30.91 3.64 19.52 1.04 20.06 2.28
774.8 1709.6 2404.4 3079.2
2 6 4 15
NA 18.3 12.8 8.2
NA 3989 5600 7185
NA 61.0 42.5 27.3
Sludge þ Nutrient Zero-strength Quarter-strength Half-strength Full-strength
NA 2.8 0.2 4.0 0.6 7.2 0.8
NA 24.85 2.10 17.72 2.75 16.44 1.88
658 1353.6 916.6 761.8
7 5 5 2
NA 14.5 4.9 2.0
NA 3159 2139 1778
NA 48.3 16.2 6.8
Treatment
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and biomass-C. Thus, we concluded that the cell yield value used here is overestimated, especially for low oily sludge loading rates (Table 2). This phenomenon can be attributed to the possibility of presence of growth inhibitory agents in oily sludge. Furthermore, Maier (2000) suggested that utilization of the substrate can occur without production of new cells in some cases, namely low substrate concentration or low substrate bioavailability. In this case the energy from substrate utilization is used to meet the maintenance requirements of the cell under non-growth conditions. Bacterial counts in the soil were 6.07 106 and 3.63 106 cfu g1 soil for THBC and HCDBC, respectively (Table 1). Incubating soil without the addition of external carbon source (control) caused reduction of both THBC and HCDBC to 1.90 105 and 2.10 105 cfu g1 soil, respectively (Table 3). On the other hand, the addition of oily sludge to incubated soil caused a reduction in THBC, and an increase in HCDBC compared to original soil counts. For instance, THBC in quarter- and half-strength treatments decreased from 6.07 106 cfu g1 soil to 2.23 106 and 5.21 106 cfu g1 soil, respectively. However, HCDBD was 8.89 106 and 33.5 106 cfu g1 soil after the addition of oily sludge for quarter- and half-strength treatments, respectively. This was an expected result, as supplying hydrocarbons contained in the added TPH enriches HCDBC. Surprisingly, the ratio of HCDBC:HCDB exceeded 100% in some cases, especially those with active hydrocarbon mineralization activity. It is well documented that hydrocarbon degraders are heterotrophs, thus, they are a part of THBC. The reason for this apparent discrepancy is probably related to culturing conditions. Hydrocarbon degraders were incubated for 2 weeks and produced tiny colonies, which gave room on the plate for the growth of large numbers. On the other hand, heterotrophs were incubated on a complex medium which allowed the formation of large colonies with limited number. In addition, hydrocarbon degraders are in part oligotrophs characterized by slow growth which gives them the ability to grow on a diluted medium such as the medium used here, but they do not grow well on R2A agar. By increasing the oily sludge loading rate, the HCDBC numbers increased, thus, increasing the amount of mineralized-TPH. Therefore, there was a positive correlation between the HCDBC numbers and the biodegradation or mineralization capacity of oily sludge. For instance, C–CO2 evolution, TPH removal, and HCDBC were 1.7 g kg1 soil, 3.4 g kg1 (Table 2), and 8.89 106 cfu g1 soil (Table 3), respectively, in quarter-strength treatment that received oily sludge alone; and increased to 2.4 g kg1 C–CO2, 4.4 g kg1 of TPH (Table 2), and 33.5 106 cfu g1 soil (Table 3), respectively, when oily sludge was applied at half-strength rate. These results were to be expected, as the cell yield produced from the oxidation of petroleum hydrocarbons is high, and the increase in the numbers
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of cells reflected the utilization of the added oily sludge as a substrate to build new cell mass. 3.3. Effect of nutrients on biodegradation of oily sludge Addition of nitrogen (NH4NO3) and phosphorous (KH2PO4) inhibited TPH mineralization for all oily sludge loading rates. For instance, after addition of nutrients, the evolved C–CO2 decreased from 1.7, 2.4, and 3.1 g kg1 to 1.4, 0.9, and 0.76 g kg1 for quarter-, half-, and full-strength treatments, respectively (Table 2). The inhibitory effect of the added nutrients on TPH mineralization increased with increasing the loading rate of oily sludge. This inhibitory effect is probably due to the increase in the added nutrients concentrations with increasing TPH concentration. This situation had an even stronger impact on TPH-mineralization efficiencies, where TPH-mineralization efficiencies for quarter-, half-, and full-strength treatments were 14.5%, 4.9%, and 2.0%, respectively (Table 2). Fig. 2 shows the effect of nutrients on cumulative C–CO2 evolution from refinery soil amended with different rates of oily sludge. It is clearly seen that C–CO2 evolved from the quarterstrength treatment is greater than that from half- and full-strength treatments. C–CO2 evolution from soil amended with different levels of oily sludge was characterized by an initial period of 50 days, where the C–CO2 evolution rate was almost the same for all treatments. This indicates that the source of carbon for all treatments in this stage was similar and common to all; i.e. soil organic matter. A second phase, in which C-mineralization rate is greater, was identified for quarter- and half-strength treatments. This phase started after 50 days and 80 days for quarter- and half-strength treatments, respectively. The full-strength treatment exhibited a pattern similar to that of the control. TPH extraction results also showed a reduction in TPH removal with the addition of nutrients. The impact of nutrients addition on TPH removal, however, was less than its impact on TPH mineralization. For instance, TPH removal in quarter-strength treatment was 3.4 g kg1 soil, which declined to 2.8 g kg1 soil with nutrients addition (Table 2). In addition, TPH removal in the half-strength treatment declined from 4.4 to 4.0 g kg1 soil with the addition of nutrients (Table 2). While TPH mineralization is strictly microbial, TPH removal is probably a mixture of both biotic and abiotic processes. Hence, if high nutrient concentrations are inhibitory, then the impact of nutrients will be more effective against biotic processes, i.e. mineralization. This may explain why the impact of inhibition was greater on mineralization compared to removal. TPH mineralization and TPH-removal efficiencies were proportional to both THBC and HCDBC. Nutrients increased the THBC and HCDBC in the quarter-strength treatment, but decreased them in
Table 3 THBC and HCDBC of unamended refinery soil (control), and soil amended with different rates of oily sludge and nutrients evaluated at the end of incubation period. Treatment
THBC CFU 106 g1 soil SD
HCDBC CFU 106 g1 soil SD
HCDBC/THBC, %
Control Sludge alone Quarter-strength Half-strength Full-strength
0.19 0.04
0.21 0.04
115.57
2.23 1.17 5.21 2.44 2.09 0.68
8.89 2.59 33.5 4.04 2.42 0.62
398.30 642.43 115.55
Sludge þ Nutrient Zero-strength Quarter-strength Half-strength Full-strength
0.17 9.13 3.43 0.56
0.23 0.13 9.12 4.17 0.82 0.51 TFTC
136.34 99.88 23.91 NA
TFTC: Too Few To Count. NA: Not Applicable.
0.02 1.72 0.52 0.15
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1600
Cumulative C-CO2 (mg kg-1 soil)
1400 1200 1000 800 600 400 200 0 0
20
40
60
80
100
120
Time (days) Fig. 2. Organic carbon mineralization from nutrients-amended refinery soil (zerostrength), and soil amended with nutrients and different rates of oily sludge, measured by C–CO2 evolution. C full-strength, B half-strength, ; quarter-strength, 7 zerostrength. Error bars represent the standard deviation.
Adding SDS at both concentrations (sub- and supra-SDS) to unamended refinery soil further increased CO2 evolution by an amount equivalent to the amount of carbon added from SDS. Hence, a further experiment was conducted to evaluate SDS biodegradability. SDS was completely biodegradable (data not shown), thus contributing to the total CO2 output. However, it is evident from oily sludge amended experiments that SDS enhances oily sludge biodegradation. TPH removal increased with increasing oily sludge loading rate. For instance, TPH removal was 3.6, 4.8, and 6.2 g kg1 in quarter-, half-, and full-strength treatments, respectively, that received supra-SDS concentration (Table 5). There are no significant differences in TPH removals between the different SDS concentrations tested. For example, TPH removal was the same (3.6 g kg1) at subSDS and supra-SDS in quarter-strength treatments. In comparison to refinery soil amended with sludge and nutrients only, the addition of SDS enhanced TPH removal for quarter- and half-strength treatments. However, TPH removals decreased in full-strength treatments. The addition of SDS caused an increase in both THBC and HCDBC compared to control (data not shown). However, adding SDS to unamended soil decreased THBC and HCDBC. It is evident that bacterial counts in soil are associated with mineralization activity. 3.6. Preferential biodegradability of TPH fractions
half- and full-strength treatments. THBC and HCDBC in the refinery soil were 6.07 106 and 3.63 106 cfu g1 soil (Table 1), respectively; and increased to 9.13 106 and 9.12 106 cfu g1 soil (Table 3), respectively in the quarter-strength treatment. THBC and HCDBC decreased to 3.43 106 and 0.82 106 cfu g1 soil, respectively, in the half-strength treatment at the end of the incubation period (Table 3). This could be attributed to a possibility of biodegradation inhibition by high concentrations of nutrients, where nutrients may inhibit microorganisms that are adapted to an originally oligotrophic soil environment. 3.4. Effect of Triton X-100 on biodegradation of oily sludge Triton X-100 at both concentrations (sub- and supra-Triton) had no effect on oily sludge biodegradation. The patterns of TPH removal, mineralization, and even microbial growth were similar to those treatments without the addition of Triton X-100 at both concentrations tested. Most reports showed that Triton X-100 is not biodegradable (Tiehm, 1994). Furthermore, Triton X-100 (being a non-ionic surfactant) experiences strong interaction with hydrophobic organic compounds (such as TPH) and microorganisms in soil aqueous systems (Edwards et al., 1992). Such interactions may render the surfactant inactive. In addition, Triton X-100 interacts strongly with soil. A soil contains 79% sand and 10% clay sorbed 22.1 mmol kg1 of Triton X-100 (Cuypers et al., 2002). The reported CMC value in that system was 0.42 mM. In our system, the clay content was high, while the rate of addition of Triton X-100 at supra-Triton concentration was 0.48 mM. It can be postulated here that the added concentration of Triton X-100 was inactive because of interactions with both TPH and the soil system. 3.5. Effect of SDS on the biodegradation of oily sludge SDS generally enhanced TPH biodegradation especially for halfstrength treatment (Table 5). For instance, C–CO2 evolution was 2.2 and 2.9 g kg1 for sub- and supra-SDS treatments in the halfstrength treatment, respectively. Compare that to C–CO2 evolution from soil amended with half-strength oily sludge and nutrients only were mineralization amounted to 0.9 g kg1 only (Table 2). This trend was common to all other treatments but to a lesser extent.
Alkanes fraction was biodegraded more than all other fractions (aromatics, NSO-containing compounds, and asphaltenes) in almost all treatments. Incubating sludge-amended soil without the addition of nutrients and surfactants enhanced the removal of the alkanes fraction. For instance, incubation of oily-sludge-amended soil caused a decrease in alkanes concentration from 66.65% in the original oily sludge to 47.62, 61.36, and 38.00% for quarter-, half-, and full-strength treatments, respectively (Table 4). Many reports
Table 4 TPH-fractions from refinery soil amended with different rates of oily sludge nutrients, and synthetic surfactants. Treatment
TPH-fractions (%) Alkanes
Aromatics
Original oily-sludge Sludge alone Quarter-strength Half-strength Full-strength
66.7
14.7
7.8
12.0
47.6 61.4 38.0
22.0 11.4 32.0
36.6 18.2 14.0
12.1 15.9 14.0
58.1 66.7 53.7
14.5 14.3 25.9
16.1 19.1 18.5
16.1 9.5 9.3
Sludge þ Nutrient þ Sub-Triton X-100 Quarter-strength 59.3 17.0 Half-strength 63.0 15.2 Full-strength 62.2 17.8
18.6 19.6 20.0
13.6 8.7 6.7
Sludge þ Nutrient þ Supra-Triton X-100 Quarter-strength 63.0 16.7 Half-strength 56.9 19.6 Full-strength 64.4 17.8
18.5 19.6 17.8
7.4 9.8 6.7
Sludge þ Nutrient þ Sub-SDS Quarter-strength 65.5 Half-strength 58.0 Full-strength 62.2
14.6 18.0 17.8
16.4 18.0 15.2
7.3 10.0 6.7
Sludge þ Nutrient þ Supra-SDS Quarter-strength 64.3 Half-strength 59.1 Full-strength 61.4
16.1 15.9 18.2
16.1 18.2 20.5
8.9 13.6 11.4
Sludge þ Nutrient Quarter-strength Half-strength Full-strength
NSO
Asphaltenes
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Table 5 Carbon mineralization and TPH removal from refinery soil amended with oily sludge, nutrients, and SDS. Treatment
TPH removal, g kg1 soil SD
Control NA Sludge þ Nutrient þ Sub-SDS Zero-strength NA Quarter-strength 3.60 0.20 Half-strength 3.20 0.40 Full-strength 6.00 0.80 Sludge þ Nutrient þ Supra-SDS Zero-strength NA Quarter-strength 3.60 0.20 Half-strength 4.80 0.40 Full-strength 6.20 0.40
TPH removal, % SD NA
Mineralized TPHa, C–CO2 mg kg1 soil SD 774.8 2.0
Mineralized TPH C–CO2, % SD
C-Biomass, mg kg1 soil
C–CO2 þ Cbiomass, %
NA
NA
NA
NA 33.33 2.10 14.11 2.08 13.42 1.88
824.6 1823.8 2195.4 1081.0
3.0 2.0 2.0 16.0
NA 19.53 11.64 2.88
NA 3779.2 4646.2 2045.6
NA 59.99 36.28 8.32
NA 33.33 2.10 21.32 2.08 14.03 0.90
1307.0 2362.8 2866.4 2148.8
2.0 11.0 2.2 92.2
NA 25.3 15.2 5.72
NA 3607.8 4782.8 3108.4
NA 63.93 40.56 14
SD: Standard Deviation. NA: Not Applicable. a Carbon evolved from the mineralization of the SDS was subtracted from the total C–CO2 evolved.
demonstrated that alkanes are usually the easiest hydrocarbons to be degraded (Admon et al., 2001; Dibble and Bartha, 1979; Leahy and Colwell, 1990; Mishra et al., 2001a). Aromatics and NSO-containing compounds were not biodegraded. For example, the aromatic fraction concentration was 14.71% in the original oily sludge and 14.52% (Table 4) for the quarter-strength nutrients treatment. In addition, NSO-containing compounds concentration increased from 7.77% in the original sludge to 16.13% for the same treatment. Van Hamme and Ward (1999) found an unexpected reduction in NSO-containing compounds fraction, since this fraction has been shown to increase during the treatment of oil waste as a result of the production of polar metabolites. NSO-containing compounds could have had internal enrichment source as a result of the biodegradation process. These compounds may be produced as intermediary compounds of alkane and aromatic/asphaltic compounds degradation mainly through oxygenation processes (Dibble and Bartha, 1979). Surfactants (Triton X-100 and SDS at sub- and supra-CMC concentrations) promote the biodegradation of the asphaltenes fraction. For instance, the asphaltenes fraction decreased from 12 to 7.4% after the addition of Triton X-100 at supra-CMC concentration (Table 4). Van Hamme and Ward (1999), reported that optimal surfactant concentration contributed in diminished enrichment of asphaltenes, which was attributed to partial degradation of these compounds.
4. Conclusions TPH mineralization and removal efficiencies were the greatest for the low oily sludge loading rate. However, net amounts of TPH mineralized or removed increased as oily sludge loading rates increased. Adding nutrients caused oily sludge biodegradation inhibition, probably because of the high nutrient concentration present originally in oily sludge. The inhibitory effect of nutrients increased with increasing the amount of nitrogen and phosphorous added. Alkanes are preferentially biodegraded owing to the abundance of their degradation enzymes and ease of biodegradation. Asphaltenes fraction was generally not degraded, and NSO-containing compounds and aromatics were not removed. This could be attributed to the recalcitrance and possible enrichment of these compounds. Biodegradation kinetics of TPH in soil is a function of concentration. Depending on the purpose of the operation, economical and time constraints, a choice can be made between adding high concentration of TPH which would result in higher
absolute degradation but lower efficiency, or lower concentrations which give the opposite results. References Admon, S., Green, M., Avnimelech, Y., 2001. Biodegradation kinetics of hydrocarbons in soil during land treatment of oily sludge. Bioremediation Journal 5, 193–209. Ali, M.A., Hassan, A., 2002. Hydrocarbon group types analysis of petroleum products: a comparative evaluation of HLPC and TLC analytical performance. Petroleum Science and Technology 20, 751–762. Bossert, I., Kachel, W.M., Bartha, R., 1984. Fate of hydrocarbons during oily sludge disposal in soil. Applied and Environmental Microbiology 47, 763–767. Chaineau, C.H., Rougeux, G., Yepremian, C., Oudot, J., 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biology and Biochemistry 37, 1490–1497. Cuypers, C., Pancras, T., Grotenhuis, T., Rulkens, W., 2002. The estimation of PAH bioavailability in contaminated sediments using hydroxypropyl-b-cyclodextrin and Triton X-100 extraction techniques. Chemosphere 46, 1235–1245. Dibble, J.T., Bartha, R., 1979. Effect of environmental parameters on the biodegradation of oil sludge. Applied and Environmental Microbiology 37, 729–739. Edwards, D.A., Laha, S., Liu, Z., Luthy, R.G., 1992. Solubilization and biodegradation of hydrophobic organic compounds in soil–aqueous systems with nonionic surfactants. In: Comstock, M.J. (Ed.), Transport and Remediation of Subsurface Contaminants: Colloidal, Interfacial, and Surfactant Phenomena. American Chemical Society, Washington, DC, pp. 159–168. Garcia-Blanco, S., Venosa, A.D., Suidan, M.T., Lee, K., Cobanli, S., Haines, J.R., 2007. Biostimulation for the treatment of an oil-contaminated coastal SALT march. Biodegradation 18, 1–15. Gee, G.W., Bauder, J.W., 1986. Particle size analysis. In: Klute, A. (Ed.), Methods of Soil Analysis, Part 1: Physical and Mineralogical Methods, second ed. Soil Science Society of America, Wisconsin, pp. 383–409. Herman, D.C., Zhang, Y., Miller, R.M., 1997. Rhamnolipid (biosurfactant) affects on cell aggregation and biodegradation of residual hexadecane under saturated flow conditions. Applied and Environmental Microbiology 63, 3622–3627. Jones, M.N., Chapman, D., 1995. Micelles, Monolayers, and Biomembranes. WileyLiss, New York, pp. 1–65. Lazar, I., Dobrota, S., Voicu, A., Stefanescu, M., Sandulescu, L., Petrisor, I.G., 1999. Microbial degradation of waste hydrocarbons in oily sludge from some Romanian oil fields. Journal of Petroleum Science & Engineering 22, 151–160. Leahy, J.G., Colwell, R.R., 1990. Microbial degradation of hydrocarbon in the environment. Applied and Environmental Microbiology 54, 305–315. Maier, R.M., 2000. Bacterial growth. In: Maier, R.M., Pepper, I.L., Gebra, C.P. (Eds.), Environmental Microbiology. Academic Press, UK, pp. 43–59. Margesin, R., Schinner, F., 2001. Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an Alpine Glacier skiing area. Applied and Environmental Microbiology 67, 3127–3133. Mishra, S., Jyot, J., Kuhad, R.C., Lal, B., 2001a. Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil. Applied and Environmental Microbiology 67, 1675–1681. Mishra, S., Jyot, J., Kuhad, R.C., Lal, B., 2001b. In situ bioremediation potential of an oily sludge-degrading bacterial consortium. Current Microbiology 43, 328–335. Nelson, D.W., Sommers, L.E., 1996. Total carbon, organic carbon, and organic matter. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3: Chemical Methods. Soil Science Society of America, Wisconsin, pp. 961–1006. Nikitina, E.V., Yakusheva, O.I., Zaripov, S.A., Galiev, R.A., Garusov, A.V., Naumova, R.P., 2003. Distribution and physiological state of microorganisms in petrochemical oily sludge. Microbiology 72, 621–627. Sukatsch, D.A., Johnson, M.J., 1972. Bacterial cell production from hexadecane at high temperatures. Applied Microbiology 23, 543–546.
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Tiehm, A., 1994. Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants. Applied and Environmental Microbiology 60, 258–263. Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3: Chemical Methods. Soil Science Society of America, Wisconsin, pp. 475–490. U.S. Environmental Protection Agency, 2004. How to Evaluate Alternative Cleanup Technologies for Underground Storage Tank Sites: a Guide for Corrective Action Plan Reviewers, Solid Waste and Emergency Response 5401G, EPA 510-R-04– 002 pp XII1-XII68. U.S. Environmental Protection Agency. Available from URL: www.epa.gov/oust/pubs/tums.htm. Van Hamme, J.D., Ward, O.P., 1999. Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture. Canadian Journal of Microbiology 45, 130–137.
Vasudevan, N., Rajaram, P., 2001. Bioremediation of oil sludge-contaminated soil. Environment International 26, 409–411. Yerushalmi, L., Rocheleau, S., Cimpoia, R., Sarrazin, M., Sunahara, G., Peisajovich, A., 2003. Enhanced biodegradation of petroleum hydrocarbon in contaminated soil. Bioremediation Journal 7, 37–51. Zibilski, L.M., 1994. Carbon mineralization. In: Weaver, R.W., Angle, J.S., Bottomley, P.S. (Eds.), Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties. Soil Science Society of America, Wisconsin, pp. 835–863. Zubber, D.A., 1994. Recovery and enumeration of viable bacteria. In: Weaver, R.W., Angle, J.S., Bottomley, P.S. (Eds.), Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties. Soil Science Society of America, Wisconsin, pp. 119–144.
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil Ragheb A. Tahhan*, Rouba Youssef Abu-Ateih Department of Natural Resources and Environment, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 March 2009 Received in revised form 4 September 2009 Accepted 4 September 2009 Available online 9 October 2009
The main objective of this study was to evaluate the effect of oily sludge concentration on its biodegradability in soil. Oily sludge was collected and applied to microcosms at full-, half-, or quarterstrength concentrations equivalent to 44.2, 22.2, and 11.1 g kg1 soil, respectively, of total petroleum hydrocarbons (TPH) contained in oily sludge. The biodegradability of oily sludge was evaluated by measuring CO2 evolution and by measuring removal of TPH as well as its main composing fractions; namely; alkanes, aromatics, NSO-compounds, and asphaltenes. The collected soil contained 3.63 106 cfu g1 soil of hydrocarbon-degrading bacteria, which is satisfactory to drive successful biodegradation of hydrocarbons in soil. These numbers increased significantly with oily sludge addition at a rate proportional to the added TPH reaching 3.35 107 cfu g1 soil in the half-strength treatment. TPH mineralization rate followed the same pattern. However, TPH-mineralization efficiency was the greatest in quarter-strength treatment at 18.3%. TPH-removal efficiency was also highest in quarterstrength treatment at 30.9%. Nutrients addition caused mineralization inhibition. Since nutrients were added as a ratio of the added carbon, inhibition was the greatest with the highest TPH treatment. While alkanes were degraded, aromatics and asphaltenes were not, and NSO-compounds were enriched. Although SDS was completely biodegradable in soil, its addition promoted mineralization and removal of TPH from soil. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Oily sludge Biodegradation TPH TPH-extraction TPH-fractionation
1. Introduction Petroleum refining unavoidably generates considerable volumes of oily sludge during oil production and processing activities. Total petroleum hydrocarbons (TPH) are the most deleterious components in oily sludge, because of their potential hazard to human health and the environment. TPH are primarily composed of alkanes, aromatics, nitrogen-, sulfur-, and oxygen- (NSO)-containing compounds, and asphaltene fractions (Admon et al., 2001; Ali and Hassan, 2002; Mishra et al., 2001a). Bioremediation techniques are sound alternative methods for the treatment of TPH-contaminated soil. Such techniques are economically and politically attractive and have shown promising results in the treatment of soil contaminated with organic compounds, particularly with petroleum hydrocarbons (Chaineau et al., 2005; Garcia-Blanco et al., 2007; Yerushalmi et al., 2003).
* Corresponding author. Tel.: þ962 2 7201000x22050; fax: þ962 2 7201078. E-mail address: [email protected] (R.A. Tahhan). 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.09.001
However, there are still several technical aspects regarding bioremediation of oily sludge that are yet to be resolved. The addition of oily sludge to soil (5–30%; equivalent to 9–61 mg TPH/g soil) resulted in 70–90% degradation of TPH during two months regardless of their initial concentrations (Admon et al., 2001). However, Lazar et al. (1999) found that oily sludge concentration has an effect on the percentage of its biodegradation; the best biodegradation occurred for the treatment that received 10% oily sludge (equivalent to 3.2% initial TPH). Nutrient deficiency might be a limiting factor in the biodegradation process. Admon et al. (2001) found that hydrocarbon loss was observed only after nutrients were amended to oily sludgecontaminated soil at C:N:P ratio equivalent to 50:10:1. Jordanian oil refinery operations end up with storage of the produced oily sludge in concrete lined pools. This process will eventually lead to accumulation of large quantities of this material beyond their storage capacity or safe disposal processes. In addition, the operations involved in production, transport, and disposal of oily sludge result in spills of oily sludge on large soil areas. This study was designed to evaluate the effect of TPH concentration on its biodegradation in soil. The study also proposes a possible approach for remediating TPH-contaminated soil.
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2. Materials and methods 2.1. Soil collection and preparation The soil used in this study was collected from an orchard area inside the oil refinery campus in Zarka. This soil is regularly irrigated with treated oily wastewater, which is characterized with high salinity (6 dS m1) and an inhibitory effect on soil carbon mineralization activity (data not shown). Briefly, surface soil layer (15 cm deep) was collected in plastic bags. A small portion of the soil was packed on ice for immediate analysis of microbiological characteristics. The soil was brought to the laboratory, mixed thoroughly, and passed through a 6.3 mm sieve in order to remove large roots and plant residues. This preparation was intended for use in biodegradation studies. For physical, chemical, and biological analysis, however, the soil was passed through a 2 mm sieve (Vasudevan and Rajaram, 2001). 2.2. Soil analyses Organic matter was determined by measuring organic carbon, using Walkley Black method (Nelson and Sommers, 1996). pH was determined in 1:1 soil:water mixture by standard soil analytical methods (Thomas, 1996). Soil texture was determined by hydrometer method (Gee and Bauder, 1986). Total heterotrophic bacterial counts (THBC) and hydrocarbon-degrading bacterial counts (HCDBC) in soil were determined by spread plating method (Zubber, 1994). R2A agar plates (Margesin and Schinner, 2001) were used to enumerate heterotrophic microorganisms. Plates were incubated for 48 h at 28 C. Hydrocarbon utilizing bacteria were quantified on MSM agar plates. MSM had the following composition, in g/L DD H2O:KH2PO4, 1.0; Na2HPO4, 1.0; NH4NO3, 0.5; (NH4)2SO4, 0.5; MgSO4$7H2O, 0.2; CaCl2$2H2O, 0.02; FeCl3, 0.002; MnSO4$2H2O, 0.002 and agar, 16 (Herman et al., 1997). MSM plates were spread with the appropriate dilution and then sprayed with diethyl ether-dissolved oily sludge as a carbon source. Plates were incubated for two weeks at 28 C. All measurements were done in triplicate. 2.3. Oily sludge collection and analyses Oily sludge samples were collected from oily sludge storage basins inside the refinery campus. Moisture content was determined by oven drying overnight at 105 C (Nikitina et al., 2003). To determine TPH content in oily sludge, a triplicate of 1 g of air dried oily sludge was extracted with 100 ml of each hexanes, dichloromethane, and chloroform, successively. The extracts were pooled and air dried to evaporate the solvents. TPH was then determined gravimetrically (Mishra et al., 2001b). We collected several samples of oily sludge over 2 years. Considerable variations in content were observed between these samples. However, only one sample was used in the biodegradability experiments for this study. The composition of this sample was as follows: 64.91 0.16% moisture; 24.45 1.46% TPH; and 10.76 1.05% residue. This analysis was one in triplicate.
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(100 ml each). After evaporation of the solvents, each fraction (including asphaltenes) was determined gravimetrically (Mishra et al., 2001b). TPH composition for the sample used in this study was as follows: 66.65 2.93% alkanes, 14.71 1.72% aromatics, 7.77 5.33% NSO-compounds, and 11.99 1.83% asphaltenes. These analyses were done in triplicate. 2.5. Oily sludge biodegradability study Oily sludge biodegradability was evaluated via C-mineralization experiments (Zibilski, 1994). These experiments (treatability studies) were conducted in order to determine the biodegradability of various concentrations of oily sludge in soil. Four rates of oily sludge were tested. TPH concentrations with the applied oily sludge were; control (no addition), 11.1 (quarter-strength), 22.2 (halfstrength), and 44.2 g kg1 soil (full-strength). In addition, nutrients (NH4NO3 and KH2PO4) were added to achieve C:N:P ratio equivalent to 50:10:1 (Admon et al., 2001; Yerushalmi et al., 2003). Fifty grams of air-dried soil were mixed with oily sludge, and then incubated in sealed jars. Carbon dioxide evolution was periodically collected in alkali and evaluated by titration with HCl. Moisture content was monitored gravimetrically during the experimental period and maintained at 60% of the water holding capacity. At the end of the incubation period, all treatments were subjected to TPH extraction as mentioned below, TPH fractionation, total heterotrophic bacterial counts and hydrocarbon-degrading bacterial counts. This experiment was replicated four times. 2.6. Synthetic surfactants aided biodegradation Two different synthetic surfactants; Triton X-100 (non-ionic surfactant) and Sodium dodecyl sulfate, SDS (anionic surfactant) were used in this study as they were recommended previously in the biodegradation of hydrocarbons. The Critical Micelle Concentration (CMC) was considered to be 0.24 mM for Triton X-100 and 8.1 mM for SDS (Jones and Chapman, 1995). Oily sludge rates were similar to the oily sludge biodegradability study described above. Surfactants were added at two rates to achieve the following concentrations in soil water; below the critical micelle concentration (sub-CMC ¼ ½ CMC) and above the critical micelle concentration (supra-CMC ¼ 2 CMC). In addition, separate surfactants biodegradability experiments were conducted. All these experiments were done in four replicates. 2.7. TPH extraction from soil TPH was extracted from 25 g soil with two portions of 100 ml of each hexane, dichloromethane, and chloroform, successively, as described above for TPH extraction from oily sludge. Solvents were evaporated to a constant weight before TPH was determined gravimetrically. 3. Results and discussion 3.1. Soil properties
2.4. TPH fractionation TPH was extracted as described by Mishra et al. (2001b). Extracted TPH (0.2–0.5 g) were dissolved in n-pentane and separated into soluble and insoluble fractions (asphaltenes). The soluble fraction was loaded onto a silica gel column. Alkane fraction was eluted with 100 ml of hexanes, aromatic fraction was eluted with 100 ml benzene, and finally, NSO-containing compounds (resins) fraction was eluted with a mixture of chloroform and methanol
Soil properties are presented in Table 1. Organic carbon level (1.3%) is typical for an area such as Zarka, which is characterized by a hot dry climate. pH of the collected soil is slightly alkaline, which is also typical for this area. Such pH values are not reported to be inhibitory for biodegradative abilities of microorganisms to hydrocarbons. Electrical conductivity of the soil is fairly high. This is mostly a result of irrigation with the saline oily wastewater. High salinity in the soil probably imposes stress on microorganisms.
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Table 1 Physical, chemical, and microbiological properties of soil collected from the oil refinery campus.
Cumulative C-CO2 (mg kg-1 soil)
3500
Property Organic carbon SD (%) pH SD EC (dS m1) Soil texture Bulk density (g/cm3) Porosity (%) THBC SD (cfu 106) HCDBC SD (cfu 106)
1.31 7.44 12.12 Clay 1.124 57.58 6.07 3.63
0.095 0.021
1.29 1.39
EC: Electrical Conductivity. THBC: Total Heterotrophic Bacterial Count. HCDBC: Hydrocarbon-Degrading Bacterial Count. cfu: colony forming unit. SD: Standard Deviation.
3000
2500
2000
1500
1000
500
0
However, it seems that soil bacteria are adapted to this salinity level because they have shown high numbers (6.07 106 cfu g1 soil). Total heterotrophic bacterial counts in this soil are high enough, and above the recommended range for successful hydrocarbon degradation (U.S. EPA, 2004). Moreover, the soil contains high counts of hydrocarbon-degrading bacteria (3.63 106 cfu g1 soil). This is probably a result of adaptation to the continuous addition of hydrocarbons contained in the treated oily wastewater applied to the soil. According to Mishra et al. (2001a), hydrocarbon-degrading bacteria that are indigenous to soil with populations exceeding 105 cfu g1 soil are usually enough to satisfactorily carry out the degradation process without the need for bioaugmentation.
0
20
40
60
80
100
120
Time (days) Fig. 1. Organic carbon mineralization from unamended refinery soil (7 zero-strength; control), and soil amended with different rates of oily sludge, measured by C–CO2 evolution. C full-strength, B half-strength, ; quarter-strength. Error bars represent the standard deviation.
pattern was common to all oily sludge loading rates studied. It was characterized by an initial period of 40 days, where C–CO2 evolution rate was low. This period was considered as an acclimation period. A second phase started after 40 days indicating an active TPH mineralization. A third phase that started after 90 days was roughly identified. It was characterized by lower rate of C-mineralization especially for half- and full-strength application rates. Bossert et al. (1984), noted that an active phase of CO2 evolution was followed by a late phase (closure phase) that showed ‘‘greatly decreased’’ CO2 evolution activity. TPH removal is defined as the amount of extracted TPH subtracted from the amount of added TPH. TPH-removal efficiency is the ratio of TPH removal to the amount of added TPH. TPH removal in the quarter-, half-, and full-strength treatments that received oily sludge alone also followed the same trend as that in TPH mineralization. TPH removal was 3.4, 4.4 and 8.8 g kg1 soil, for quarter-, half-, and full-strength treatments, respectively (Table 2). In addition, TPH-removal efficiency in quarter-strength treatment was the greatest (31%). TPH removal was about 20% in both half- and fullstrength treatments. The carbon used to build new cell mass was calculated based on documented cell yield value of 1.12 (Sukatsch and Johnson, 1972). If we consider that TPH in oily sludge is identical to TPH in petroleum oil, then, the amount of TPH removal should be equivalent or greater than the summation of mineralized-C and biomass-C. However, our results show exactly the opposite of that, where removal was smaller than the summation of mineralized-C
3.2. Effect of oily sludge loading rate on biodegradation As oily sludge loading rate increases, TPH concentration and any other potentially toxic compound increase as well. Thus, it is necessary to test the effect of diluting oily sludge on the rate and extent of biodegradation. TPH mineralization is represented by the amount of carbon evolved as carbon dioxide. TPH-mineralization efficiency is the ratio of carbon evolved as carbon dioxide to the amount of TPH-carbon added to the soil. The amount of mineralized TPH increased with increasing oily sludge loading rate. For instance, when soil was amended with oily sludge alone, the amounts of carbon dioxide evolved were 3.1, 2.4, and 1.7 g kg1 in full-, half-, and quarter-strength treatments, respectively (Table 2). However, mineralization efficiency was the greatest for quarterstrength treatment, and generally, inversely proportional to the amount of TPH added. This shows that the mineralized carbon is proportional to the amount of TPH added. Fig. 1 shows the cumulative C–CO2 evolved from refinery soil amended with different levels of oily sludge. Application of oily sludge increased the rate of C–CO2 evolution by 2.2, 3.1, and 3.9 fold for quarter-, half-, and full-strength treatments. A mineralization
Table 2 Mineralization from refinery soil (control), and mineralization, and TPH removal from refinery soil amended with different rates of oily sludge with or without nutrients. TPH removal (g kg1 soil) SD
TPH removal (%) SD
Mineralized C, C–CO2 (mg kg1 soil) SD
Mineralized C, C–CO2 (%)
C-Biomass (mg kg1 soil)
C–CO2 þ C-biomass (%)
Sludge alone Control (no sludge) Quarter-strength Half-strength Full-strength
NA 3.4 0.4 4.4 0.2 8.8 1.0
NA 30.91 3.64 19.52 1.04 20.06 2.28
774.8 1709.6 2404.4 3079.2
2 6 4 15
NA 18.3 12.8 8.2
NA 3989 5600 7185
NA 61.0 42.5 27.3
Sludge þ Nutrient Zero-strength Quarter-strength Half-strength Full-strength
NA 2.8 0.2 4.0 0.6 7.2 0.8
NA 24.85 2.10 17.72 2.75 16.44 1.88
658 1353.6 916.6 761.8
7 5 5 2
NA 14.5 4.9 2.0
NA 3159 2139 1778
NA 48.3 16.2 6.8
Treatment
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and biomass-C. Thus, we concluded that the cell yield value used here is overestimated, especially for low oily sludge loading rates (Table 2). This phenomenon can be attributed to the possibility of presence of growth inhibitory agents in oily sludge. Furthermore, Maier (2000) suggested that utilization of the substrate can occur without production of new cells in some cases, namely low substrate concentration or low substrate bioavailability. In this case the energy from substrate utilization is used to meet the maintenance requirements of the cell under non-growth conditions. Bacterial counts in the soil were 6.07 106 and 3.63 106 cfu g1 soil for THBC and HCDBC, respectively (Table 1). Incubating soil without the addition of external carbon source (control) caused reduction of both THBC and HCDBC to 1.90 105 and 2.10 105 cfu g1 soil, respectively (Table 3). On the other hand, the addition of oily sludge to incubated soil caused a reduction in THBC, and an increase in HCDBC compared to original soil counts. For instance, THBC in quarter- and half-strength treatments decreased from 6.07 106 cfu g1 soil to 2.23 106 and 5.21 106 cfu g1 soil, respectively. However, HCDBD was 8.89 106 and 33.5 106 cfu g1 soil after the addition of oily sludge for quarter- and half-strength treatments, respectively. This was an expected result, as supplying hydrocarbons contained in the added TPH enriches HCDBC. Surprisingly, the ratio of HCDBC:HCDB exceeded 100% in some cases, especially those with active hydrocarbon mineralization activity. It is well documented that hydrocarbon degraders are heterotrophs, thus, they are a part of THBC. The reason for this apparent discrepancy is probably related to culturing conditions. Hydrocarbon degraders were incubated for 2 weeks and produced tiny colonies, which gave room on the plate for the growth of large numbers. On the other hand, heterotrophs were incubated on a complex medium which allowed the formation of large colonies with limited number. In addition, hydrocarbon degraders are in part oligotrophs characterized by slow growth which gives them the ability to grow on a diluted medium such as the medium used here, but they do not grow well on R2A agar. By increasing the oily sludge loading rate, the HCDBC numbers increased, thus, increasing the amount of mineralized-TPH. Therefore, there was a positive correlation between the HCDBC numbers and the biodegradation or mineralization capacity of oily sludge. For instance, C–CO2 evolution, TPH removal, and HCDBC were 1.7 g kg1 soil, 3.4 g kg1 (Table 2), and 8.89 106 cfu g1 soil (Table 3), respectively, in quarter-strength treatment that received oily sludge alone; and increased to 2.4 g kg1 C–CO2, 4.4 g kg1 of TPH (Table 2), and 33.5 106 cfu g1 soil (Table 3), respectively, when oily sludge was applied at half-strength rate. These results were to be expected, as the cell yield produced from the oxidation of petroleum hydrocarbons is high, and the increase in the numbers
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of cells reflected the utilization of the added oily sludge as a substrate to build new cell mass. 3.3. Effect of nutrients on biodegradation of oily sludge Addition of nitrogen (NH4NO3) and phosphorous (KH2PO4) inhibited TPH mineralization for all oily sludge loading rates. For instance, after addition of nutrients, the evolved C–CO2 decreased from 1.7, 2.4, and 3.1 g kg1 to 1.4, 0.9, and 0.76 g kg1 for quarter-, half-, and full-strength treatments, respectively (Table 2). The inhibitory effect of the added nutrients on TPH mineralization increased with increasing the loading rate of oily sludge. This inhibitory effect is probably due to the increase in the added nutrients concentrations with increasing TPH concentration. This situation had an even stronger impact on TPH-mineralization efficiencies, where TPH-mineralization efficiencies for quarter-, half-, and full-strength treatments were 14.5%, 4.9%, and 2.0%, respectively (Table 2). Fig. 2 shows the effect of nutrients on cumulative C–CO2 evolution from refinery soil amended with different rates of oily sludge. It is clearly seen that C–CO2 evolved from the quarterstrength treatment is greater than that from half- and full-strength treatments. C–CO2 evolution from soil amended with different levels of oily sludge was characterized by an initial period of 50 days, where the C–CO2 evolution rate was almost the same for all treatments. This indicates that the source of carbon for all treatments in this stage was similar and common to all; i.e. soil organic matter. A second phase, in which C-mineralization rate is greater, was identified for quarter- and half-strength treatments. This phase started after 50 days and 80 days for quarter- and half-strength treatments, respectively. The full-strength treatment exhibited a pattern similar to that of the control. TPH extraction results also showed a reduction in TPH removal with the addition of nutrients. The impact of nutrients addition on TPH removal, however, was less than its impact on TPH mineralization. For instance, TPH removal in quarter-strength treatment was 3.4 g kg1 soil, which declined to 2.8 g kg1 soil with nutrients addition (Table 2). In addition, TPH removal in the half-strength treatment declined from 4.4 to 4.0 g kg1 soil with the addition of nutrients (Table 2). While TPH mineralization is strictly microbial, TPH removal is probably a mixture of both biotic and abiotic processes. Hence, if high nutrient concentrations are inhibitory, then the impact of nutrients will be more effective against biotic processes, i.e. mineralization. This may explain why the impact of inhibition was greater on mineralization compared to removal. TPH mineralization and TPH-removal efficiencies were proportional to both THBC and HCDBC. Nutrients increased the THBC and HCDBC in the quarter-strength treatment, but decreased them in
Table 3 THBC and HCDBC of unamended refinery soil (control), and soil amended with different rates of oily sludge and nutrients evaluated at the end of incubation period. Treatment
THBC CFU 106 g1 soil SD
HCDBC CFU 106 g1 soil SD
HCDBC/THBC, %
Control Sludge alone Quarter-strength Half-strength Full-strength
0.19 0.04
0.21 0.04
115.57
2.23 1.17 5.21 2.44 2.09 0.68
8.89 2.59 33.5 4.04 2.42 0.62
398.30 642.43 115.55
Sludge þ Nutrient Zero-strength Quarter-strength Half-strength Full-strength
0.17 9.13 3.43 0.56
0.23 0.13 9.12 4.17 0.82 0.51 TFTC
136.34 99.88 23.91 NA
TFTC: Too Few To Count. NA: Not Applicable.
0.02 1.72 0.52 0.15
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1600
Cumulative C-CO2 (mg kg-1 soil)
1400 1200 1000 800 600 400 200 0 0
20
40
60
80
100
120
Time (days) Fig. 2. Organic carbon mineralization from nutrients-amended refinery soil (zerostrength), and soil amended with nutrients and different rates of oily sludge, measured by C–CO2 evolution. C full-strength, B half-strength, ; quarter-strength, 7 zerostrength. Error bars represent the standard deviation.
Adding SDS at both concentrations (sub- and supra-SDS) to unamended refinery soil further increased CO2 evolution by an amount equivalent to the amount of carbon added from SDS. Hence, a further experiment was conducted to evaluate SDS biodegradability. SDS was completely biodegradable (data not shown), thus contributing to the total CO2 output. However, it is evident from oily sludge amended experiments that SDS enhances oily sludge biodegradation. TPH removal increased with increasing oily sludge loading rate. For instance, TPH removal was 3.6, 4.8, and 6.2 g kg1 in quarter-, half-, and full-strength treatments, respectively, that received supra-SDS concentration (Table 5). There are no significant differences in TPH removals between the different SDS concentrations tested. For example, TPH removal was the same (3.6 g kg1) at subSDS and supra-SDS in quarter-strength treatments. In comparison to refinery soil amended with sludge and nutrients only, the addition of SDS enhanced TPH removal for quarter- and half-strength treatments. However, TPH removals decreased in full-strength treatments. The addition of SDS caused an increase in both THBC and HCDBC compared to control (data not shown). However, adding SDS to unamended soil decreased THBC and HCDBC. It is evident that bacterial counts in soil are associated with mineralization activity. 3.6. Preferential biodegradability of TPH fractions
half- and full-strength treatments. THBC and HCDBC in the refinery soil were 6.07 106 and 3.63 106 cfu g1 soil (Table 1), respectively; and increased to 9.13 106 and 9.12 106 cfu g1 soil (Table 3), respectively in the quarter-strength treatment. THBC and HCDBC decreased to 3.43 106 and 0.82 106 cfu g1 soil, respectively, in the half-strength treatment at the end of the incubation period (Table 3). This could be attributed to a possibility of biodegradation inhibition by high concentrations of nutrients, where nutrients may inhibit microorganisms that are adapted to an originally oligotrophic soil environment. 3.4. Effect of Triton X-100 on biodegradation of oily sludge Triton X-100 at both concentrations (sub- and supra-Triton) had no effect on oily sludge biodegradation. The patterns of TPH removal, mineralization, and even microbial growth were similar to those treatments without the addition of Triton X-100 at both concentrations tested. Most reports showed that Triton X-100 is not biodegradable (Tiehm, 1994). Furthermore, Triton X-100 (being a non-ionic surfactant) experiences strong interaction with hydrophobic organic compounds (such as TPH) and microorganisms in soil aqueous systems (Edwards et al., 1992). Such interactions may render the surfactant inactive. In addition, Triton X-100 interacts strongly with soil. A soil contains 79% sand and 10% clay sorbed 22.1 mmol kg1 of Triton X-100 (Cuypers et al., 2002). The reported CMC value in that system was 0.42 mM. In our system, the clay content was high, while the rate of addition of Triton X-100 at supra-Triton concentration was 0.48 mM. It can be postulated here that the added concentration of Triton X-100 was inactive because of interactions with both TPH and the soil system. 3.5. Effect of SDS on the biodegradation of oily sludge SDS generally enhanced TPH biodegradation especially for halfstrength treatment (Table 5). For instance, C–CO2 evolution was 2.2 and 2.9 g kg1 for sub- and supra-SDS treatments in the halfstrength treatment, respectively. Compare that to C–CO2 evolution from soil amended with half-strength oily sludge and nutrients only were mineralization amounted to 0.9 g kg1 only (Table 2). This trend was common to all other treatments but to a lesser extent.
Alkanes fraction was biodegraded more than all other fractions (aromatics, NSO-containing compounds, and asphaltenes) in almost all treatments. Incubating sludge-amended soil without the addition of nutrients and surfactants enhanced the removal of the alkanes fraction. For instance, incubation of oily-sludge-amended soil caused a decrease in alkanes concentration from 66.65% in the original oily sludge to 47.62, 61.36, and 38.00% for quarter-, half-, and full-strength treatments, respectively (Table 4). Many reports
Table 4 TPH-fractions from refinery soil amended with different rates of oily sludge nutrients, and synthetic surfactants. Treatment
TPH-fractions (%) Alkanes
Aromatics
Original oily-sludge Sludge alone Quarter-strength Half-strength Full-strength
66.7
14.7
7.8
12.0
47.6 61.4 38.0
22.0 11.4 32.0
36.6 18.2 14.0
12.1 15.9 14.0
58.1 66.7 53.7
14.5 14.3 25.9
16.1 19.1 18.5
16.1 9.5 9.3
Sludge þ Nutrient þ Sub-Triton X-100 Quarter-strength 59.3 17.0 Half-strength 63.0 15.2 Full-strength 62.2 17.8
18.6 19.6 20.0
13.6 8.7 6.7
Sludge þ Nutrient þ Supra-Triton X-100 Quarter-strength 63.0 16.7 Half-strength 56.9 19.6 Full-strength 64.4 17.8
18.5 19.6 17.8
7.4 9.8 6.7
Sludge þ Nutrient þ Sub-SDS Quarter-strength 65.5 Half-strength 58.0 Full-strength 62.2
14.6 18.0 17.8
16.4 18.0 15.2
7.3 10.0 6.7
Sludge þ Nutrient þ Supra-SDS Quarter-strength 64.3 Half-strength 59.1 Full-strength 61.4
16.1 15.9 18.2
16.1 18.2 20.5
8.9 13.6 11.4
Sludge þ Nutrient Quarter-strength Half-strength Full-strength
NSO
Asphaltenes
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Table 5 Carbon mineralization and TPH removal from refinery soil amended with oily sludge, nutrients, and SDS. Treatment
TPH removal, g kg1 soil SD
Control NA Sludge þ Nutrient þ Sub-SDS Zero-strength NA Quarter-strength 3.60 0.20 Half-strength 3.20 0.40 Full-strength 6.00 0.80 Sludge þ Nutrient þ Supra-SDS Zero-strength NA Quarter-strength 3.60 0.20 Half-strength 4.80 0.40 Full-strength 6.20 0.40
TPH removal, % SD NA
Mineralized TPHa, C–CO2 mg kg1 soil SD 774.8 2.0
Mineralized TPH C–CO2, % SD
C-Biomass, mg kg1 soil
C–CO2 þ Cbiomass, %
NA
NA
NA
NA 33.33 2.10 14.11 2.08 13.42 1.88
824.6 1823.8 2195.4 1081.0
3.0 2.0 2.0 16.0
NA 19.53 11.64 2.88
NA 3779.2 4646.2 2045.6
NA 59.99 36.28 8.32
NA 33.33 2.10 21.32 2.08 14.03 0.90
1307.0 2362.8 2866.4 2148.8
2.0 11.0 2.2 92.2
NA 25.3 15.2 5.72
NA 3607.8 4782.8 3108.4
NA 63.93 40.56 14
SD: Standard Deviation. NA: Not Applicable. a Carbon evolved from the mineralization of the SDS was subtracted from the total C–CO2 evolved.
demonstrated that alkanes are usually the easiest hydrocarbons to be degraded (Admon et al., 2001; Dibble and Bartha, 1979; Leahy and Colwell, 1990; Mishra et al., 2001a). Aromatics and NSO-containing compounds were not biodegraded. For example, the aromatic fraction concentration was 14.71% in the original oily sludge and 14.52% (Table 4) for the quarter-strength nutrients treatment. In addition, NSO-containing compounds concentration increased from 7.77% in the original sludge to 16.13% for the same treatment. Van Hamme and Ward (1999) found an unexpected reduction in NSO-containing compounds fraction, since this fraction has been shown to increase during the treatment of oil waste as a result of the production of polar metabolites. NSO-containing compounds could have had internal enrichment source as a result of the biodegradation process. These compounds may be produced as intermediary compounds of alkane and aromatic/asphaltic compounds degradation mainly through oxygenation processes (Dibble and Bartha, 1979). Surfactants (Triton X-100 and SDS at sub- and supra-CMC concentrations) promote the biodegradation of the asphaltenes fraction. For instance, the asphaltenes fraction decreased from 12 to 7.4% after the addition of Triton X-100 at supra-CMC concentration (Table 4). Van Hamme and Ward (1999), reported that optimal surfactant concentration contributed in diminished enrichment of asphaltenes, which was attributed to partial degradation of these compounds.
4. Conclusions TPH mineralization and removal efficiencies were the greatest for the low oily sludge loading rate. However, net amounts of TPH mineralized or removed increased as oily sludge loading rates increased. Adding nutrients caused oily sludge biodegradation inhibition, probably because of the high nutrient concentration present originally in oily sludge. The inhibitory effect of nutrients increased with increasing the amount of nitrogen and phosphorous added. Alkanes are preferentially biodegraded owing to the abundance of their degradation enzymes and ease of biodegradation. Asphaltenes fraction was generally not degraded, and NSO-containing compounds and aromatics were not removed. This could be attributed to the recalcitrance and possible enrichment of these compounds. Biodegradation kinetics of TPH in soil is a function of concentration. Depending on the purpose of the operation, economical and time constraints, a choice can be made between adding high concentration of TPH which would result in higher
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