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The effect of soil type on the bioremediation of petroleum contaminated soils
Journal of Environmental Management 180 (2016) 197e201
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
The effect of soil type on the bioremediation of petroleum contaminated soils Ali Haghollahi a, Mohammad Hassan Fazaelipoor a, b, *, Mahin Schaffie a, b a b
Mineral Industries Research Center, Shahid Bahonar University of Kerman, Iran Department of Chemical Engineering, Faculty of Engineering, Shahid Bahonar University of Kerman, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2016 Received in revised form 13 May 2016 Accepted 14 May 2016
In this research the bioremediation of four different types of contaminated soils was monitored as a function of time and moisture content. The soils were categorized as sandy soil containing 100% sand (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and 32% sand (type III), and coarse grained with high clay content containing 40% gravel, 20% sand, and 40% clay (type IV). The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg, and left on the floor for the evaporation of light hydrocarbons. A full factorial experimental design with soil type (four levels), and moisture content (10 and 20%) as the factors was employed. The soils were inoculated with petroleum degrading microorganisms. Soil samples were taken on days 90, 180, and 270, and the residual total petroleum hydrocarbon (TPH) was extracted using soxhlet apparatus. The moisture content of the soils was kept almost constant during the process by intermittent addition of water. The results showed that the efficiency of bioremediation was affected significantly by the soil type (Pvalue < 0.05). The removal percentage was the highest (70%) for the sandy soil with the initial TPH content of 69.62 g/kg, and the lowest for the clay soil (23.5%) with the initial TPH content of 69.70 g/kg. The effect of moisture content on bioremediation was not statistically significant for the investigated levels. The removal percentage in the clay soil was improved to 57% (within a month) in a separate experiment by more frequent mixing of the soil, indicating low availability of oxygen as a reason for low degradation of hydrocarbons in the clay soil. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Total petroleum hydrocarbon (TPH) Bioremediation Moisture Soil type Sandy soil Clay soil
1. Introduction Petroleum hydrocarbons are persistent pollutants in the environment. Uncontrolled release of these compounds affects soil, water, and air negatively (Ulrici, 2008). Soil pollution with hydrocarbons is caused by the leakage from underground reservoirs, petroleum refineries, storage facilities, and accidental spillage from production units and transport pipelines. The presence of petroleum hydrocarbons affects physical, physiological and biochemical properties of soil (Margesin et al., 2003; Head et al., 2006). Plants are susceptible to oil exposure due to phytotoxic nature of hydrocarbons, and immobilization of nutrients in the soil (De Jong, 1980; Udo and Fayemi, 1995). Inherent mutagenic properties of some
* Corresponding author. Department of Chemical Engineering, Faculty of Engineering, Shahid Bahonar University of Kerman, Iran. E-mail addresses: [email protected], [email protected] (M.H. Fazaelipoor). http://dx.doi.org/10.1016/j.jenvman.2016.05.038 0301-4797/© 2016 Elsevier Ltd. All rights reserved.
hydrocarbons and their low degradation rates require special attention to remediate these pollutants (Johnsen et al., 2007). Various physical, chemical and thermal methods have been employed to clean up oil contaminated sites (Frick et al., 1999). More recently, bioremediation has been suggested as a cost effective and environmental friendly method for soil cleaning. In bioremediation the capability of biological agents is exploited to degrade hydrocarbons. A large body of literature exist that suggest bioremediation as a cost effective and environmental friendly method of soil cleaning (Kwok and Loh, 2003; Glick, 2003; Zhuang et al., 2007; Gerhardt et al., 2009; Dindar et al., 2013; Sarma Roy et al., 2014). Moisture content of soil, microbial population, nutrient availability, soil type, salinity, and oxygen transport in soil are among the factors affecting the process of bioremediation. The moisture content of soil should be at an optimum range. Low levels of moisture content decrease microbial activity, while excess water may create resistance to oxygen transfer and may also produce an unwanted leachate (Schjønning et al., 2011). In bioremediation the
198
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Table 1 Bandar Abbas gasoil components. Water and sediments
Ash
Total sulfur
Density
Compound
0.05%Vol
0.01%wt
1%wt
840 kg/m3
C6eC16
moisture content is normally adjusted to a fraction of water holding capacity of the soil. The optimum value of moisture, however, is a function of soil type, pore size distribution, and soil texture. The information in the literature on the optimum value of the moisture content of soil for the purpose of bioremediation is scarce, and more investigation is needed to address this case. In general, optimum microbial activity is achieved by the maximum water content that does not restrict oxygen diffusion. Nitrogen, phosphorous, sulfur and some other nutrients are necessary for microbial growth and activity. Therefore contaminated soils should contain ample amounts of these elements for a successful bioremediation process. As a rule of thumb the ratio of Carbon: Nitrogen: phosphorous should be 100:10:1 to ensure a balanced medium for microbial growth in term of nutrients (Prescott et al., 2002). Soil type and texture can affect bioremediation. Fine grained soils like clay have low permeability and retard oxygen and nutrients transport in the soil. Controlling the moisture content in fine grained soils is difficult due to having small pores and high surface area (Balba et al., 1998). Clays can also catalyze humic acid formation and protect organic materials from decomposition within aggregates (Stott and Martin, 1990). Bioremediation of clays is therefore a challenging task. The ratio of external to total surface area of soil particles is also an important factor since only external surfaces are accessible to microorganism (Kwok and Loh, 2003). The effect of soil type is among the less investigated factors affecting bioremediation. The aim of the present study is to determine the role of soil type and moisture content in the bioremediation of highly contaminated soils. The effects of soil type and moisture content have been investigated using a two factor factorial experimental design. 2. Materials and methods 2.1. Soil Four different types of soil were selected. Sandy soil containing 100% sand (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and 32% sand (type III), and coarse grained soil with high clay content containing 40% gravel, 20% sand, and 40% clay (type IV). The soils were categorized based on the Unified Soil Classification System (USCS). Soils types I,II, and III were obtained from the suburb of Yazd, Iran (31.8972 N, 54.3678 E), and soil type IV was obtained from the suburb of Kerman, Iran (30.2907 N, 57.0679 E). The soils were stored in closed buckets at room conditions before the tests. An investigation
on the origin of the soils indicated negligible organic content. The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg, and left on the floor at room temperature. After evaporation of the light hydrocarbons, the residual gasoil in the soils was quantified, and the soils were stored for the next step. The gasoil was obtained from Bandar Abbas refinery (Table 1). 2.2. Design of experiment for bioremediation A full factorial experimental design was applied to investigate the effects of soil type, and moisture content on bioremediation. Soil type in four levels as mentioned above, and moisture content in two levels of 10 and 20% were investigated. The experiment contained 8 runs which were performed in duplicate. 30 kg soil was used for each run. The soils were placed in buckets.110 g (NH4)2SO4 and 108 g KH2PO4 were added to each bucket as nutrient supplements. The chemicals were purchased from Kiankaveh Azma pharmaceutical & chemicals complex Inc. These amounts resulted in the approximate ratio of 100:1:1 for Carbon: Nitrogen: phosphorous in soils at the beginning of the bioremediation process. The amount of (NH4)2SO4 was considered lower than usual to avoid possible excessive osmotic pressure in the microenvironments of the soils. A group of unidentified petroleum degrading microorganisms were used in this research. The microorganisms were isolated from a petroleum contaminated soil, previously undergone a bioremediation process. The microorganisms were grown in a basal mineral medium with the composition presented in Table 2, and with gasoil as the substrate. 500 mL of this microbe containing medium was used as the inoculums for each bucket. The moisture content of the soils was measured on monthly basis and adjusted with tap water. The soils were blended thoroughly after the addition of water. Due the low removal percentage of TPH in clay soil, in a complementary experiment, the soil was subjected to more frequent water adjustment and mixing. In this experiment, two factors were examined: water content and mixing. Water content was examined at five levels of 5, 10, 20, 30, and 40%. Thorough mixing of the soil every 48 h after moisture adjustment was another factor (versus intact samples during bioremediation). The experiment was designed as a two factor factorial experiment having 10 runs. The runs were performed in duplicate. The samples weighed 500 g. For this experiment the bioremediation continued for one month. 2.3. Quantification of residual TPH in soil For quantification of residual TPH is soil, samples (2 g) were taken and dried in an oven at the temperature of 70 C for 5 h. The TPH content of the soil samples were extracted by Soxhlet apparatus and quantified based on the EPA Method 9071B (EPA, Method 9071B, 1998). A blank test was done to determine the efficiency TPH
Table 2 Composition of the basal mineral medium used for the initial microbial growth. Compound (g L
1
)
CaCl2$7H2O (0.04)
MgCl2$7H2O (0.2)
K2HPO4 (4.3)
KH2PO4 (3.4)
(NH4)2SO4 (4)
Compound (g L
1
)
MnCl2 (0.001)
NaMoO4 (0.00175)
CuSO4 (0.00015)
H3BO3 (0.000375)
ZnSO4 (0.0017)
FeSO4 (0.03)
Table 3 Removal percentage of TPH in soil after 270 days of bioremediation. Moisture
10% 20%
Soil Type Sandy soil
Clay soil
Coarse grained soil
Coarse grained soil with high clay content
63% 70%
23.5% 17%
62.5% 57%
65% 66.5%
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
199
Table 4 Analysis of variance for the effects of soil type and moisture on bioremediation. Source of variation
Degree of freedom
Sum of squares
Mean sum of squares
F
Pvalue
Soil type Moisture Soil type- moisture interaction Error
3 1 3 8
5851.69 14.06 109.69 77.50
1950.56 14.06 36.56 9.69
201.35 1.45 3.77
0.000 0.263 0.059
100
TPH Removal(%)
extraction from the soil samples. For this purpose 5 g gasoil was blended with 10 g dried soil, and put the sample in an oven at 70 C for 24 h for the evaporation of light hydrocarbons. The sample was then weighed to determine the amount of residual gasoil in the soil. Subsequently the sample was subjected to extraction and quantification by the EPA Method 9071B. This blank test verified that more than 95% of the residual gasoil in the soil could be extracted.
10% Moisture Con.
80
20% Moisture Con.
60 40 20
0
2.4. GC (Gas chromatograph) and FTIR (Fourier Transform Infrared spectroscopy) analysis
60
120
180
240
300
Time (Day) Fig. 1. TPH removal in sandy soil (type I) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.625 g/kg.
100
TPH Removal(%)
The extracted TPH from the sandy soil was analyzed by FTIR and GC to monitor the changes in the composition of the TPH qualitatively. For GC analysis the extracted TPH from the soil sample was diluted with chloroform and 2 microliter of the resulting solution was injected to the GC. The temperature of injector and detector were 250 C and 200 C, respectively. The temperature of the column was 100 C initially, and increased by the rate of 15 C/min to 300 C. The column was capillary Varian CP-Sil5CB 30 m 0.32 mm 0.10 mm e CP8740. Nitrogen was the carrier gas. For FTIR analysis the extracted oil from the soil was made into pellets using KBr in the ratio of 100:1(100 mg KBr and 1 mg sample), and spectra were taken in the range of 500e4000 cm 1.
0
80 60
10% Moisture Con.
20% Moisture Con.
40 20 0
0
60
120
180
240
300
Time(Days)
2.5. Statistics Analysis of variance (ANOVA) was performed to determine the significance of the factors using Minitab software.
Fig. 2. TPH removal in clay soil (type II) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.700 g/kg.
100
Table 3 shows the results of bioremediation on day 270. The bioremediation was most successful in sandy soil with more than 70% degradation of TPH. The removal percentage was relatively low in clay. In Table 4 the results of the analysis of variance have been presented. The results shows that soil type significantly affects bioremediation (Pvalue < 0.05), and the variation in moisture content does not significantly affect the TPH removal in the tested range. The removal percentages in other soils were comparable to each other. The results confirm that the presence of sand in the soil is advantageous in bioremediation. The low bioremediation in clay could be due to inefficient oxygen transfer in the soil. Fine grained clay with high surface area formed a sticky texture in the presence of water, blocking efficient oxygen transfer through the soil. Sandy soils on the other hand are more porous than clays. Higher porosity allows better oxygen transfer in the soil which is essential to biodegradation of hydrocarbons. Larger pores provide also enough space for microbial growth. Akbari and Ghoshal (2015) demonstrated that pores smaller than 3 micrometers are not accessible to bacteria. Another reason for low degradation of hydrocarbons in clay could be strong adsorption of the pollutants on the surface of soil particles. Figs. 1e4 show the results of bioremediation for the soils as a function of time and moisture content. For the sandy soil (Type I)
80 10% Moisture Con.
60
20% Moisture Con.
40 20 0 0
60
120
180
240
300
Time(Days) Fig. 3. TPH removal in coarse grained soil (type III) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 71.610 g/kg.
100
TPH Removal(%)
3.1. The effects of soil type and moisture content on the bioremediation of gasoil
TPH Removal(%)
3. Result and discussions
80 60 10% Moisture Con.
20% Moisture Con.
40 20 0 0
60
120
180
240
300
Time(Days)
Fig. 4. TPH removal in coarse grained soil with high clay content (type IV) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.525 g/kg.
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A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Table 5 TPH removal percentage in the clay soil as a function of moisture content and Mixing in the complementary experiment. Moisture content
Water addition & mixing
5% 10% 20% 30% 40%
35% 57% 45% 35% 16%
± ± ± ± ±
Water addition 30% ± 0.06 47% ± 0.08 43% ± 0.03 30% ± 0.01 7% ± 0.03
0.04 0.03 0.04 0.03 0.06
the gasoil residual was 69.63 g/kg at the beginning and decreased to 20.6 g/kg on day 270. For the clay soil (Type II) the gasoil residual was 69.7 g/kg at the beginning and decreased to 52.9 g/kg on day 270. For the coarse soil (Type III) the gasoil residual was 71.61 g/kg at the beginning and decreased to 26.35 g/kg on day 270, and finally for the coarse soil with high clay content (Type IV) the gasoil residual was 69.52 g/kg at the beginning and decreased to22.55 g/kg on day 270. Overall the results of this part showed that sandy soils are the best candidate for bioremediation while clay soils resist bioremediation. The results of the complementary experiment on the clay soil have been presented in Table 5. The results indicate that by
frequent mixing the TPH removal increased to 57% in one month. The removal in unmixed soil samples was lower than the samples with mixing. The best result here was for the moisture content of 10%. Higher moisture content negatively affected the TPH removal in the samples. Table 6 shows the results of the analysis of variance for this experiment. The results confirm that the moisture content and blending both significantly affect the TPH removal in clay soils. Comparing the results of this experiment with the previous one, it can be concluded that the main reason for low degradation of TPH in the previous experiment was low availability of oxygen to the microbes. The large size of the sample (30 kg) made it difficult to mix it efficiently and this prevented proper exposure of the soil aggregates with air. In the latter experiment small size of the samples (500 g) and frequent mixing allowed efficient exposure to the air and improved the rate of biodegradation. 3.2. GC and FTIR analysis Fig. 5 compares the GC chromatograms for the extracted TPH from the sandy soil on days 0 and 270. The chromatogram for the extracted oil on day 270 has lower number of peaks indicating near complete degradation of some components. Other peaks appeared smaller than the corresponding peaks in the chromatogram
Table 6 Analysis of variance for the effects of moisture content and mixing on the TPH removal in clay. Source of variation
Degree of freedom
Sum of squares
Mean sum of squares
F
Pvalue
Moisture Mixing Moisture- mixing interaction Error
4 1 4 10
3737.69 192.20 42.80 80.00
933.50 192.20 10.70 8.00
116.69 24.02 1.34
0.000 0.001 0.322
Fig. 5. Chromatograms of the extracted hydrocarbons from the sandy soil on days 0 (top), and 270 (down).
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
201
Fig. 6. FTIR Spectra of the extracted hydrocarbons from the sandy soil on days 0 and 270.
obtained for the extracted residual oil on day 0 indicating the biodegradability of most components of the gasoil. Another point form comparing the chromatograms is that the total area of the peaks for the chromatogram of the sample on day 270 is 76.5% smaller than the total area of the peaks for the chromatogram of the sample extracted on day 0. This figure is reasonably close to the removal percentage of TPH (70%) obtained by the gravimetric method (EPA Method 9071B). The reduction in total area of peaks, therefore, can be considered as an approximate measure for the reduction in TPH content of the soils. The FTIR spectra of the extracted hydrocarbons from the sandy soil on days 0 and 270 have been presented in Fig. 6. Inspection of the spectra shows considerable change in the extracted sample after bioremediation. All spectra show strong absorptions in the wavelength range of 3000e2840 cm 1. The absorptions arise from CeH stretching in the alkanes. This indicates that the remaining hydrocarbons after bioremediation are mainly alkanes. Many of absorption peaks at lower wavelengths have been weakened or disappeared. 4. Conclusion Bioremediation is a feasible method for the remediation of sand containing soils highly contaminated with gasoil. More than 70% of TPH could be removed from sandy soil in 270 days. The initial TPH content was 7%. The bioremediation of clay soil was not successful due to the low bioavailability of hydrocarbons to microorganisms. Bioremediation of soils containing both clay and sand gave comparable results to the bioremediation of sandy soils. The moisture content of 10% was sufficient for bioremediation and greater moisture contents did not result in better performance. For quantification of the removal percentage of TPH in soil the EPA Method 9071B which is a gravimetric method, and comparison of GC chromatograms of the extracted TPH before and after the process gave similar results. Our future research in this area is focused on the ways for better bioremediation of clay soils. Application of surfactants is a possible way to desorb hydrocarbons from clay and improve their bioavailability. Inclusion of cheap inducers such as waste organic materials is another possible method to improve bioremediation of clay soils. Microbes can proliferate using the inducers and consume the hydrocarbons upon the exhaustion of the inducers with a large population. Other research needs in this area are quantification of oxygen transport in clay soils, and the effect of oxidation reduction potential on bioremediation of
contaminated clay soils.
References Akbari, A., Ghoshal, S., 2015. Bioaccessible porosity in soil aggregates and implications for biodegradation of highly molecular weight petroleum compounds. Environ. Sci. Technol. 49, 14368e14375. Balba, M., Al-Awadhi, N., Al-Daher, R., 1998. Bioremediation of oil contaminated soil: microbiology methods for feasibility assessment and field evaluation. J. Microbiol. Methods 32, 155e164. De Jong, E., 1980. Effect of a crude oil spill on cereals. Environ. Pollute 22, 187e307. Dindar, E., S¸a_gban, F.O.T., Bas¸kaya, H.S., 2013. Bioremediation of petroleum contaminated soil. J. Biodivers. Environ. Sci. 7, 39e47. EPA Method 9071B, 1998. N-Hexane Extractable Material (HEM) for Sludge, Sediment, and Solid Samples, Revision 2. Frick, C.M., Farrell, R.E., Germida, J.J., 1999. Assesment of Phytoremediation as an Insitu Technique for Cleaning Oil Contaminated Sites. PTAC Petroleum Technology Alliance, Canada,Calgary. Gerhardt, K., Huang, X.D., Glick, B.R., Greenberg, B.M., 2009. Phytoremediation and rhizoremediation of organic soil contaminants: potential and challenges. Plant Sci. 176, 20e30. Glick, B.R., 2003. Phytoremediation: synergistic use of plants and bacteria to clean up the environment. Biotechnol. Adv. 21, 383e393. Head, I., Jones, D., Roling, W., 2006. Marine microorganisms make a meal of oil. Nat. Rev. Microbiol. 4, 173e182. Johnsen, A.R., Schmidt, S., Hybholy, T.K., Hendriksen, S., Jacobsen, C.S., Andersen, O., 2007. Strong impact on the polycyclic aromatic hydrocarbon (PAH)-degrading Community of a PAH-polluted soil but marginal effect on PAH degradation when priming with bioremediated soil dominated by mycobacteria. Appl. Environ. Microbiol. 73, 1474e1480. Kwok, C., Loh, K., 2003. Effects of Singapore soil type on bioavailability of nutrients in soil bioremediation. Adv. Environ. Res. 7, 889e900. Margesin, R., Labbe, D., Schinner, F., Greer, C., Whyte, L., 2003. Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine alpine soils. Appl. Environ. Microbiol. 69, 3085e3092. Prescott, Harley, Klein, 2002. Microbiology, fifth ed. Mcgraw-hill. Sarma Roy, A., Baruah, R., Borah, M., Kumar Singh, A., Boruah, Hari Prasanna Deka, Saikia, Neelima, Deka, Manab, Dutta, Nipu, Bora, Tarun Chandra, 2014. Bioremediation potential of native hydrocarbon degrading bacterial strains in crude oil contaminated soil under microcosm study. Int. Biodeterior. Biodegrad. 94, 79e89. Schjønning, P., Thomsen, I.K., Petersen, S.O., Kristensen, K., Christensen, B.T., 2011. Relating soil microbial activity to water content and tillage-induced differences in soil structure. Geoderma 163, 256e264. Stott, D.E., Martin, J.P., 1990. Synthesis and degradation of natural and synthetic humic material in soils. In: McCarty, P., Clapp, C.E., Malcolm, R.L., Bloom, P.R. (Eds.), Humic Substances in Soils and Corp Sciences: Selected Readings. AsASSSA, Madison, Wl, pp. 37e63. Udo, E.J., Fayemi, A.A.A., 1995. The effect of oil pollution on soil germination, growth and nutrient uptake of corn. J. Environ. Qual. 4, 537e540. Ulrici, W., 2008. Contaminant soil areas, different countries and contaminant monitoring of contaminants., Environmental Process II. Soil Decontam. Biotechnol. 11, 5e42 (WILEY-VCH Verlag GmbH). Zhuang, X., Chen, J., Shim, H., Bai, Z., 2007. New advances in plant growth promoting rhizobacteria for bioremediation. Environ. Int. 33, 406e413.
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
The effect of soil type on the bioremediation of petroleum contaminated soils Ali Haghollahi a, Mohammad Hassan Fazaelipoor a, b, *, Mahin Schaffie a, b a b
Mineral Industries Research Center, Shahid Bahonar University of Kerman, Iran Department of Chemical Engineering, Faculty of Engineering, Shahid Bahonar University of Kerman, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 February 2016 Received in revised form 13 May 2016 Accepted 14 May 2016
In this research the bioremediation of four different types of contaminated soils was monitored as a function of time and moisture content. The soils were categorized as sandy soil containing 100% sand (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and 32% sand (type III), and coarse grained with high clay content containing 40% gravel, 20% sand, and 40% clay (type IV). The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg, and left on the floor for the evaporation of light hydrocarbons. A full factorial experimental design with soil type (four levels), and moisture content (10 and 20%) as the factors was employed. The soils were inoculated with petroleum degrading microorganisms. Soil samples were taken on days 90, 180, and 270, and the residual total petroleum hydrocarbon (TPH) was extracted using soxhlet apparatus. The moisture content of the soils was kept almost constant during the process by intermittent addition of water. The results showed that the efficiency of bioremediation was affected significantly by the soil type (Pvalue < 0.05). The removal percentage was the highest (70%) for the sandy soil with the initial TPH content of 69.62 g/kg, and the lowest for the clay soil (23.5%) with the initial TPH content of 69.70 g/kg. The effect of moisture content on bioremediation was not statistically significant for the investigated levels. The removal percentage in the clay soil was improved to 57% (within a month) in a separate experiment by more frequent mixing of the soil, indicating low availability of oxygen as a reason for low degradation of hydrocarbons in the clay soil. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Total petroleum hydrocarbon (TPH) Bioremediation Moisture Soil type Sandy soil Clay soil
1. Introduction Petroleum hydrocarbons are persistent pollutants in the environment. Uncontrolled release of these compounds affects soil, water, and air negatively (Ulrici, 2008). Soil pollution with hydrocarbons is caused by the leakage from underground reservoirs, petroleum refineries, storage facilities, and accidental spillage from production units and transport pipelines. The presence of petroleum hydrocarbons affects physical, physiological and biochemical properties of soil (Margesin et al., 2003; Head et al., 2006). Plants are susceptible to oil exposure due to phytotoxic nature of hydrocarbons, and immobilization of nutrients in the soil (De Jong, 1980; Udo and Fayemi, 1995). Inherent mutagenic properties of some
* Corresponding author. Department of Chemical Engineering, Faculty of Engineering, Shahid Bahonar University of Kerman, Iran. E-mail addresses: [email protected], [email protected] (M.H. Fazaelipoor). http://dx.doi.org/10.1016/j.jenvman.2016.05.038 0301-4797/© 2016 Elsevier Ltd. All rights reserved.
hydrocarbons and their low degradation rates require special attention to remediate these pollutants (Johnsen et al., 2007). Various physical, chemical and thermal methods have been employed to clean up oil contaminated sites (Frick et al., 1999). More recently, bioremediation has been suggested as a cost effective and environmental friendly method for soil cleaning. In bioremediation the capability of biological agents is exploited to degrade hydrocarbons. A large body of literature exist that suggest bioremediation as a cost effective and environmental friendly method of soil cleaning (Kwok and Loh, 2003; Glick, 2003; Zhuang et al., 2007; Gerhardt et al., 2009; Dindar et al., 2013; Sarma Roy et al., 2014). Moisture content of soil, microbial population, nutrient availability, soil type, salinity, and oxygen transport in soil are among the factors affecting the process of bioremediation. The moisture content of soil should be at an optimum range. Low levels of moisture content decrease microbial activity, while excess water may create resistance to oxygen transfer and may also produce an unwanted leachate (Schjønning et al., 2011). In bioremediation the
198
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Table 1 Bandar Abbas gasoil components. Water and sediments
Ash
Total sulfur
Density
Compound
0.05%Vol
0.01%wt
1%wt
840 kg/m3
C6eC16
moisture content is normally adjusted to a fraction of water holding capacity of the soil. The optimum value of moisture, however, is a function of soil type, pore size distribution, and soil texture. The information in the literature on the optimum value of the moisture content of soil for the purpose of bioremediation is scarce, and more investigation is needed to address this case. In general, optimum microbial activity is achieved by the maximum water content that does not restrict oxygen diffusion. Nitrogen, phosphorous, sulfur and some other nutrients are necessary for microbial growth and activity. Therefore contaminated soils should contain ample amounts of these elements for a successful bioremediation process. As a rule of thumb the ratio of Carbon: Nitrogen: phosphorous should be 100:10:1 to ensure a balanced medium for microbial growth in term of nutrients (Prescott et al., 2002). Soil type and texture can affect bioremediation. Fine grained soils like clay have low permeability and retard oxygen and nutrients transport in the soil. Controlling the moisture content in fine grained soils is difficult due to having small pores and high surface area (Balba et al., 1998). Clays can also catalyze humic acid formation and protect organic materials from decomposition within aggregates (Stott and Martin, 1990). Bioremediation of clays is therefore a challenging task. The ratio of external to total surface area of soil particles is also an important factor since only external surfaces are accessible to microorganism (Kwok and Loh, 2003). The effect of soil type is among the less investigated factors affecting bioremediation. The aim of the present study is to determine the role of soil type and moisture content in the bioremediation of highly contaminated soils. The effects of soil type and moisture content have been investigated using a two factor factorial experimental design. 2. Materials and methods 2.1. Soil Four different types of soil were selected. Sandy soil containing 100% sand (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and 32% sand (type III), and coarse grained soil with high clay content containing 40% gravel, 20% sand, and 40% clay (type IV). The soils were categorized based on the Unified Soil Classification System (USCS). Soils types I,II, and III were obtained from the suburb of Yazd, Iran (31.8972 N, 54.3678 E), and soil type IV was obtained from the suburb of Kerman, Iran (30.2907 N, 57.0679 E). The soils were stored in closed buckets at room conditions before the tests. An investigation
on the origin of the soils indicated negligible organic content. The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg, and left on the floor at room temperature. After evaporation of the light hydrocarbons, the residual gasoil in the soils was quantified, and the soils were stored for the next step. The gasoil was obtained from Bandar Abbas refinery (Table 1). 2.2. Design of experiment for bioremediation A full factorial experimental design was applied to investigate the effects of soil type, and moisture content on bioremediation. Soil type in four levels as mentioned above, and moisture content in two levels of 10 and 20% were investigated. The experiment contained 8 runs which were performed in duplicate. 30 kg soil was used for each run. The soils were placed in buckets.110 g (NH4)2SO4 and 108 g KH2PO4 were added to each bucket as nutrient supplements. The chemicals were purchased from Kiankaveh Azma pharmaceutical & chemicals complex Inc. These amounts resulted in the approximate ratio of 100:1:1 for Carbon: Nitrogen: phosphorous in soils at the beginning of the bioremediation process. The amount of (NH4)2SO4 was considered lower than usual to avoid possible excessive osmotic pressure in the microenvironments of the soils. A group of unidentified petroleum degrading microorganisms were used in this research. The microorganisms were isolated from a petroleum contaminated soil, previously undergone a bioremediation process. The microorganisms were grown in a basal mineral medium with the composition presented in Table 2, and with gasoil as the substrate. 500 mL of this microbe containing medium was used as the inoculums for each bucket. The moisture content of the soils was measured on monthly basis and adjusted with tap water. The soils were blended thoroughly after the addition of water. Due the low removal percentage of TPH in clay soil, in a complementary experiment, the soil was subjected to more frequent water adjustment and mixing. In this experiment, two factors were examined: water content and mixing. Water content was examined at five levels of 5, 10, 20, 30, and 40%. Thorough mixing of the soil every 48 h after moisture adjustment was another factor (versus intact samples during bioremediation). The experiment was designed as a two factor factorial experiment having 10 runs. The runs were performed in duplicate. The samples weighed 500 g. For this experiment the bioremediation continued for one month. 2.3. Quantification of residual TPH in soil For quantification of residual TPH is soil, samples (2 g) were taken and dried in an oven at the temperature of 70 C for 5 h. The TPH content of the soil samples were extracted by Soxhlet apparatus and quantified based on the EPA Method 9071B (EPA, Method 9071B, 1998). A blank test was done to determine the efficiency TPH
Table 2 Composition of the basal mineral medium used for the initial microbial growth. Compound (g L
1
)
CaCl2$7H2O (0.04)
MgCl2$7H2O (0.2)
K2HPO4 (4.3)
KH2PO4 (3.4)
(NH4)2SO4 (4)
Compound (g L
1
)
MnCl2 (0.001)
NaMoO4 (0.00175)
CuSO4 (0.00015)
H3BO3 (0.000375)
ZnSO4 (0.0017)
FeSO4 (0.03)
Table 3 Removal percentage of TPH in soil after 270 days of bioremediation. Moisture
10% 20%
Soil Type Sandy soil
Clay soil
Coarse grained soil
Coarse grained soil with high clay content
63% 70%
23.5% 17%
62.5% 57%
65% 66.5%
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199
Table 4 Analysis of variance for the effects of soil type and moisture on bioremediation. Source of variation
Degree of freedom
Sum of squares
Mean sum of squares
F
Pvalue
Soil type Moisture Soil type- moisture interaction Error
3 1 3 8
5851.69 14.06 109.69 77.50
1950.56 14.06 36.56 9.69
201.35 1.45 3.77
0.000 0.263 0.059
100
TPH Removal(%)
extraction from the soil samples. For this purpose 5 g gasoil was blended with 10 g dried soil, and put the sample in an oven at 70 C for 24 h for the evaporation of light hydrocarbons. The sample was then weighed to determine the amount of residual gasoil in the soil. Subsequently the sample was subjected to extraction and quantification by the EPA Method 9071B. This blank test verified that more than 95% of the residual gasoil in the soil could be extracted.
10% Moisture Con.
80
20% Moisture Con.
60 40 20
0
2.4. GC (Gas chromatograph) and FTIR (Fourier Transform Infrared spectroscopy) analysis
60
120
180
240
300
Time (Day) Fig. 1. TPH removal in sandy soil (type I) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.625 g/kg.
100
TPH Removal(%)
The extracted TPH from the sandy soil was analyzed by FTIR and GC to monitor the changes in the composition of the TPH qualitatively. For GC analysis the extracted TPH from the soil sample was diluted with chloroform and 2 microliter of the resulting solution was injected to the GC. The temperature of injector and detector were 250 C and 200 C, respectively. The temperature of the column was 100 C initially, and increased by the rate of 15 C/min to 300 C. The column was capillary Varian CP-Sil5CB 30 m 0.32 mm 0.10 mm e CP8740. Nitrogen was the carrier gas. For FTIR analysis the extracted oil from the soil was made into pellets using KBr in the ratio of 100:1(100 mg KBr and 1 mg sample), and spectra were taken in the range of 500e4000 cm 1.
0
80 60
10% Moisture Con.
20% Moisture Con.
40 20 0
0
60
120
180
240
300
Time(Days)
2.5. Statistics Analysis of variance (ANOVA) was performed to determine the significance of the factors using Minitab software.
Fig. 2. TPH removal in clay soil (type II) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.700 g/kg.
100
Table 3 shows the results of bioremediation on day 270. The bioremediation was most successful in sandy soil with more than 70% degradation of TPH. The removal percentage was relatively low in clay. In Table 4 the results of the analysis of variance have been presented. The results shows that soil type significantly affects bioremediation (Pvalue < 0.05), and the variation in moisture content does not significantly affect the TPH removal in the tested range. The removal percentages in other soils were comparable to each other. The results confirm that the presence of sand in the soil is advantageous in bioremediation. The low bioremediation in clay could be due to inefficient oxygen transfer in the soil. Fine grained clay with high surface area formed a sticky texture in the presence of water, blocking efficient oxygen transfer through the soil. Sandy soils on the other hand are more porous than clays. Higher porosity allows better oxygen transfer in the soil which is essential to biodegradation of hydrocarbons. Larger pores provide also enough space for microbial growth. Akbari and Ghoshal (2015) demonstrated that pores smaller than 3 micrometers are not accessible to bacteria. Another reason for low degradation of hydrocarbons in clay could be strong adsorption of the pollutants on the surface of soil particles. Figs. 1e4 show the results of bioremediation for the soils as a function of time and moisture content. For the sandy soil (Type I)
80 10% Moisture Con.
60
20% Moisture Con.
40 20 0 0
60
120
180
240
300
Time(Days) Fig. 3. TPH removal in coarse grained soil (type III) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 71.610 g/kg.
100
TPH Removal(%)
3.1. The effects of soil type and moisture content on the bioremediation of gasoil
TPH Removal(%)
3. Result and discussions
80 60 10% Moisture Con.
20% Moisture Con.
40 20 0 0
60
120
180
240
300
Time(Days)
Fig. 4. TPH removal in coarse grained soil with high clay content (type IV) as a function of time. Each point represents the average value of duplicate samples. Initial TPH 69.525 g/kg.
200
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Table 5 TPH removal percentage in the clay soil as a function of moisture content and Mixing in the complementary experiment. Moisture content
Water addition & mixing
5% 10% 20% 30% 40%
35% 57% 45% 35% 16%
± ± ± ± ±
Water addition 30% ± 0.06 47% ± 0.08 43% ± 0.03 30% ± 0.01 7% ± 0.03
0.04 0.03 0.04 0.03 0.06
the gasoil residual was 69.63 g/kg at the beginning and decreased to 20.6 g/kg on day 270. For the clay soil (Type II) the gasoil residual was 69.7 g/kg at the beginning and decreased to 52.9 g/kg on day 270. For the coarse soil (Type III) the gasoil residual was 71.61 g/kg at the beginning and decreased to 26.35 g/kg on day 270, and finally for the coarse soil with high clay content (Type IV) the gasoil residual was 69.52 g/kg at the beginning and decreased to22.55 g/kg on day 270. Overall the results of this part showed that sandy soils are the best candidate for bioremediation while clay soils resist bioremediation. The results of the complementary experiment on the clay soil have been presented in Table 5. The results indicate that by
frequent mixing the TPH removal increased to 57% in one month. The removal in unmixed soil samples was lower than the samples with mixing. The best result here was for the moisture content of 10%. Higher moisture content negatively affected the TPH removal in the samples. Table 6 shows the results of the analysis of variance for this experiment. The results confirm that the moisture content and blending both significantly affect the TPH removal in clay soils. Comparing the results of this experiment with the previous one, it can be concluded that the main reason for low degradation of TPH in the previous experiment was low availability of oxygen to the microbes. The large size of the sample (30 kg) made it difficult to mix it efficiently and this prevented proper exposure of the soil aggregates with air. In the latter experiment small size of the samples (500 g) and frequent mixing allowed efficient exposure to the air and improved the rate of biodegradation. 3.2. GC and FTIR analysis Fig. 5 compares the GC chromatograms for the extracted TPH from the sandy soil on days 0 and 270. The chromatogram for the extracted oil on day 270 has lower number of peaks indicating near complete degradation of some components. Other peaks appeared smaller than the corresponding peaks in the chromatogram
Table 6 Analysis of variance for the effects of moisture content and mixing on the TPH removal in clay. Source of variation
Degree of freedom
Sum of squares
Mean sum of squares
F
Pvalue
Moisture Mixing Moisture- mixing interaction Error
4 1 4 10
3737.69 192.20 42.80 80.00
933.50 192.20 10.70 8.00
116.69 24.02 1.34
0.000 0.001 0.322
Fig. 5. Chromatograms of the extracted hydrocarbons from the sandy soil on days 0 (top), and 270 (down).
A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
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Fig. 6. FTIR Spectra of the extracted hydrocarbons from the sandy soil on days 0 and 270.
obtained for the extracted residual oil on day 0 indicating the biodegradability of most components of the gasoil. Another point form comparing the chromatograms is that the total area of the peaks for the chromatogram of the sample on day 270 is 76.5% smaller than the total area of the peaks for the chromatogram of the sample extracted on day 0. This figure is reasonably close to the removal percentage of TPH (70%) obtained by the gravimetric method (EPA Method 9071B). The reduction in total area of peaks, therefore, can be considered as an approximate measure for the reduction in TPH content of the soils. The FTIR spectra of the extracted hydrocarbons from the sandy soil on days 0 and 270 have been presented in Fig. 6. Inspection of the spectra shows considerable change in the extracted sample after bioremediation. All spectra show strong absorptions in the wavelength range of 3000e2840 cm 1. The absorptions arise from CeH stretching in the alkanes. This indicates that the remaining hydrocarbons after bioremediation are mainly alkanes. Many of absorption peaks at lower wavelengths have been weakened or disappeared. 4. Conclusion Bioremediation is a feasible method for the remediation of sand containing soils highly contaminated with gasoil. More than 70% of TPH could be removed from sandy soil in 270 days. The initial TPH content was 7%. The bioremediation of clay soil was not successful due to the low bioavailability of hydrocarbons to microorganisms. Bioremediation of soils containing both clay and sand gave comparable results to the bioremediation of sandy soils. The moisture content of 10% was sufficient for bioremediation and greater moisture contents did not result in better performance. For quantification of the removal percentage of TPH in soil the EPA Method 9071B which is a gravimetric method, and comparison of GC chromatograms of the extracted TPH before and after the process gave similar results. Our future research in this area is focused on the ways for better bioremediation of clay soils. Application of surfactants is a possible way to desorb hydrocarbons from clay and improve their bioavailability. Inclusion of cheap inducers such as waste organic materials is another possible method to improve bioremediation of clay soils. Microbes can proliferate using the inducers and consume the hydrocarbons upon the exhaustion of the inducers with a large population. Other research needs in this area are quantification of oxygen transport in clay soils, and the effect of oxidation reduction potential on bioremediation of
contaminated clay soils.
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