Removal of polycyclic aromatic hydrocarbons from soil: A comparison ...

Chemosphere 86 (2012) 985–993

Contents lists available at SciVerse ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Removal of polycyclic aromatic hydrocarbons from soil: A comparison between bioremoval and supercritical fluids extraction M.A. Amezcua-Allieri a,⇑, M.A. Ávila-Chávez a, A. Trejo a,⇑, J. Meléndez-Estrada b a b

Instituto Mexicano del Petróleo, Eje Central Lázaro Cárdenas Norte 152, 07730 México, DF, Mexico Universidad Autónoma Metropolitana-Azcapotzalco, Departamento de Ciencias Básicas, Av. San Pablo #180, Col Reynosa Tamaulipas, 02200 México, DF, Mexico

a r t i c l e

i n f o

Article history: Received 20 October 2011 Received in revised form 12 November 2011 Accepted 15 November 2011 Available online 24 December 2011 Keywords: Bioremediation Certified soil Contaminated soil Ethane Polycyclic aromatic hydrocarbons Supercritical fluid extraction

a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic substances which are resistant to environmental degradation due to their highly hydrophobic nature. Soils contaminated with PAHs pose potential risks to human and ecological health, therefore concern over their adverse effects have resulted in extensive studies on their removal from contaminated soils. The main purpose of this study was to compare experimental results of PAHs removal, from a natural certified soil polluted with PAHs, by biological methods (using bioaugmentation and biostimulation in a solid-state culture) with those from supercritical fluid extraction (SFE), using supercritical ethane as solvent. The comparison of results between the two methods showed that maximal removal of naphthalene, acenaphthene, fluorene, and chrysene was performed using bioremediation; however, for the rest of the PAHs considered (fluoranthene, pyrene, and benz(a)anthracene) SFE resulted more efficient. Although bioremediation achieved higher removal ratios for certain hydrocarbons and takes advantage of the increased rate of natural biological processes, it takes longer time (i.e. 36 d vs. half an hour) than SFE and it is best for 2–3 PAHs rings. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Polycyclic Aromatic Hydrocarbons (PAHs) constitute a group of priority pollutants (WHO, 2001), which are present in the soils of many industrially contaminated sites, particularly those associated with the petroleum industry (Samanta et al., 2002). The majority of these substances are highly persistent, easily adsorb onto the organic matter of solid particles, forming persistent micropollutants in the environment, less degradable chemical forms because of their very low water solubility (Reza et al., 2002; Reza and Trejo, 2004; Arias-González et al., 2010), their intrinsic chemical stability, and high recalcitrance to degradation (Gan et al., 2009). Therefore, hydrocarbon-polluted sites may represent a long-term source of pollution and pose a severe risk to environmental health (Andreoni and Gianfreda, 2007). Biostimulation and bioaugmentation are widely known technologies to remediate hydrocarbon-polluted sites. Biostimulation or enhanced biodegradation is the stimulation of microbial degradation of organic contaminants by the addition of microorganisms, nutrients, or optimization of environmental factors on-site or in situ. Bioaugmentation is an approach whereby microorganisms are added to a contaminated site to hasten the degradation of ⇑ Corresponding authors. Tel.: +52 55 9175 8496 (M.A. Amezcua-Allieri), tel.: +52 55 9175 8373 (A. Trejo). E-mail addresses: [email protected] (M.A. Amezcua-Allieri), [email protected] (A. Trejo). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.11.032

pollutants. It is through the introduction of excess, active, degrader microorganisms that the pollutant is mineralized, thereby providing remediation at a faster rate than would otherwise occur by means of the indigenous microorganisms (Singer et al., 2005). The biodegradation of PAHs with less than three aromatic rings has been well documented for bacteria and fungi (Zhang et al., 2006). Fungi play an important role in the hydrocarbon-oxidizing activities of the soil, and they seem to be at least as versatile as bacteria in metabolising aromatics. Fungi (e.g. Penicillium and Cunninghamella) exhibit greater hydrocarbon biodegradation than bacteria. Phenanthrene, a 3 ring-PAH is considered as a model substrate for studies on metabolism of carcinogenic PAHs (Samanta et al., 2002). The fungi Penicillium frequentans has been suggested as a suitable microorganism for phenanthrene removal from soil under certain culture conditions (Amezcua-Allieri et al., 2003; Leitao, 2009). Four-ring PAHs, e.g. fluoranthene, pyrene, chrysene, and benz(a)anthracene, have been investigated to various degrees in the biodegradation literature (Abdulsalam and Omale, 2009). Under aerobic conditions, bacteria and fungi utilize PAHs as carbon and energy sources for the degrading organisms (assimilative biodegradation) or for intracellular detoxification. Substances more amenable to biodegradation than the target contaminant can be added to the soil to stimulate the microbial cometabolic transformation of the pollutants, otherwise not degraded (Andreoni and Gianfreda, 2007).

986

M.A. Amezcua-Allieri et al. / Chemosphere 86 (2012) 985–993

Bioremediation has been demonstrated as a potential technique for PAHs removal from soil, due to the fact that it usually does not produce toxic by-products, it destroys the target chemicals, it can be used in situ and it is usually less expensive than other technologies (Abdulsalam and Omale, 2009; Gan et al., 2009; Madueño et al., 2011). However, there are other non-biological techniques that are useful to extract PAHs from soils, such as the supercritical fluid extraction (SFE). A supercritical fluid is any compound at temperature and pressure above its critical values (i.e. above the gas– liquid critical point). Above the critical point the substances are named fluids. The fluid, as it is termed, is neither a gas nor a liquid and is best described as intermediate of the two states. Supercritical solvents have been used in a very wide range of scientific and technological fields because of several advantages, such as high efficiency, ease of separation of solutes from solvent, fast mass transfer rate, low energy consumption for solvent recovery, and elimination of conventional toxic solvents. Therefore, in recent years the use of supercritical fluids (SCFs) has been proposed as an efficient alternative for the remediation of contaminated soils (Gan et al., 2009) as well as in the recovery of hydrocarbons from different types of matrices. Supercritical extraction in the environmental area has been applied mostly for PAH extraction (Saeed et al., 2009). There are some important thermodynamic parameters that are necessary to control in SFE in order to obtain high extraction ratios. Langenfeld et al. (1993) mentioned that temperature has a larger effect than pressure over the extraction capacity, for a given solvent. Since density of the SCF is a variable that is function both of pressure and temperature (Langenfeld et al., 1995) maintaining equal density of the SCF observed that higher extractions were obtained at highest values of pressure and temperature. Another element that is important in the extraction is the solute molar mass; Andrews et al. (1990) observed that the extraction capacity decreases as molar mass of PAH increases. Although several supercritical solvents, such as carbon dioxide, argon, ethane, ethylene, and propane have been used in different investigations, in general carbon dioxide is the most common solvent used both in solubility and SFE studies. Nonetheless, some authors have mentioned that the extraction capacity of CO2 could be increased adding a cosolvent. Wright et al. (1989) mentioned that CO2 has not enough solvation power to extract high molar mass hydrocarbons. Kothandaraman et al. (1992) observed that CO2 is not a good solvent due to its low polar interaction with PAHs; hence, to increase the desorption capacity they proposed to employ a cosolvent, such as water or methanol; however, Becnel and Dooley (1998) conclude that a cosolvent does not increase substantially the extraction of HAPs with three and four benzene rings. Other alternative to increase the extraction yield is to apply a solvent similar to the solute that will be extracted, i.e. the like dissolves the like; for instance Rose et al. (2001) using ethane as supercritical solvent extracted a Peace River bitumen (Alberta, Canada) blended with sand and (Ávila-Chávez et al., 2007) used supercritical ethane to extract saturated, branched, and aromatic hydrocarbons from crude oil tank-bottom sludges. On the other hand, there are different techniques focused on the remediation of polluted soils in the specialized literature, each technique has different advantages, for that reason some authors have made comparisons between different techniques. Saim et al. (1997) compared four extraction techniques against supercritical CO2 (24.5 MPa and 343.15 K) using methanol as a cosolvent, better extractions were obtained with Soxhlet extraction using dicloromethane. In the same way Hawthorne et al. (2000) compared extraction efficiencies between different extraction methods, in general quantitatively extractions were similar among the different techniques; however, they observed that different hydrocarbon fractions were obtained in each method. Hawthorne et al. (2001) compared supercritical extraction with CO2 against bioremedia-

tion. The soil obtained from a gas plant was extracted at 20 MPa, 323.15 K and 200 min, whereas the bioremediation technique was carried out during 340 d. Reduction of PAH in both techniques was similar. It was observed that in both treatments high molar mass polycyclic hydrocarbons were difficult to remove and low molar mass polycyclic hydrocarbons were easily extracted. Therefore, in this work we present the results of PAHs removal from a natural certified soil using two methods: bioremediation (using both biostimulation and bioaugmentation) and SFE with supercritical ethane as solvent. It is worth to say that both techniques are quite different and have different advantages and disadvantages. However, it is useful to compare both in terms of efficiency of removal and time. 2. Material and methods 2.1. Soil characterization A certified reference soil (CRM115-100, lot JC115) was used in this study with the following PAHs: naphthalene, acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, benzo(a)anthracene, and chrysene. To establish the physical and chemical characteristics the certified soil was homogenized (gentle blending) and characterized before treatment (Table 1). The CRM is natural matrix whose certified concentration or reference value is established through analysis in 20 different laboratories to meet the requirements specified by the USEPA/AALA RM-03 and ISO guides 34 and 35. 2.2. PAHs bioremoval 2.2.1. Inoculum production The fungi P. frequentans was grown in modified liquid Wunder media (Wunder et al., 1994) over 4 d in order to obtain the fungal pellets. The medium consisted of (in g L1): glucose, 10.0; (NH4)2SO4, 1.0; MgSO47H2O, 0.5; KH2PO4, 0.875; K2HPO4, 0.125; CaCl2.2H2O, 0.1; NaCl, 0.1; and FeSO4, 0.001. The final pH was 5.3. In order to propagate the fungal strain, 0.02 g of mycelium was added to 0.4 g bagasse with 0.2 cm3 deionized sterilized water and incubated in 125 cm3 sealed vials for 15 d, at 28 °C. This period of time corresponds to the time at which instantaneous CO2 evolution leveled off. On day 15, a sample of 4 g of the certified soil with polyaromatic hydrocarbons was added to the vial in order to adjust the moisture content to 30% (Amezcua-Allieri et al., 2003). The C:N and C:P ratio from the soil original concentration and bulking agent (bagasse) were adjusted with ammonium sulfate and monobasic ammonium phosphate, respectively. All this was carried out by triplicate. All chemicals used were at least 99% pure. The main bagasse characteristics were obtained through the following methods: total nitrogen by Kjeldhal method (AOAC, 1970), total phosphorus by Bray-I method (Bray and Kurtz, 1945), organic matter by Walkley–Black method (Jackson, 1970), and total heterotrophs by standard spread plate technique (Seeley and VanDenmark, 1981). 2.2.2. Experimental design In order to evaluate the effect of biaugmentation and biostimulation on PAHs removal (response variable), a combined experimental design, composedpffiffiffiby 22 factorial with both central and axial points, where a ¼ 1 2 was used. In all the treatments (from 1 to 13), bioaugmentation and bioestimulation was included. C:N and C:P ratios were the independent variables. The coded values of the independent variables were: a, 1, 0 (central point), +1, +a (Table 2). Central points values for the C:N and C:P ratios were selected according to a literature review and water content value. Axial values were obtained by the experimental design equation.

987

M.A. Amezcua-Allieri et al. / Chemosphere 86 (2012) 985–993 Table 1 Main physicochemical characteristics of the studied certified soil. Parameter

Method

Value

Texture Sand (%) Silt (%) Clay (%) Organic carbon (%) Water holding capacity (%) pH Total phoshorous (mg kg1) Total nitrogen (%) Cationic exchange capacity (meq 100 g1) Organic matter (%) Naphthalene (mg kg1) Acenaphthene (mg kg1) Fluorene (mg kg1) Phenanthrene (mg kg1) Fluoranthene (mg kg1) Pyrene (mg kg1) Benz(a)anthracene (mg kg1) Chrysene (mg kg1) Total bacteria (MPN) Hydrocarbonoclastic (MPN) Total fungus (colony-forming units, CFU)

Laser particle size analyzer

Sandy loam 83.65 14.21 2.14 3.38 15.00 5.92 0.82 0.25 4.80 5.83 1.34 4.60 13.02 0.08 22.10 7.66 12.11 16.81 20 105 1 103 Absent

a b

Walkley–Black (Jackson, 1970)a Ignition (Jackson, 1970) Potentiometric (Page et al., 1982) Bray-I (Bray and Kurtz, 1945) Kjeldhal (AOAC, 1970) EDTA (Jackson, 1970) Walkley–Black (Jackson, 1970)b EPA 3540C for Soxhlet extraction and EPA 8270C for GC/MS EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) EPA 3540C (Soxhlet extraction), EPA 8270C (GC/MS analysis) Most probable number (MPN) method Most probable number (MPN) method Serial dilution method (Page et al., 1982)

Recovery factor = 1.330 (Schumacher, 2002). A conversion factor of 1.724 was used to convert organic matter to organic carbon.

Table 2 Factorial experimental design with axial and central points, including independent variables (C:N and C:P ratios). Treatment

1 2 3 4 5 6 7 8 9 10 11 12 13

Coded variables

Independent variables

X

Y

C:N

C:P

0 0 0 0 0 0 1 0 1.414 1 1.414 1 1

1.414 0 0 0 0 0 1 1.414 0 1 0 1 1

47.58 90 90 90 90 90 120 132 90 60 90 60 120

14 15 15 15 15 15 10 8 15 6 15 11 10

a = 1.414. Coded variables were fixed according to the factorial experimental design.

The experimental error was calculated from central points of the composite experimental design (Montgomery, 2000). Different controls were run for each treatment: (1) soil without adding P. frequentans inoculum, to quantify the abiotic removal, (2) soil with P. frequentans and without any nutrient adjustment to evaluate the real removal improvement by biostimulation, (3) soil and fungi in order to evaluate CO2 production attributed to the use of soil organic matter as a carbon source, (4) soil with bagasse without fungi, and (5) soil with polyaromatic hydrocarbons, although without fungi nor bagasse to evaluate the real removal improvement by bioaugmentation (soil contains only soil native microflora). 2.2.3. CO2 evolution Heterotrophic activity was measured as an indirect method using CO2 evolution (Mitchell, 1992a,b). Microbial activity was monitored by analyzing CO2 accumulation in the headspace by gas chromatography. Headspace samples of 2 cm3 were taken from microcosms with a gas-tight syringe and were injected into a 580

gas chromatograph (Gow-Mac) equipped with a thermal conductivity detector and a CTRI stainless steel column. The carrier gas was helium at a flow rate of 66.5 cm3 min1. The oven, injection port, and detector temperatures were 22, 40, and 99 °C, respectively. CO2 evolution was reported as lg CO2 IDM1 (Saucedo et al., 1994), using the general gas law. IDM is initial dry matter, and includes all total solids that were used in the microcosm (PAHs, organic matter, and bagasse). PAHs removal was measured at the end of the experiment; CO2 evolution was measured every 3 d. Vials were aerated for 15 min after CO2 measurement to maintain oxygen levels in the system. 2.2.4. PAHs extraction and analysis PAHs removal was evaluated after 36 d of incubation, by triplicate, when instantaneous CO2 evolution was constant. A 4-g sample of soil samples was removed from each bottle and extracted in a Soxhlet apparatus with acetone/dichloromethane (50:50 v/v) for 16 h (Dean and Xiong, 2000). The average efficiency of PAHs extraction from soil was 95.0 ± 3.7%. The resulting extract was concentrated in a Kuderna-Danish evaporative concentrator until approximately 5 cm3 of solvent in order to change to acetonitrile (Fisher, 99% pure), adding 10 cm3 of it, concentrating again according to the standard EPA method 3540C and cleaning according to the EPA method 3600C. Extracts were analyzed by gas chromatography/mass spectrometry (GC/MS), using a GC 8000 Top Fisons with an AS800 auto-sampler, fused-silica capillary column Excellence (0.25 mm x 60 m, ID-BPS 0.25 mm, phase BP5 non-polar). The initial column temperature was 60 °C for 4 min, and the temperature was increased to 160 °C at a rate of 20 °C min1 followed by an increase to 270 °C at 5 °C min1. Then, 2 ll of the sample was injected in the splitless mode. The mass spectrometer was operated in the SIM mode (mass range, 76–288 m z1) with electron impact ionization at 70 e V. Helium was used as carrier gas at a constant flow rate of 13.2 cm3 min1. The injector temperature was 260 °C, the interface temperature was 280 °C while that of the ion source was 230 °C. PAHs were identified on the basis of its fragmentation peak in the GC/MS library. Identification was validated by comparison of the retention time using the respective standard (99% pure). PAHs quantification was accomplished by comparing its mass spectra

988


with the electron impact spectra of the respective standard purchased as certified. The base peak ion from the specific internal standard was used as the primary ion for quantification, which was accomplished by comparing the response of a major (quantification) ion relative to the internal standard using a five-point calibration curve. The method had a detection limit of 0.002 mg kg1 and ± 8% for reproducibility. The quantification limits (lg kg1) of the PAHs were 0.02 for fluorene, phenanthrene, fluoranthene, pyrene, benz(a)anthracene; 0.2 for naphthalene, 0.03 for acenaphthene and 0.01 for chrysene. The limits were determined as three times the minimum detection level (MDL) and ranged from 0.01 to 0.2 lg kg1. The MDL was set as three times the observed standard deviation of three replicate analyses of the lowest standard. 2.2.5. Statistical analysis The analysis of variance (ANOVA), regression and surface response calculations were performed using the software STATISTICAÒ 6.0. 2.3. SFE extraction and analysis 2.3.1. SFE Extraction The apparatus used in this work to carry out the supercritical extractions with ethane was constructed in our laboratory. The device and the extraction procedure have been described in detail by Ávila-Chávez et al. (2007). Fig. 1 is a schematic diagram of the complete experimental device. In general, the apparatus is divided in three main subsystems: feeding system, extraction system, and separation of solutes from the solvent system. The feeding system is composed of an ethane commercial cylinder (1) which feeds solvent into two stainless steel storage cells (2 and 3). Ethane (Praxair Mexico with > 99 mol% purity) from the storage cells is sent to a high-pressure positive displacement pump (4), which pressurizes ethane up to the desired working pressure into the extraction cell. The pump feeds ethane into the extraction system, which is placed into an insulated constant temperature air bath (5). The stainless steel extraction cell (6) contains the soil sample to be studied at the temperature and pressure selected. The extraction method was dynamic, i.e. solvent was continuously fed into the extraction cell, always attaining thermodynamic equilibrium. Once the solvent is in contact with the sample both the solvent together with the extract are sent to the separation system. The separation system is composed of separation or recovery cell (7), which is under conditions of low pressure; hence these conditions ensure total separation of the extracted PAHs from ethane. Finally, ethane is

sent to the calibrated wet test meter (8) to measure the total volume used in each experimental run. The extractions were performed through a dynamic process, i.e., the solvent is continuously fed into the extraction or equilibrium cell. The solvent together with the extract proceed to the separation system which is composed of separation or recovery cell, which is under conditions of low pressure, these conditions ensure total separation of the extracted hydrocarbons from ethane. Finally, ethane is sent to the wet test meter to measure the total volume. Supercritical extraction was carried out at (27.10 ± 0.18) MPa and (308.18 ± 0.02) K on three samples of the certified soil. Previous work from the literature and from our laboratory agree on the best conditions of temperature and pressure to reach high extraction yields and low extraction time. The volume of ethane used in the extraction was 6.23 L; this value was obtained from the wet test meter and corrected at standard conditions, i.e. 293.15 K and 101.325 kPa. Soil sample was 7.2 g for the extraction experiments, therefore the solvent to sample mass ratio was 0.94. 2.3.2. Quantitative analysis of PAHs The certified soil was extracted using Soxhlet devices according to EPA method 3540C. Three samples of 7.5 g were placed in extraction thimbles with 1 g of Na2SO4 (dried at 150 °C overnight). The thimbles containing the soil samples were extracted with 200 cm3 of a mixture of acetone–dichloromethane (50:50 v/v) during 16 h. After the extraction the liquid samples were concentrated in a Kuderna-Danish apparatus. The quantitative analysis of PAHs was made following EPA method 3600C. The concentration obtained of PAHs was compared with the corresponding values reported in the certificate. Identification and quantification of PAHs in the certified soil sample were carried out, based on EPA method 8100, using dual detection with a Waters HPLC system consisting of a solvent delivery system, model 626, with a controller, model 600S, a fluorescence detector, model 474, a photodiode array detector, model 996, and a solvent degasser unit, model 6324. For the separation of the PAHs a stainless steel analytical column of 125 mm length 4.6 mm internal diameter, with a proprietary polymeric bonded phase of 5 lm particle size, Phenomenex Envirosep PP, was used. The best chromatographic conditions to separate and quantify the studied PAHs were established to be the following: isocratic elution with acetonitrile–water 40:60 (v/v) for 5 min was performed, changing to a gradient for 25 min to reach acetonitrile– water 100:0 (v/v). The flow was kept constant throughout at

Fig. 1. Experimental system for hydrocarbons extraction from solid matrices using supercritical solvents.

989


1.2 cm3 min1. These conditions yielded the best separation and resolution of the different PAHs. Identification of each PAH was performed with the UV detector (photodiode array detector), whereas quantification was carried out with a fluorescence detector (scanning fluorescent detector). The wavelengths of excitation and emission (kex and kem, respectively) of the fluorescence detector were changed throughout the elution process to obtain the highest resolution: 0–17 min, 280 kex and 340 kem; 17–21 min, 240 kex and 425 kem; 21–25 min, 265 kex and 385 kem. The concentration of each of seven PAHs in the certified natural soil sample was obtained by interpolation from an external five-point calibration curve which was prepared from a purchased standard mixture of different PAHs (CSM-8310 rpm, from ChemService) dissolved in acetonitrile. Each calibration point is the average of at least five chromatographic injections. The concentration obtained in this work for five of the seven PAHs considered is in agreement with both the confidence and prediction intervals given in the certificate of the CRM; furthermore for naphthalene, pyrene, and benzo(a)anthracene the concentration agrees, within experimental uncertainty, with the corresponding reference value included in Table 1. The concentration of acenaphtene and fluorene of this work is in agreement with the prediction interval of the certificate. 3. Results 3.1. Soil analysis The results obtained in this work, presented in Table 1, show that the soil had a loamy texture with a predominance of sand, 400 Controls*

350

1 2

300

3

250

4

200

5

µ

6

150

7

100

8 9

50

10 11

0 0

3

6

9

12 15 18 21 24 27 30 33 36

12 13

Time (days)

Fig. 2. Heterotrophic microbial activity during soil treatment by solid state culture, as a function of C:N and C:P ratio. Arrow indicates the time when soil and nutrients were added, which coincided with CO2 evolution increases. IDM = Initial dry matter. ⁄ Controls included sterilized soil and sterilized bagasse. Uncertainty bars are too small to be shown with clarity.

was slightly acidic and the organic matter concentration was relatively high. The concentration of the different PAHs in the soil, given in the certificate as reference values on a dry weight basis, was variable depending on the type of PAH. The most probable number (MPN) of hydrocarbonoclastic bacteria was 1 103, while fungi were absent; therefore bioaugmentation and biostimulation were suitable for this soil. The main bagasse characteristics are: total nitrogen and phosphorus (mg kg1) 1603 and 32, respectively; organic matter (%), 90.3; total heterotrophs (colony-forming units, CFU g1), 13 102. 3.2. CO2 production As indicator of microbial activity, cumulative CO2 evolution was measured during 36 d (Fig. 2). CO2 production correlated well with PAHs removal in most of the cases (i.e. higher CO2 production (lg CO2/IDM) corresponds to higher PAHs removal). 3.3. PAHs removal The amount of PAHs that P. frequentans was able to remove without any nutrient addition is shown in Table 3. The amount of PAHs that P. frequentans was able to remove after the adjustment of C/N and C/P ratios (biostimulation), under different treatments, is shown in Fig. 3. Surface response for phenanthrene is omitted because total removal was obtained through bioaugmentation. Table 3 also includes removal results using bioaugmentation and biostimulation and those obtained from SFE. In terms of C/N and C/P ratios, both had a positive statistically significant effect (p < 0.0003 and p < 0.002, respectively) on PAHs removal. As it was expected, depending on the PAH, there is an optimal ratio (higher content of C than both N and P). ANOVA carried out on the changes in total PAHs concentrations with time showed that treatment 8 was the most effective in removing naphthalene and pyrene, treatment 10 for acenaphthene, treatment 2 for fluorene, treatment 3 for fluoranthene, treatment 6 for benz(a)anthracene, and treatment 1 for chrysene. Overall, at p 0.05, all treatments that received nutrient amendment and bulking were more effective than those that were not amended. Supercritical fluid extraction using ethane was able to reduce quantitatively the concentration of the different PAHs considered. It is observed that the removal of PAHs was around 80%, with the maximum reduction obtained for fluoranthene (88.3%), whereas naphthalene reduction was 59%. It is interesting to underline that supercritical extraction was carried out in one step during 30.68 min and the volume of ethane used in the extraction was 10.0 L, under laboratory conditions, although this value was corrected to standard pressure and temperature obtaining 6.23 ± 0.16 L.

Table 3 Comparison of PAHs removal results by bioremoval and SFE.

a b

PAH

Removal (%)a by fungal bioaugmentation and without bioestimulation

Naphthalene Acenaphthene Fluorene Phenanthrene Fluoranthene Pyrene Benz(a)anthracene Chrysene

12 14 6 100 11 6 11 14

Removal (%)a by bioaugmentation and biostimulation Min

Max

82 88 86 100b 62 57 21 73

92 100 95 100b 85 77 56 86

C:N for maximal removal

C:P for maximal removal

Removal (%)a by SFE

132 60 90 – 90 132 90 90

8 6 15 – 15 8 15 15

59 88 86 – 88 83 83 65

Removal in percent with respect to the reference values in the certificate of the soil sample. After 15 d of essay, 100% of phenanthrene removal was obtained through bioaugmentation, therefore biostimulation was not needed.

990


Naphthalene

(a)

z=84.78+-0.262*x+0.067*y

83.888 84.782 85.676 86.57 87.465 88.359 89.253 90.147 91.041 91.935 above

(b)

Acenaphthene

(d)

z=102.778+-0.534*x+-0.022*y

Fluoranthene z=73.187+0.69*x+-0.044*y

91.381 92.165 92.949 93.733 94.517 95.301 96.085 96.869 97.653 98.437 above

(c)

71.591 72.746 73.901 75.056 76.211 77.366 78.522 79.677 80.832 81.987 above

(e) Fluorene

Pyrene

z=93.612+-0.126*x+-0.013*y

z=72.686+-0.613*x+0.032*y

89.947 90.2 90.453 90.706 90.958 91.211 91.464 91.717 91.969 92.222 above

64.528 65.492 66.456 67.42 68.384 69.348 70.312 71.276 72.241 73.205 above

Fig. 3. Surface response of naphthalene (a), acenaphthene (b), fluorene (c), fluoranthene (d), pyrene (e), benz(a)anthracene (f), and chrysene (g) removal as a function of C:N and C:P ratio. The value of the independent variables was calculated according to the combined factorial experimental design, composed by 22 factorial with both central and axial points (Table 2).

The SFE results show that the reduction of concentration for acenaphthene, fluorene, fluoranthene, pyrene, and benzo(a)anthracene is higher than 80% with 0.94 g of ethane g1 of soil.

Fig. 4 shows the concentration reduction for each PAH studied. The first bar corresponds to the concentration given in the certificate as reference values, on a dry weight basis, and the second bar is the concentration after SFE.

991

M.A. Amezcua-Allieri et al. / Chemosphere 86 (2012) 985–993 Chrysene

Benz[a]anthracene

(f)

(g)

z=19.099+-0.425*x+0.234*y

z=89.932+-0.149*x+-0.058*y

79.969 80.659 81.349 82.039 82.728 83.418 84.108 84.798 85.488 86.177 above

23.815 26.405 28.994 31.584 34.174 36.763 39.353 41.943 44.532 47.122 above

Fig. 3 (continued)

Fig. 4. Concentration reduction of seven PAHs using supercritical ethane.

4. Discussion 4.1. PAHs bioremoval As expected, after sugarcane bagasse pith was added to the soil, CO2 evolution and O2 uptake increased significantly. Soil respiration is a very valuable measurement in soil microbial ecology, giving information about the energetic state of the soil microbial biomass (biomass specific respiration). Except for controls, all treatments showed an increase in CO2 production (115 lg CO2 g1 IDM) after 15 d (Fig. 1). This increase might be explained by the high carbohydrate content of bagasse (Padley et al., 2000), used by microorganisms as one of the main carbon and energy source. The total nitrogen and total phosphorus content as well as organic matter content were high, showing features that are suitable for PAHs biodegradation process, with the advantage that bagasse contain appreciable densities of autochthonous microorganisms (approximately 102 CFU g1) that could influence the biodegradation process.

When contaminated soil was spiked with bagasse on day 15, the heterotrophic activity increased in all microcosms. After day 15, heterotrophic activity of soil did correlate with PAHs removal, which is attributed to the capacity of fungus to remove more available carbon sources, contained in the Wunder media (such as glucose) and once depleted, microorganisms were able to use PAHs. This finding is in agreement with previous reports, which describe that the presence of co-substrates enhances PAHs degradation (Gan et al., 2009). A variety of non-actinomycete bacteria have also been reported to metabolize fluoranthene, pyrene, chrysene, and benz(a)anthracene. P. putida, P. aeruginosa and Flavobacterium sp. were capable of metabolizing fluoranthene and pyrene when supplemented with other forms of organic carbon (Haritash and Kaushik, 2009). These strains, when recombined into a mixed culture, were found to degrade PAHs in a fashion similar to that of the original culture. The potential of P. frequentans to degrade aromatic compounds has received limited attention (Amezcua-Allieri et al., 2003; Leitao, 2009). In this study, we have shown that the selected fungus removes PAHs and that removal is a function of the culture

992


conditions. In particular, C:N and C:P ratio were dominant in optimizing PAH degradation. Under optimal conditions, P. frequentans is promising as a mechanism for the PAHs removal. With biostimulation and bioagmentation PAHs removal is 3–4 times higher compared with the soil without optimization. The 2-ring PAH naphthalene was almost completely removed (ranges from 82% to 92%) from the nutrient supplemented treatments (Table 3). The 3-ring PAHs acenaphthene, fluorene, and phenanthrene were actively removed. In particular, phenanthrene was easily removed by P. frequentans as it was previously demonstrated (Amezcua-Allieri et al., 2003). Fluoranthene was removed depending on the nutrient content (removal ranged from 57% to 77%), which is in agreement with the results obtained in an earlier study by Atagana et al. (2003), using a consortium of microorganisms, in which phenanthrene removal reached 98% and the 4- and 5- ring PAHs continued to remain in reasonable amounts at the end of the experimental period with pyrene, fluoranthene, and benzo(a)pyrene being more actively degraded by the organisms than chrysene. Overall, the treatments that received nutrient and fungal supplements showed more PAHs removal from soil. It has been generally accepted that the larger the number of aromatic rings in the PAHs, the more recalcitrant to biodegradation. However, this was not observed from our results with chrysene (Table 3), since having the same number of rings as benz(a)anthracene, and the same molar mass, the bioremoval was higher for the former. It is evident that not only the number of rings for chrysene but also the molecular structure could impact positively on its toxicity, that is, diminishing toxicity so that it is easier to be bioremoved. No doubt this behavior has to see with molecular parameters, such as, structure and molecular interactions, which influence the accessibility to microorganisms to break down the aromatic rings. Further toxicity bioessays are needed to probe this explanation. Experiments with certified soils have many important analytical advantages, such as tuning different analytical methods, as mentioned above; although some additional limitations are acknowledged. Soils are complex, multi-component systems with a range of different types of contaminants co-existing in different physical and chemical forms. Despite this, organic contaminants and toxic metals are frequently studied separately and their interaction, and the effect that remediation procedures may have on both, is neglected (Amezcua-Allieri et al., 2005). Therefore, a comparison between bioremoval and supercritical fluids extraction using natural contaminated soils will be helpful. 4.2. Comparison between methods The objective of this work is not only to direct the reader towards the most efficient method for PAHs removal, but also to show the advantages and disadvantages of the methods studied. The comparison between the results from two different methods showed that maximal removal of naphthalene, acenaphthene, fluorene, and chrysene was performed using bioremediation; however for the rest of the PAHs (i.e. fluoranthene, pyrene, and benz(a)anthracene), SFE resulted more efficient. It should be noted that the SFE result for fluorene, 85.8%, is essentially equal to the minimum value, 85.61%, obtained by bioremediation. The results from bioremoval could be explained in terms of toxicity, due to in general terms, the more PAHs rings more toxicity for microorganisms. This behavior is in agreement with previous reports that describe that high-molar-mass PAHs benz(a)anthracene and benz(a)pyrene showed a considerably slower removal rate and less overall degradation than low-molar-mass PAHs in soil microcosms (Silva et al., 2009). The final user needs to take into account that although bioremediation was better for certain hydrocarbons and takes advantage of

the increased rate of natural biological processes, it takes more time (i.e. 36 d vs. half hour) and it is useful for 2–3 PAHs rings. It is also very relevant to note that the biotransformation of PAHs to less carcinogenic and toxic compounds through bioremediation is an important advantage in terms of health risk assessment. 5. Conclusions It was found that P. frequentans was able to remove PAHs from a contaminated soil sample, under optimal culture conditions. Further work is required to elucidate the enzymatic pathways used in the biodegradation process as well as to compare both techniques included in this work in terms of cost. SFE is a very promising technique for application in different technological fields, which in the present study can be considered as complementary for the biological treatment of polluted soils, particularly to remove high molar mass PAHs. Acknowledgements M.A. Ávila-Chávez thanks both the Instituto Mexicano del Petróleo and the Consejo Nacional de Ciencia y Tecnología (CONACyT-México) for a Grant to pursue postgraduate studies. The rest of the authors are grateful for SNI-CONACyT fellowships. References Abdulsalam, S., Omale, A.B., 2009. Comparison of biostimulation and bioaugmentation techniques for the remediation of used motor oil contaminated soil. Braz. Arch. Biol. Technol. 52, 747–754. Amezcua-Allieri, M.A., Lead, J.R., Rodríguez-Vázquez, R., Meléndez-Estrada, J., 2003. Phenanthrene removal in a selected Mexican soil by the fungus Penicillium frequentans: role of C:N ratio and water content. Soil Sediment. Contam. 12, 387–399. Amezcua-Allieri, M.A., Lead, J., Rodríguez-Vázquez, R., 2005. Impact of microbial activity on copper, lead and nickel mobilization during the bioremediation of soil PAHs. Chemosphere 61, 484–491. Andreoni, V., Gianfreda, L., 2007. Bioremediation and monitoring of aromaticpolluted habitats. Appl. Microbiol. Biotechnol. 76, 287–308. Andrews, A.T., Ahlert, R.C., Kosson, D.S., 1990. Supercritical fluid extraction of aromatic contaminants from a sandy loam soil. Environ. Prog. 9, 204–210. AOAC, 1970. Official Methods of Analysis of the Association of Official Analytical Chemists. In: Horwitz, W. (Ed.), 13thedition. Washington, DC. Arias-González, I., Reza, J., Trejo, A., 2010. Temperature and sodium chloride effects on the solubility of anthracene in water. J. Chem. Thermodyn. 42, 1386–1392. Atagana, H.I., Haynes, R.J., Wallis, F.M., 2003. Co-composting of soil heavily contaminated with creosote with cattle manure and vegetable waste for the bioremediation of creosote-contaminated soil. Soil Sediment Contam. 12, 885– 899. Ávila-Chávez, M.A., Eustaquio-Rincón, R., Reza, J., Trejo, A., 2007. Extraction of hydrocarbons from crude oil tank-bottom sludges using supercritical ethane. Separ. Sci. Technol. 42, 2327–2345. Becnel, J.M., Dooley, K.M., 1998. Supercritical fluid extraction of polycyclic aromatic hydrocarbon mixtures from contaminated soils. Ind. Eng. Chem. Res. 37, 584– 594. Bray, R.H., Kurtz, L.T., 1945. Determination of total, organic, and available forms of phosphorus in soils. Soil Sci. 59, 39–45. Dean, J.R., Xiong, G., 2000. Extraction of organic pollutants from environmental matrices: selection of extraction technique. Trends Anal. Chem. 19, 553–564. EPA 8270C Semivolatile organic compounds by gas chromatography/mass spectrometry (GC/MS); US Environmental Protection Agency: Washington, DC, 1996. EPA 8100 Polynuclear aromatic hydrocarbons; US Environmental Protection Agency: Washington, DC, 1986. EPA 3600C Cleanup; US Environmental Protection Agency: Washington, DC, 1996. EPA 3540C Soxhlet extraction; US Environmental Protection Agency: Washington, DC, 1996. Gan, S., Lau, E.V., Ng, H.K., 2009. Remediation of soils contaminated with polycyclic aromatic hydrocarbons (PAHs). J. Hazard. Mater. 172, 532–549. Haritash, A.K., Kaushik, C.P., 2009. Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review. J. Hazard. Mater. 169, 1–15. Hawthorne, S.B., Grabanski, C.B., Martin, E., Miller, D.J., 2000. Comparison of Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction for environmental solids: recovery, selectivity and effects on sample matrix. J. Chromatogr. A. 892, 421–433. Hawthorne, S.B., Poppendiek, D., Grabanski, C.B., Loehr, R.C., 2001. PAH release during water desorption, supercritical carbon dioxide extraction, and field bioremediation. Environ. Sci. Technol. 35, 4577–4583.

M.A. Amezcua-Allieri et al. / Chemosphere 86 (2012) 985–993 Jackson, M.L., 1970. Soil Analysis. McGraw-Hill, New York. Kothandaraman, S., Ahlert, R.C., Venkataramani, E.S., Andrews, A.T., 1992. Supercritical extraction of polynuclear aromatic hydrocarbons from soil. Environ. Prog. 11, 220–222. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., Pawliszyn, J., 1993. Effects of temperature and pressure on supercritical fluid extraction efficiencies of polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Anal. Chem. 65, 338–344. Langenfeld, J.J., Hawthorne, S.B., Miller, D.J., Pawliszyn, J., 1995. Kinetic study of supercritical fluid extraction of organic contaminants from heterogeneous environmental samples with carbon dioxide and elevated temperature. Anal. Chem. 67, 1727–1736. Leitao, A.L., 2009. Potential of Penicillium species in the bioremediation field. Int. J. Environ. Res. Public Health. 6, 1393–1417. Madueño, L., Coppotelli, B.M., Alvarez, H.M., Morellia, I.S., 2011. Isolation and characterization of indigenous soil bacteria for bioaugmentation of PAH contaminated soil of semiarid Patagonia, Argentina. Int. Biodeter. Biodegr. 65, 345–351. Mitchell, D.A., 1992a. Biomass determination in solid-state cultivation. In: Doelle, H., Mitchell, D.A., Rolz, C.E. (Eds.), Solid Substrate Cultivation. Elsevier, London. p. 55. Mitchell, D.A., 1992b. Growth patterns, growth kinetics and the modeling of growth in solid-state cultivation. In: Doelle, H., Mitchell, D.A., Rolz, C.E. (Eds.), Solid Substrate Cultivation. Elsevier, London. P 55. Montgomery, D.C., 2000. Design and analysis of experiments, fifth ed. John Wiley and Sons Inc., New York. Padley, A., Soccol, C.R., Nigan, P., Soccol, V.T., 2000. Biotechnological potential of agro-industrial residues. I: sugarcane bagasse. Bioresource Technol. 74, 69–80. Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. Academic Press, Wisconsin, USA. Reza, J., Trejo, A., Vera-Ávila, L.E., 2002. Determination of the temperature dependence of water solubilities of polycyclic aromatic hydrocarbons by a generator column-on line solid phase extraction-liquid chromatographic method. Chemosphere 47, 933–945. Reza, J., Trejo, A., 2004. Temperature dependence of the infinite dilution activity coefficient and Henry’s law constant of polycyclic aromatic hydrocarbons in water. Chemosphere 56, 537–547.

993

Rose, J.L., Monnery, W.D., Chong, K., Svrcek, W.Y., 2001. Experimental data for the extraction of Peace River bitumen using supercritical ethane. Fuel 80, 1101– 1110. Saeed, S., Shams Zeinab, H., Venous, R., 2009. Investigation of operating conditions for soil remediation by subcritical water. Chem. Ind. Chem. Eng. Q. 15, 89–94. Saim, N., Dean, J.R., Abdullah, M.P., Zakaria, Z., 1997. Extraction of polycyclic aromatic hydrocarbons from contaminated soil using Soxhlet extraction, pressurized and atmospheric microwave-assisted extractions, supercritical fluid extraction and accelerated solvent extraction. J. Chromatogr. A. 791, 361–366. Samanta, S.K., Singh, O.V., Jain, R.K., 2002. Polycyclic aromatic hydrocarbons: environmental pollution and bioremediation. Trends Biotechnol. 20, 243–248. Saucedo, C.J., Trejo, H.M., Lonsane, B.K., 1994. On line-automated monitoring and control systems for CO2 and O2 in aerobic and anaerobic solid-state fermentations. Process Biochem. 29, 13–24. Schumacher, B.A., 2002. Methods for the determination of total organic carbon (TOC) in soils and sediments. In: Ecological Risk Assessment Support Center Office of Research and Development. US Environmental Protection Agency. Seeley, W.H., VanDenmark, P.J., 1981. Microbes in Action. A Laboratory Manual of Microbiology, WH Freeman and Co, Pennsylvania, USA. Silva, S., Grossman, M., Durrant, L., 2009. Degradation of polycyclic aromatic hydrocarbons (2–7 rings) under microaerobic and very low oxygen conditions by soil fungi. Int. Biodeter. Biodegr. 63, 224–229. Singer, A.C., Van der Gast, C.J., Thompson, I.P., 2005. Perspectives and vision for strain selection in bioaugmentation. Trends Biotechnol. 23, 74–77. WHO, 2001. Environmental Health Criteria 202: Selected non-heterocyclic polycyclic aromatic hydrocarbons. . Wright, B.W., Wright, C.W., Fruchter, J.S., 1989. Supercritical fluid extraction of coal tar contaminated soil samples. Energy Fuel 3, 474–480. Wunder, T., Kremer, S., Sterne, C., Anke, H., 1994. Metabolism of the polycyclic aromatic hydrocarbon pyrene by Aspergillus niger SK 9317. Appl. Microbiol. Biotechnol. 42, 636–641. Zhang, X., Cheng, S., Zhu, C., Sun, S., 2006. Microbial PAH-degradation in soil: degradation pathways and contributing factors. Pedosphere 16, 555–565.