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Modification of PAHs Biodegradation withHumic CompoundsRoshanak Rezaei Kalantary a , Ahmad Badkoubi b , AnoushiravanMohseni-Bandpi c , Ali Esrafili a , Sahand Jorfi d , Emad Dehghanifarda & Mohammad Mehdi Baneshi aa Department of Environmental Health Engineering, School of PublicHealth , Tehran University of Medical Sciences , Tehran , Iranb Department of Civil Engineering, Environmental EngineeringDivision , Tarbiat Modares University , Tehran , Iranc Department of Environmental Health Engineering, School of PublicHealth , Mazandaran University of Medical Sciences , Sari , Irand Department of Environmental Health Engineering, School ofPublic Health, Environmental Technology Research Center , AhvazJondishapur University of Medical Sciences , Ahwaz , IranAccepted author version posted online: 18 Sep 2012.Publishedonline: 10 Jan 2013.

To cite this article: Roshanak Rezaei Kalantary , Ahmad Badkoubi , Anoushiravan Mohseni-Bandpi , AliEsrafili , Sahand Jorfi , Emad Dehghanifard & Mohammad Mehdi Baneshi (2013) Modification of PAHsBiodegradation with Humic Compounds, Soil and Sediment Contamination: An International Journal,22:2, 185-198, DOI: 10.1080/15320383.2013.722139

To link to this article: http://dx.doi.org/10.1080/15320383.2013.722139

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Soil and Sediment Contamination, 22:185–198, 2013Copyright © Taylor & Francis Group, LLCISSN: 1532-0383 print / 1549-7887 onlineDOI: 10.1080/15320383.2013.722139

Modification of PAHs Biodegradation withHumic Compounds

ROSHANAK REZAEI KALANTARY,1 AHMAD BADKOUBI,2

ANOUSHIRAVAN MOHSENI-BANDPI,3 ALI ESRAFILI,1

SAHAND JORFI,4 EMAD DEHGHANIFARD,1

AND MOHAMMAD MEHDI BANESHI1

1Department of Environmental Health Engineering, School of Public Health,Tehran University of Medical Sciences, Tehran, Iran2Department of Civil Engineering, Environmental Engineering Division, TarbiatModares University, Tehran, Iran3Department of Environmental Health Engineering, School of Public Health,Mazandaran University of Medical Sciences, Sari, Iran4Department of Environmental Health Engineering, School of Public Health,Environmental Technology Research Center, Ahvaz Jondishapur University ofMedical Sciences, Ahwaz, Iran

The effect of extractable humic substances (EHS) on the bioremediation of phenanthrenein a slurry phase was investigated using adapted microorganisms with polycyclic aro-matic hydrocarbons (PAHs). Two concentrations of EHS were used: 150 and 30 mg/kgsoil. The phenanthrene concentration was 500 mg/kg soil. The results showed that thetrend of biodegradation was increased after four weeks retardation. These tests showedthat humic compounds could overcome the bond between the soil and phenanthrenein the presence of the bacterial consortium. The bacterial density in the medium withEHS was about six-fold greater in magnitude than in the medium without the humiccompounds. The chemical relationship between phenanthrene and the humic substancesin the form of a phenanthrene-humic-soil complex or phenanthrene-humic is loosely as-sociated and reversible. Therefore, after the initial inhibition by humic substances, thebioavailability of phenanthrene increases.

Keywords Phenanthrene, bioavailability, humic substance, soil slurry

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants that are of major concernbecause of their persistence in the environment. PAHs are widely distributed in the soilvia natural or anthropogenic combustion processes (Hamdi et al., 2007) from land-appliedwastes (e.g., biosolids, sludge, and compost) and have increasingly accumulated in amendedsoils (Overcash et al., 2005). PAHs exhibit cytotoxic, mutagenic, and carcinogenic proper-ties and pose a serious hazard to human health and environment (Karimi-Lotfabad et al.,

Address correspondence to Roshanak Rezaei Kalantary, Department of Environmental HealthEngineering, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. E-mail:r.rezaei@tums.ac.ir

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1996; Yerushalmi and Guiot, 2001; Muckian et al., 2009). Phenanthrene is a three-ringPAH which may be found in relatively high concentrations in coal-tar-contaminated sites,wood-preserving sites, gas work sites (Juhasz and Naidu, 2000), and petroleum refiningareas (Blakely et al., 2002).

Among the different methods to remove pollutants from the environment, bioremedia-tion serves as one of the most efficient technologies to restore contaminated sites and preventdeterioration of the ecosystem (Ramirez et al., 2001). However, bioremediation is oftenlimited by the availability of pollutants to undergo biological transformations (Ramirezet al., 2001; Park et al., 2002; Venkata Mohan et al., 2009). One of the main factors limitingbioavailability is the low aqueous solubility of PAHs (Van Stempvoort et al., 2002; Yu et al.,2007).

Many studies have been conducted to explain and to overcome problems related to thepoor availability of PAHs. Mixed results have been reported concerning the effect on thebiodegradation of PAHs by the addition of organic matter (OM) or humic substances (HS)to soil (Nayak et al., 2009). Humic substances consist of three main fractions: fulvic acid(FA), humic acid (HA), and humin (Kastner, 2000; Mecozzi et al., 2002). FA, HA, and clayoften coexist in soil through hydrophobic interactions, electrostatic interactions, hydrogenbonding, or coordinate bonding (Nakashima et al., 2007). Although their interactions havebeen studied in detail, limited information is available about how OM and clay interactionsaffect the biodegradation of hydrophobic organic pollutants (Ortega-Calvo et al., 1997).

Some investigators have discussed that using humic substances may result in a declineor reduction in the contaminants’ concentration with a potential risk of remobilizationand redistribution of the hazardous compounds (Kastner, 2000), notably low degradablecompounds, such as PAHs. Studies have shown that using OM or HS can increase thesolubility of PAHs (Grasso et al., 2001; Van Stempvoort et al., 2002) or the desorption ofthe compounds (Reid et al., 2000; Van Gestel et al., 2003). Laor et al. (1996; 1999) reportedthat the sorption of microorganisms to phenanthrene-enriched surfaces might stimulatemineralization rates.

The bond between the contaminants and humic substances can have a negative effecton the biodegradation of PAHs, making them less available (Dercova et al., 2007). Thesebonds can make the contaminants more resistant against desorption or extraction by solventsand biodegradation by microorganisms (Plaza et al., 2009). By increasing humic substanceconcentrations in the soil, pyrene mineralization has been more retarded (Macleod andSemple, 2002). Heywood et al. (2006) reported that there was not any relationship betweenthe OM content of the soil and PAHs biodegradation.

Although PAHs removal via binding with humic compounds has been the subject of anumber of studies (Niederer et al., 2007), the literature does not provide a clear explanationof the binding behavior between PAHs and soil containing OM (Pan et al., 2008). Thus,more biodegradation of PAHs in the presence of humic substances is still unresolved. Theobjective of this study was to investigate the effect of humic substances that were extractedfrom forest soils on the bioremediation of phenanthrene in contaminated soils.

Material and Methods

Chemicals

Phenanthrene (96%), methanol, acetone (HPLC grade), sulfuric acid, sodium chloride,sodium hydroxide, and HCl were purchased from Merck. Nutrient agar and nutrient brothwere purchased from Hi-Media and Difco, respectively. Chemical materials for mineralsalt medium (MSM) were purchased from Sigma-Aldrich (analytical grade).

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The MSM contained the following (per liter): 0.8 g K2HPO4, 0.2 g KH2PO4, 1 gKNO3, 0.2 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.1 g NaCl, 0.01 g FeCl3.6H2O, and 1 mLtrace element solution. The trace element solution contained the following (per liter): 23 mgMnCl2.2H2O, 30 mg MnCl4, 32 mg H3BO3, 39 mg CoCl2.2H2O, 50 mg ZnCl2, 30 mgNaMnO4.2H2O, and 20 mg NiCl2 (Ressler et al., 1999).

Soils

The contaminated soil was collected near the Tehran Petroleum Refinery because of itsprolonged contact with oil, and it has suitable properties for preparing PAHs microbialculture. The forestry soil for preparing the humic substances was collected from the northernpart of Iran. The extractable humic substances (EHS) consist of fulvic acid (FA) and humicacid (HA) fractions that were obtained according to Mecozzi et al. (2002). The dry amountof EHS and the organic compound fraction were determined to be 0.35 mg in 1 mL and30%, respectively, according to standard methods (APHA et al., 2005).

Adaptation

Bacteria were grown in MSM. The pH of the medium was 6.8 ± 0.2. To prepare the microbialculture, 100 mL of sterile distilled water (DW) was added to 10 grams of contaminated soil(10% w:v). The mixture was stirred for 24 hours using a magnetic stirrer. After settling for20 minutes, 1 mL of supernatant was added to 50 mL of sterile MSM plus phenanthrene asa carbon source.

The MSM was freshened every week to prevent any deficiency of nutrients and carbonsource. This procedure continued for 10 weeks. The adapted microorganisms, which werenamed mixPh based on their carbon source, were then brought on the nutrient agar for laterexperiments.

The other mix culture was enriched with naphthalene as the carbon source, whichhad better efficiency in our previous study (Rezaei Kalantary et al., 2003). The bacte-rial consortium consisted of Microbacterium lacticum, Bacillus mycoides, Sphingomonasparapaucimobilis, Acidovorax (Pseudomonas) facilis, Capnocytophaga ochracea (presum-ably), and Staphylococcus hominis (Rezaei Kalantary and Badkoubi, 2006), which wereidentified on morphological and physiological characteristics according to Winn et al.(2006).

Degradation Investigation

The uncontaminated soil was collected from uncontaminated sites near the TehranPetroleum Refinery in order to have the same texture as the contaminated soil, and it waswashed with acetone several times to prepare organic-free contaminated soil (Vosoughiet al., 2002). The texture and mineral constituents were determined using sieves and X-raydiffraction (XRD). Two grams of clean soil was placed into a 50 mL Erlenmeyer flask. Thevessels containing clean soil were autoclaved. To prepare soil containing 500 mg phenan-threne/kg of dry soil, a measured weight of phenanthrene was dissolved in acetone at aconcentration of 1 g/L and was used to spike the soil at a ratio of 500 mL/kg of dry soil.After evaporation of acetone, the soil was inoculated with a consortium of bacteria in MSMwith an optical density of 1 at 630 nm (Nasseri et al., 2010).

The EHS solution, which was extracted in NaOH, was added to each vessel accordingto Table 1. The pH was adjusted to 6.8 ± 0.2. At the end, the soil liquid ratio was 10% w:v(Ramirez et al., 2001). Based on three different variables and related blank samples (without

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Table 1Operational conditions of study

Mixed Humic concentration Phenanthrene concentrationRun culture (w:w) (mg/kg soil)

1 N∗ H∗∗∗ 5002 Ph∗∗ H 5003 — H 5004 N L∗∗∗∗ 5005 Ph L 5006 — L 5007 N 0∗∗∗∗∗ 5008 Ph 0 5009 — 0 500

10 N H 011 Ph H 012 N L 013 Ph L 014 N 0 015 Ph 0 0

∗N: Mixed culture of bacteria with carbon source as naphthalene.∗∗Ph: Mixed culture of bacteria with carbon source as phenanthrene.∗∗∗H: High level of HS (150 mgHS/kg soil) (Liang et al., 2008).∗∗∗∗L: Low level of HS (30 mgHS/kg soil) (Liang et al., 2008).∗∗∗∗∗0: Without HS (0 mgHS/kg soil).

bacterial inoculums; no addition of humic substances and phenanthrene), 15 experiments(runs) were carried out in 12 weeks. Measurements of microbial density and residualconcentration of the pollutant had been done every two weeks.

To achieve the effect of mixed culture and humic concentration for removal of phenan-threne from the soil, a statistical factorial design in three levels was performed in triplicate.In Table 1, the details of experimental design runs are shown.

Determination of Microbial Population

To determine the population of the inoculated culture, the most probable number (MPN)method with some modifications was used (Juhasz et al., 2000). One milliliter of themicrobial suspension, which was diluted ten-fold to 10−10 in a ringer solution (8.5 g NaClper 1 L DW), was added to 9 mL of the sterile nutrient broth in five replicates in 10 series.

Extraction and Analysis

The residual phenanthrene in the soil was extracted according to EPA 3550B (EPA) withmethanol by ultrasonic treatment (Bandelin HD 2070). The extracted sample was thencentrifuged (Hettich D7200) for 10 minutes at 6000 rpm and filtered through 2–3 cm ofglass wool. A portion of the filtered solution was removed for analysis (USEPA, 1996).

The extract was quantified by gas chromatography (GC; Chrompack CP 9001) usinga flame ionization detector (FID) with an HP5 capillary column (length of 30 m, inside

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PAHs Biodegradation with Humic Compounds 189

diameter of 0.32 mm, and a coated-film thickness of 0.2 μm). α-Naphthol was used asan internal standard. The oven temperature was initially set at 100◦C for one minute, thenincreased to 250◦C at a rate of 10◦C/min. The injector and detector temperatures were setat 250◦C and 290◦C, respectively.

Differences in the experimental data between different treatments groups were deter-mined by SPSS software (Statistical Package for the Social Sciences).

Results

After adaptation, a consortium of bacteria was separated and denoted as mixPh in referenceto phenanthrene as the carbon source. The culture consisted of five types of bacteria.The identified bacteria were Bacillus sporogenes., Bacillus licheniformis, Capnocytophagaochracea (presumably), Acinetobacter sporogenes, and Staphylococcus xylosus. The MPNfor the inoculated population of the mixed culture was about 2 × 107/100 mL (2 × 105

cfu/mL). The texture of the soil was lean clay, and the mineralization of the soil, asdetermined by XRD, was quartz, calcite, clinochlore, muscovite, and montmorillonite.

The results in the absence of bacterial inoculums are presented in Figure 1. It wasobserved that the concentration of the remaining phenanthrene after 84 days of contacttime did not decrease considerably.

Analysis of variance (ANOVA) was used to estimate the significance of the maineffects and interactions. The sum of squares is the information used to estimate the F-ratios (considering the respective mean square effect and the mean square error). The Pvalue

indicates when the effect of each factor was statistically significant (Pvalue < 0.05).The ANOVA results showed that the most important factor contributing to the removal

percent was factor A (Mixed Culture, 81%), followed by factor B (Humic concentration,15%) and lastly, AB (Interaction, 3%).

The effect of the humic compounds on the removal of phenanthrene is presented inFigures 2 and 3. Runs 1, 4, and 7 with the same inoculation (mix N) are characterized bytheir HS content as high, low, and none. Run 4 gave the best removal efficiency of 78.4%

Figure 1. Variations of phenanthrene concentration in runs 3, 6, and 9. Run 3: Inoculation (-), HS(H), Phenanthrene (500 mg/kg); Run 6: Inoculation (-), HS (L), Phenanthrene (500 mg/kg); Run 9:Inoculation (-), HS (0), Phenanthrene (500 mg/kg) (Color figure available online).

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Figure 2. Variations of phenanthrene in runs 1, 4, and 7. Run 1: Inoculation (N), HS (H), Phenan-threne (500 mg/kg); Run 4: Inoculation (N), HS (L), Phenanthrene (500 mg/kg); Run 7: Inoculation(N), HS (0), Phenanthrene (500 mg/kg) (Color figure available online).

for phenanthrene. The removal efficiency of run 1 was 53%. On the other hand, the removalefficiency for run 7 in the absence of HS was only 42.6%, which verifies the positive effectof the optimal amount of humic compounds for phenanthrene removal.

Phenanthrene removal in runs with HS was evident until 28 days. In runs 3 and 6 (withHS and without bacteria), the phenanthrene concentration was approximately constant. Inthe other runs (1, 2, 4, and 5), the degradation of phenanthrene continued until the end ofthe experiment.

At the end of four weeks, the phenanthrene concentration in the runs with HS rangedfrom 16% to 28% (360–420 mg/kg soil), but the amounts at the twelfth week were very

Figure 3. Variations of phenanthrene in runs 2, 5, and 8. Run 2: Inoculation (Ph), HS (H), Phenan-threne (500 mg/kg); Run 5: Inoculation (Ph), HS (L), Phenanthrene (500 mg/kg); Run 8: Inoculation(Ph), HS (0), Phenanthrene (500 mg/kg) (Color figure available online).

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PAHs Biodegradation with Humic Compounds 191

different for runs 3 and 6 as compared with the others (1, 2, 4, and 5) (Figure 4). Thesedata show that phenanthrene concentration in the runs with HS and bacteria significantlydecreased from the fourth week until the twelfth week (Pvalue < 0.05). The biodegradationefficiency in this period was about 28–50%.

The trend of phenanthrene concentration in runs 5 and 8 with the same inoculum wasdifferent. In runs 7 and 8 (without humic compounds), the concentration of phenanthrenedecreased from the first day until the fourth week, then the trend of decrease was low.The bacterial population in these two runs was different from the others, and it was stablefrom the first day until the third week (Figure 5). After the third week, the populationdecreased slowly, while in the runs with HS and phenanthrene (runs 1, 2, 4, and 5) thebacterial population increased, and it reached the highest level on the third week and thendecreased sharply (Figure 5). The trend of the bacterial population change in the runsthat had HS (runs 1, 2, 4, and 5) shows the same behavior. In these tests, after a smallinitial decrease, increases in the population density were observed. In the tests with HS andwithout phenanthrene (runs 10–13), only decreases were observed after the initial increase(Figure 6). In the tests with the initial inoculation and without HS or phenanthrene (runs14 and 15), the bacterial population decreased from the first day until the end time of theexperiment (Figure 6).

Variations in bacterial density in the presence of phenanthrene and different amounts ofand bacterial consortium are shown in Figure 5. The population of the consortium (Runs 1and 2) was about 2 × 108 MPN/100mL and 2.1 × 108 MPN/100mL on the twenty-first dayfor mixN and mixPh, respectively. At the end of the experiments (84 days), the populationsdecreased to 1.3 × 108 MPN/100 mL and 1.2 × 108 MPN/100 mL for mixN and mixPh,respectively. These values are greater than the initial value of 2 × 107 MPN/100 mL.

The bacterial densities of mixN and mixPh (Runs 4 and 5) on the twenty-first daywere 1.7 × 108 MPN/100 mL and 1.4 × 108 MPN/100 mL, respectively. At the end of theexperiments (84 days), the bacterial densities decreased to 8 × 107 MPN/100 mL and 9 ×107 MPN/100 mL for mixN and mixPh, respectively. These values were still greater thanthe initial value of 2 × 107 MPN/100 mL. Generally, the MPN values were lower than runs1 and 2, indicating the effect of high levels of HS as an electron donor and carbon source.These amounts are relatively more than the MPN of runs 7 and 8 and the same as runs 1and 2. However, the rates of variation are different.

The MPN of bacteria (Runs 7 and 8) was 1.7 × 108/100 mL and 2.1 × 108/100 mL onday 14 for mixN and mixPh, respectively. At the end of the experiments (84 days), theseamounts decreased to 7 × 107 MPN/100 mL and 5 × 107 MPN/100 mL for mixN andmixPh, respectively. In contrast to runs 1, 2, 4, and 5, the highest MPN for runs 7 and 8was obtained on day 14.

Variations in bacterial density in the presence of different amounts of HS and in absenceof phenanthrene as the carbon source are shown in Figure 6. A bacterial population of5 × 108 MPN/100 mL was observed on day 21 for mixPh in the presence of a high amountof HS. This value gradually decreased to 3 × 107 MPN/100 mL, indicating the importanceof HS and phenanthrene as the carbon source. This result can be verified with the resultsof runs 14 and 15 in which the MPN decreased throughout the experiment and reached5 × 105 MPN/100 mL and 3 × 105 MPN/100 mL for mixN and mixPh, respectively.

Discussion

One of the most important problems in the bioremediation of PAHs is bioavailability.Substrates with low solubility, such as phenanthrene (1.29 mg/L in distilled water, 25◦C)

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Figure 4. Removal percent of phenanthrene in different levels of two parameters obtained by full fac-torial design: a) 4th week; b) 12th week. Run 1: Inoculation (N), HS (H), Phenanthrene (500 mg/kg);Run 2: Inoculation (Ph), HS (H), Phenanthrene (500 mg/kg); Run 3: Inoculation (-), HS (H), Phenan-threne (500 mg/kg); Run 4: Inoculation (N), HS (L), Phenanthrene (500 mg/kg); Run 5: Inoculation(Ph), HS (L), Phenanthrene (500 mg/kg); Run 6: Inoculation (-), HS (L), Phenanthrene (500 mg/kg);Run 7: Inoculation (N), HS (0), Phenanthrene (500 mg/kg); Run 8: Inoculation (Ph), HS (0), Phenan-threne (500 mg/kg); Run 9: Inoculation (-), HS (0), Phenanthrene (500 mg/kg) (Color figure availableonline).

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Figure 5. Variations in bacterial density during 84 days in Runs 1, 2, 4, 5, 7, and 8. Run 1: In-oculation (N), HS (H), Phenanthrene (500 mg/kg); Run 2: Inoculation (Ph), HS (H), Phenanthrene(500 mg/kg); Run 4: Inoculation (N), HS (L), Phenanthrene (500 mg/kg); Run 5: Inoculation (Ph),HS (L), Phenanthrene (500 mg/kg); Run 7: Inoculation (N), HS (0), Phenanthrene (500 mg/kg); Run8: Inoculation (Ph), HS (0), Phenanthrene (500 mg/kg) (Color figure available online).

Figure 6. Variations in bacterial density during 84 days in Runs 10–15. Run 10: Inoculation (N), HS(H), Phenanthrene (0); Run 11: Inoculation (Ph), HS (H), Phenanthrene (0; Run 12: Inoculation (N),HS (L), Phenanthrene (0); Run 13: Inoculation (Ph), HS (L), Phenanthrene (0); Run 14: Inoculation(N), HS (0), Phenanthrene (0); Run 15: Inoculation (Ph), HS (0), Phenanthrene (0) (Color figureavailable online).

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(Ressler et al., 1999), cannot be used easily by bacteria. The properties of both the soiland the contaminant can affect their interaction. The adsorption of hydrophobic organiccompounds (HOCs) onto organic matter or humic compounds in soil can influence thebioavailability and the rate of biodegradation (Das et al., 2008).

Runs 1–6 show the presence of higher levels of phenanthrene on the fourth week,indicating the lack of bioavailability as a result of binding until 28 days. The differencesbetween the runs at the fourth week were insignificant, but after that there were significantdifferences between them (Pvalue<0.05). In runs 4 and 5, the phenanthrene concentrationat the eighty-fourth day was low, and a reduction greater than 50% and 46% for mixN andmixPh biodegradation, respectively, was observed.

In run 9 (without humic or bacteria, Table 1), the spiked phenanthrene was extractable.After three months, the phenanthrene amount was approximately 454 mg/kg soil. Nophenanthrene loss was expected either through phenanthrene-consuming microorganismsor HS bonding with phenanthrene; therefore, such a decrease may be related to aging. Thereduction of 10% in run 9 may be related to the bonding between phenanthrene and soiltexture and/or aging after three months (Hwang and Cutright, 2002). Results showed that thepresence of bacteria in soil sediment has the most critical role in removal of phenanthrene.The F-ratio would also confirm this.

The biodegradation in run 8 was about 33% after one month, and it only improved by 7%three months later. This slow biodegradation may be related to the bonding of phenanthrenewith the soil, which makes it non-bioavailable over time and slows biodegradation. In runs2, 5, and 8, the same mix culture was used, but on the eighty-fourth day the phenanthreneconcentrations in these tests were 260, 159, and 303 mg/kg soil, respectively. These resultsshow that the presence of HS in tests 2 and 5 prevents phenanthrene from bonding withclay and therefore phenanthrene became available. Phenanthrene appears to adsorb first toHS, and phenanthrene becomes available to bacteria over time.

The trends of the bacterial population changes show that the population increased atfirst with similar increases in bacterial density that were observed in our earlier study inliquid medium (Rezaei Kalantary and Badkoubi, 2006). At the same time, the number ofbacteria enumerated by the MPN technique and the degradation rates for phenanthrene wereuncorrelated, confirming the results of previous studies (Rezaei Kalantary and Badkoubi,2006; Kao and Borden, 1997).

Thus, increases in the bacterial population may be related to HS. Alkorta and Garbisu(2001) showed that the microbial density in the soil around the roots of plants was two-to four-fold that of the other sites in the soil. Binet et al. (2000) and Liste and Alexander(2000) in separate studies showed enhancement in biodegradation with increasing microbialpopulation and activity around the roots in the soil. As can be seen in Table 2, the F-ratioof mixed culture was 81.06%, which confirmed the role of microbial biodegradation of thepollutant.

Some extracellular enzymes, which can be stabilized by adsorption to humic com-pounds, can catalyze HA and FA oxidants and produce active coupling between OM andHS. This activity is useful for supplying nutrients to microorganisms (Gramss et al., 1999).Nutrient supplying leads to stimulation in bacterial activity and increases in bioremediation(Rezaei Kalantary and Badkoubi, 2006). HS may be a growth substrate, which can initiatethe growth of bacteria and induce the production of catabolic enzymes (Juhasz et al., 2000).

The level of biodegradation was low at the onset of the experiments, but increasingbiodegradation was observed with increasing time. This retardation may be related to thepresence of HS. Macleod and Semple (2002) reported that the mineralization of pyrene inthe soil with HS occurred later than in soils without HS. This retardation was attributed

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to the slow desorption of HOCs as a rate-limiting factor in biodegradation (Macleod andSemple, 2002). In our research after retardation period, bacteria could use phenanthrene.This biodegradation pathway can be attributed to two factors of reversible binding be-tween contaminant and HS/or soil, and the positive effect of HS on bioavailability ofPAHs.

In the soil, the bond between PAHs and organic macromolecules can be either a non-covalent (e.g., hydrophobic sorption, charge transfer complex, hydrogen bond) or covalentbond (ester bond, ether bond, carbon-carbon bond). Covalent bonds between PAHs and HSare stable and irreversible (Richnow et al., 1998). PAHs bound covalently are the prevail-ing mechanism that prevents total bacterial mineralization of PAHs (Ressler et al., 1999).The only way to cleave the covalent bond is through alkaline hydrolysis (Richnow et al.,1998). The high level of extracted phenanthrene in this research shows that most of theHS/soil-bound phenanthrene must be reversible, which is indicative of a noncovalent bond.The noncovalent binding of pesticides and pyrene to soil has been reported by Lerch et al.(1997).

Plaza et al. (2009) reported that composting processes decrease the binding affinity andmake the binding sites of HA heterogeneous (Plaza et al., 2009). Heterogeneity in soil OMwas reported by Wen et al. (2007). HA in HS is likely to be loosely connected. The O/Catomic ratio in HA indicates that water molecules can swell HA and make it more flexibleso that adsorbed molecules can penetrate rapidly into or out of it along a concentrationgradient (Huang et al., 2003). HA may cause reversible sorption in the medium. Macleodand Semple (2002) reported that the biodegradation of pentachlorophenol is promoted bythe reversible sorption of the chemical to bulking agents used in composting strategies. HAwould be deprotonated as an acidic group in the neutral case, which would cause diffusion ofPAHs from the organic matrix in the solution phase. This model agrees with the results fromYang et al. (2001) that reported that by increasing the pH from 2 to 9, FA and HA bondingwould be less tight and that for neutral pHs the desorbed PAHs would increase. HS has acomplex and winding structure, and therefore the adsorption of HOCs on such structuresmay reduce desorption and bioavailability. When HA and FA are extracted by using NaOH(i.e., pH > 12), the functional groups, such as carboxylic groups, are predominantly in thedissociated form. This extraction procedure may produce uncoiled macromolecules of HAand FA in solution and this may influence the retention of nonpolar contaminants associatedwith this phase (Nam and Kim, 2002). Hence, the adsorbed phenanthrene to HA and FAcan be redistributed. The presence of EHS can increase desorption and allow the removalof HOCs. White et al. (1999) reported that the removal of HA and FA causes a decreasein the desorption of phenanthrene. Therefore, the existence of a looser texture with EHScauses contaminants to become more bioavailable.

The soluble parts of HS have aromatic sites that are compatible with PAHs rings,which can have an effect on solutions of PAHs (Blakely et al., 2002; Lippold et al., 2008).Some reports confirm that by increasing the solubility of phenanthrene, its bioavailabil-ity and bioremediation would increase (Van Stempvoort et al., 2002). Laor et al. (1996)indicated that increasing the solubility stimulates microbial activity. Alternatively, phenan-threne could be used by the bacteria, which can attack adsorbed PAHs. This idea couldexplain the increase in biodegradation during the last days in this research. Martins andMermoud (1998) showed that some microorganisms could use nitroaromatic compoundsin an adsorbed situation. Guerin and Boyd (1997) showed that Pseudomonas putida coulduse adsorbed naphthalene on soil organic matter. Laor et al. (1996) reported that microor-ganisms are able to use phenanthrene directly from the HA-bound phase at the same rateas the free phase.

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Conclusion

Humic substances initially cause retardation of bioremediation. Over a period of time, theresults show that humic substances increase the bioavailability of the contaminant becauseit can enhance the biodegradation of phenanthrene by more than 45% in two months.Extracted humic substances can overcome the bond between the contaminants and the soilin the presence of bacteria, but the bacteria had the most effect on bioremediation. Bacterialgrowth in media with humic substances is about six-fold greater than in media withouthumic substances.

References

Alkorta, I. and Garbisu, C. 2001. Phytoremediation of organic contaminants in soils. Bioresour.Technol., 79, 273–276.

APHA, AWWA and WEF. 2005. Standard Methods for the Examination of Water and Wastewater,American Public Health Association/American Water Works Association/Water EnvironmentFederation, Washington, DC.

Binet, P., Portal, J. and Leyval, C. 2000. Dissipation of 3–6-ring polycyclic aromatic hydrocarbonsin the rhizosphere of ryegrass. Soil Biol. Biochem., 32, 2011–2017.

Blakely, J. K., Neher, D. A. and Spongberg, A. L. 2002. Soil invertebrate and microbial communities,and decomposition as indicators of polycyclic aromatic hydrocarbon contamination. Appl SoilEcol., 21, 71–88.

Das, P., Mukherjee, S. and Sen, R. 2008. Improved bioavailability and biodegradation of a model pol-yaromatic hydrocarbon by a biosurfactant producing bacterium of marine origin. Chemosphere,72, 1229–1234.

Dercova, K., Sejakova, Z., Skokanova, M., Barancikova, G. and Makovnıkova, J. 2007. Bioremedi-ation of soil contaminated with pentachlorophenol (PCP) using humic acids bound on zeolite.Chemosphere, 66, 783–790.

Gramss, G., Voigt, K. D. and Kirsche, B. 1999. Oxidoreductase enzymes liberated by plant roots andtheir effects on soil humic material. Chemosphere, 38, 1481–1494.

Grasso, D., Subramaniam, K., Pignatello, J., Yang, Y. and Ratte, D. 2001. Micellar desorption ofpolynuclear aromatic hydrocarbons from contaminated soil. Colloids Surf. A, 194, 65–74.

Guerin, W. F. and Boyd, S. A. 1997. Bioavailability of naphthalene associated with natural andsynthetic sorbents. Water Res., 31, 1504–1512.

Hamdi, H., Benzarti, S., Manusadzianas, L., Aoyama, I. and Jedidi, N. 2007. Bioaugmentation andbiostimulation effects on PAH dissipation and soil ecotoxicity under controlled conditions. SoilBiol. Biochem., 39, 1926–1935.

Heywood, E., Wright, J., Wienburg, C. L., Black, H. I.J., Long, S. M., Osborn, D. and Spurgeon, D.J. 2006. Factors influencing the national distribution of polycyclic aromatic hydrocarbons andpolychlorinated biphenyls in British soils. Environ. Sci. Technol., 40, 7629–7635.

Huang, W., Peng, P., Yu, Z. and Fu, J. 2003. Effects of organic matter heterogeneity on sorptionand desorption of organic contaminants by soils and sediments. Appl. Geochem., 18, 955–972.

Hwang, S. and Cutright, T. 2002. Biodegradability of aged pyrene and phenanthrene in a natural soil.Chemosphere., 47, 891–899.

Juhasz, A. L. and Naidu, R. 2000. Bioremediation of high molecular weight polycyclic aromatichydrocarbons: A review of the microbial degradation of benzoo [a] pyrene. Int. Biodeterior.Biodegrad., 45, 57–88.

Juhasz, A. L., Stanley, G. A. and Britz, M. L. 2000. Degradation of high molecular weight PAHs incontaminated soil by a bacterial consortium: Effects on Microtox and mutagenicity bioassays.Biorem. J., 4, 271–283.

Kao, C. M. and Borden, R. C. 1997. Site-specific variability in BTEX biodegradation under denitri-fying conditions. Ground Water, 35, 305–311.

Dow

nloa

ded

by [

Her

iot-

Wat

t Uni

vers

ity]

at 1

0:11

06

Oct

ober

201

4

PAHs Biodegradation with Humic Compounds 197

Karimi-Lotfabad, S., Pickard, M. A. and Gray, M. R. 1996. Reactions of polynuclear aromatichydrocarbons on soil. Environ. Sci. Technol., 30, 1145–1151.

Kastner, M. 2000. “Humification” process or formation of refractory soil organic matter. Biotechnol.,11b, 89–125.

Laor, Y., Strom, P. F. and Farmer, W. J. 1996. The effect of sorption on phenanthrene bioavailability.J. Biotechnol., 51, 227–234.

Laor, Y., Strom, P. F. and Farmer, W. J. 1999. Bioavailability of phenanthrene sorbed to mineral-associated humic acid. Water Res., 33, 1719–1729.

Lerch, R. N., Thurman, E. M. and Kruger, E. L. 1997. Mixed-mode sorption of hydroxylatedatrazine degradation products to soil: A mechanism for bound residue. Environ. Sci. Technol.,31, 1539–1546.

Liang, Y., Sorensen, D., Mclean, J. and Sims, R. C. 2008. Pyrene fate affected by humic acidamendment in soil slurry systems. J. Biol. Eng., 2, 1–7.

Lippold, H., Gottschalch, U. and Kupsch, H. 2008. Joint influence of surfactants and humic matteron PAH solubility: Are mixed micelles formed? Chemosphere, 70, 1979–1986.

Liste, H. H. and Alexander, M. 2000. Accumulation of phenanthrene and pyrene in rhizosphere soil.Chemosphere, 40, 11–14.

Macleod, C. J.A. and Semple, K. T. 2002. The adaptation of two similar soils to pyrene catabolism.Environ. Pollut., 119, 357–364.

Martins, J. M. and Mermoud, A. 1998. Sorption and degradation of four nitroaromatic herbicides inmono and multi-solute saturated/unsaturated soil batch systems. J Contam. Hydrol., 33, 187–210.

Mecozzi, M., Amici, M., Pietrantonio, E. and Romanelli, G. 2002. An ultrasound assisted extractionof the available humic substance from marine sediments. Ultrason. Sonochem., 9, 11–18.

Muckian, L. M., Grant, R. J., Clipson, N. J.W. and Doyle, E. M. 2009. Bacterial community dy-namics during bioremediation of phenanthrene-and fluoranthene-amended soil. Int. Biodeterior.Biodegrad., 63, 52–56.

Nakashima, K., Maki, M., Ishikawa, F., Yoshikawa, T., Gong, Y. K. and Miyajima, T. 2007. Fluores-cence studies on binding of pyrene and its derivatives to humic acid. Spectrochim. Acta, Part A,67, 930–935.

Nam, K. and Kim, J. 2002. Role of loosely bound humic substances and humin in the bioavailabilityof phenanthrene aged in soil. Environ. Pollut., 118, 427–433.

Nasseri, S., Rezaei Kalantary, R., Nourieh, N., Naddafi, K., Mahvi, A. and Baradaran, N. 2010.Influence of bioaugmentation in biodegradation of PAHs-contaminated soil in bio-slurry phasereactor. Iran. J. Environ. Health Sci. Eng., 7, 199–208.

Nayak, A. S., Vijaykumar, M. and Karegoudar, T. 2009. Characterization of biosurfactant producedby Pseudoxanthomonas sp. PNK-04 and its application in bioremediation. Int. Biodeterior.Biodegrad., 63, 73–79.

Niederer, C., Schwarzenbach, R. P. and Goss, K. U. 2007. Elucidating differences in the sorptionproperties of 10 humic and fulvic acids for polar and nonpolar organic chemicals. Environ. Sci.Technol., 41, 6711–6717.

Ortega-Calvo, J., Lahlou, M. and Saiz-Jimenez, C. 1997. Effect of organic matter and clays on thebiodegradation of phenanthrene in soils. Int. Biodeterior. Biodegrad., 40, 101–106.

Overcash, M., Sims, R. C., Sims, J. L. and Nieman, J. K.C. 2005. Beneficial reuse and sustainability:The fate of organic compounds in land-applied waste. J. Environ. Qual, 34, 29–41.

Pan, B., Ghosh, S. and Xing, B. 2008. Dissolved organic matter conformation and its interaction withpyrene as affected by water chemistry and concentration. Environ. Sci. Technol., 42, 1594–1599.

Park, J. H., Zhao, X. and Voice, T. C. 2002. Development of a kinetic basis for bioavailability ofsorbed naphthalene in soil slurries. Water Res., 36, 1620–1628.

Plaza, C., Xing, B., Fernandez, J. M., Senesi, N. and Polo, A. 2009. Binding of polycyclic aromatichydrocarbons by humic acids formed during composting. Environ. Pollut., 157, 257–263.

Ramirez, N., Cutright, T. and Ju, L. K. 2001. Pyrene biodegradatin in aqueous solutions and soilslurries by Mycobacterium PYR-1 and enriched consortium. Chemosphere, 44, 1079–1086.

Dow

nloa

ded

by [

Her

iot-

Wat

t Uni

vers

ity]

at 1

0:11

06

Oct

ober

201

4

198 R.R. Kalantary et al.

Reid, B. J., Jones, K. C. and Semple, K. T. 2000. Bioavailability of persistent organic pollutantsin soils and sediments: A perspective on mechanisms, consequences and assessment. Environ.Pollut., 108, 103–112.

Ressler, B., Kneifel, H. and Winter, J. 1999. Bioavailability of polycyclic aromatic hydrocarbonsand formation of humic acid-like residues during bacterial PAH degradation. Appl. Microbiol.Biotechnol., 53, 85–91.

Rezaei Kalantary, R., Badkoubi, A., Ganjidoust, H. and Shojaossadati, S. A. Anthracence biodegrada-tion in aqueous solutions by contaminated soil microorganisms mixed culture. IWWM (Instituteof Water and Waste Management), The 4th IWA Specialized Conference on Assessment andControl of Hazardous Substances in Water, Aachen, Germany, December 14–17, 2003.

Rezaei Kalantary, R. and Badkoubi, A. 2006. Effect of humic compounds on bacterial growth inbioremediation of pahs. Iran. J. Environ. Health Sci. Eng., 3, 31–38.

Richnow, H. H., Eschenbach, A., Mahro, B., Seifert, R., Wehrung, P., Albrecht, P. and Michaelis,W. 1998. The use of 13C-labelled polycyclic aromatic hydrocarbons for the analysis of theirtransformation in soil. Chemosphere, 36, 2211–2224.

USEPA. 1996. Ultrasonic Extraction. Center for Environmental Research Information. Available atwww.trincoll.edu/∼henderso/textfi∼1/416%20notes/3550b.pdf

Van Gestel, K., Mergaert, J., Swings, J., Coosemans, J. and Ryckeboer, J. 2003. Bioremediation ofdiesel oil-contaminated soil by composting with biowaste. Environ. Pollut., 125, 361–368.

Van Stempvoort, D., Lesage, S., Novakowski, K., Millar, K., Brown, S. and Lawrence, J. 2002. Humicacid enhanced remediation of an emplaced diesel source in groundwater. J. Contam. Hydrol.,54, 249–276.

Venkata Mohan, S., Purushotham Reddy, B. and Sarma, P. 2009. Ex situ slurry phase bioremediationof chrysene contaminated soil with the function of metabolic function: Process evaluation by dataenveloping analysis (DEA) and Taguchi design of experimental methodology (DOE). Bioresour.Technol., 100, 164–172.

Vosoughi, M., Yaghmaei, S., Aalemzadeh, I. and Seyf, K. A. A. 2002. Some investigation on biore-mediation of PAHs contaminated soil in Iran tar refinery. Int. J. Eng., 15, 1–10.

Wen, B., Zhang, J. J., Zhang, S. Z., Shan, X. Q., Khan, S. U. and Xing, B. 2007. Phenanthrene sorptionto soil humic acid and different humin fractions. Environ. Sci. Technol., 41, 3165–3171.

White, J. C., Hunter, M., Nam, K., Pignatello, J. J. and Alexander, M. 1999. Correlation between bio-logical and physical availabilities of phenanthrene in soils and soil humin in aging experiments.Environ. Toxicol. Chem., 18, 1720–1727.

Winn, W. C., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenbereger, P. and Woods, G.2006. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Lippincott Williamsand Wilkins, Philadelphia, PA.

Yang, Y., Ratte, D., Smets, B. F., Pignatello, J. J. and Grasso, D. 2001. Mobilization of soil organicmatter by complexing agents and implications for polycyclic aromatic hydrocarbon desorption.Chemosphere, 43, 1013–1021.

Yerushalmi, L. and Guiot, S. R. 2001. Biodegradation of benzene in a laboratory-scale biobarrier atlow dissolved oxygen concentrations. Biorem. J., 5, 63–77.

Yu, H., Zhu, L. and Zhou, W. 2007. Enhanced desorption and biodegradation of phenanthrene insoil-water systems with the presence of anionic-nonionic mixed surfactants. J. Hazard. Mater.,142, 354–361.

Dow

nloa

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by [

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