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Biosorption and biodegradation of polycyclic aromatic hydrocarbons in aqueous solutions by a consortium of white-rot fungi
Journal of Hazardous Materials 179 (2010) 845–851
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Biosorption and biodegradation of polycyclic aromatic hydrocarbons in aqueous solutions by a consortium of white-rot fungi Baoliang Chen a,∗ , Yinshan Wang a , Dingfei Hu b a b
Department of Environmental Sciences, Zhejiang University, Tianmushan Road 148, Hangzhou, Zhejiang 310028, China Department of Civil and Environmental Engineering, The University of Iowa, Iowa City, IA 52242, USA
a r t i c l e
i n f o
Article history: Received 23 April 2009 Received in revised form 14 March 2010 Accepted 16 March 2010 Available online 27 March 2010 Keywords: White-rot fungi Polycyclic aromatic hydrocarbons Biosorption Biodegradation Nutrient limitation
a b s t r a c t Bioremediation is a popular approach used to abate polycyclic aromatic hydrocarbons (PAHs) in the environment. A consortium of white-rot fungi (CW-1) isolated from wood pieces was used for studying their potential of bioremediation of PAHs. Biosorption and biodegradation of PAHs by live and heatkilled white-rot fungi (CW-1) were investigated to elucidate the bio-dissipation mechanisms of PAHs. Sorption isotherms of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene to heat-killed fungal biomass were linear and non-competitive, indicating the primary mechanism of biosorption to be by partition. The carbon-normalized partition coefficients (Koc ) were linearly correlated with octanol–water partition coefficients (Kow ), i.e., log Koc = 1.13 log Kow − 0.84 (n = 5, r2 = 0.996). Biosorption and biodegradation of phenanthrene and pyrene by live white-rot fungi were quantified. In 1 week, the removal efficiency of phenanthrene (70–80%) and pyrene (90%) by live fungi from aqueous solution were comparable to those by heat-killed fungi. However, approximately 40–65% of phenanthrene and 60–85% of pyrene were still stored in organismal bodies. Biosorption might restrict biodegradation while nutrient limitation and presence of a PAH mixture might stimulate biodegradation. The apparent partition coefficients (Kd∗ ) in live fungal systems and the Kd of heat-killed fungi without biodegradation were compared, and then the Kd∗ /Kd ratios were employed to illustrate the relative contributions of biosorption and biodegradation under different nutrient conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) resulting from the combustion of fossil fuels and biomass are potentially harmful to human health due to their high bioaccumulation, potential mutagenic, carcinogenic, and terategenic properties [1]. PAHs are prevailing in surface water, sediments and soils [2]. The removal of PAHs in water and soil has been extensively studied, and bioremediation is thought to be one of the most useful and advisable measures [3–6]. Usually, a few genera of bacteria are capable of degrading low molecular weight PAHs with three or less fused benzene rings, whereas, fungi are capable of mineralizing high molecular weight PAHs with different enzymes. In particular, white-rot fungi are capable of extensively degrading lignin from lignocelluosic substrates [3,7–10]. Commonly, biosorption and biodegradation are involved in the removal of PAHs by white-rot fungi. Recently, mechanisms of biodegradation of PAHs have been studied, especially fungal metabolism of PAHs. [3,7–11].
∗ Corresponding author. Tel.: +86 571 8827 3901; fax: +86 571 8827 3693. E-mail addresses: [email protected] (B. Chen), [email protected] (Y. Wang), [email protected] (D. Hu). 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.03.082
However, the biosorption of PAHs and the relative contributions of biosorption and biodegradation are still unclear [12,13]. Biosorption is a physico-chemical process involved in the sorption of a chemical substance in/on a biological matrix/surface [14]. As one of the processes in bioremediation, biosorption plays a significant role in the removal of PAHs. Many types of biosorbents (e.g., fungi, bacteria, algae and plant cuticle) were investigated for their ability to sequester heavy metals, dyes, pesticides, and organic pollutants [14–23], but the use of biosorption for removing PAHs in environment has received considerably less attention [12–14,22]. By far, studies on biosorption and biodegradation of PAHs have mainly concentrated on bacteria rather than fungi [12,13]. Biosorption of PAHs is ubiquitous in the environment. It is well-known that soil organic matter has a major influence on transport and fate of PAHs in the environment. As an important original source of soil organic matter [24], microbial biomass also plays a significant role through biosorption and biodegradation in abating PAHs contamination. Microorganisms have been widely introduced to remediate PAHs contaminated soils and to treat refinery wastewater by activated sludge treatment systems [10–12]. Generally, biodegradation is considered to account for the removal of PAHs from water by microorganisms, while the process of biosorption and storage in biomass is ignored.
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The objective of the current study is to elucidate biosorption of PAHs to fungal biomass and the relative contributions of biosorption and biodegradation to the total removal of PAHs by white-rot fungi. Biosorption of five PAHs (naphthalene, acenaphthene, fluorene, phenanthrene and pyrene) to heat-killed white-rot fungi and bi-solute sorption of phenanthrene and pyrene in aqueous solutions were performed using batch sorption experiment. Biosorption and biodegradation of phenanthrene and pyrene by live white-rot fungi in solution with different nutrient conditions were investigated. The partition coefficients (Kd ) derived from heatkilled fungal biomass and the apparent partition coefficients (Kd∗ ) derived from live fungal system were calculated and compared, and the Kd∗ /Kd ratios were employed to illustrate the relative contribution of biosorption and biodegradation in different nutrient conditions. 2. Materials and methods 2.1. Isolation of fungi and sample preparation Culture medium (Martin broth, a selective medium for fungi) was made by combining 1 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 10 g glucose, and 10 g peptone in 1 L of distilled–deionized water for liquid shake culture. For plate culture, 17 g/L agar was added to the Martin broth liquid culture solution. The two culture media were used for isolating fungi from wood pieces. Growths of bacteria and actinomyces were restricted in the two culture media, while fungi were influenced rarely. Several fungi were recovered, but specific genera from these have not been defined. The isolation of fungi in brief was as follows: all of the equipments and containers were sterilized, and the entire experiment procedures were carried out under aseptic conditions. Initially, the white colony on wood pieces was selected and streaked with a sterilized loop, and then selective incubation was performed by the plate culture at 28 ◦ C for 3 days to develop the new colony. Subsequently, the new white colony was selected for preparing suspension solution with sterilized water, which was pipetted for further culture by spread plate method. Several of these white colonies were prepared for morphological study. The colony with white mycelium with abundant mycelia and plenty of conidiophores in the culture solution was selected. The fungi thus obtained (CW-1) were inoculated in 500 mL conical flask containing 200 mL culture solutions. After 3 days, the culture solution with fungi was divided into three conical flasks, and 200 mL fresh culture solution was added to each flask. After three cycles, fungal biomass were collected from the culture solution, and washed with distilled–deionized water to remove the residue of the culture solution. The clean fungal biomass was oven-dried at 60 ◦ C for 48 h. The dry fungal biomass was pulverized and then passed through a 0.154 mm sieve for use. On the other hand, some live fungal biomass was used for further amplification culture, and prepared for biosorption and biodegradation experiment. 2.2. Characterization of white-rot fungi Elemental (C, H, N) analyses of the fungal biomass were conducted using an EA 112 CHN elemental analyzer (Thermo Finnigan). FTIR spectra were recorded in the 4000–400 cm−1 region for a KBrpellet by a Nicolet FTIR spectrophotometer (model 560) with a resolution of 1.0 cm−1 . 2.3. Batch sorption experiment Five PAHs (naphthalene, acenaphthene, fluorene, phenanthrene and pyrene) were chosen as model sorbates due to their prevailing presence in environments. The selected properties of the PAHs are presented in Table 1. All the single-solute sorption isotherms by
white-rot fungi (CW-1) were obtained using a batch equilibration technique described elsewhere [23]. In brief, initial concentrations ranged from 0.20 to 25 mg/L for naphthalene, from 0.002 to 2.5 mg/L for acenaphthene, from 0.008 to 1.7 mg/L for fluorene, from 0.001 to 1 mg/L for phenanthrene and from 0.0002 to 0.1 mg/L for pyrene. Each isotherm consisted of 10 concentration points; each point, including the control and calibration, was run in duplicate. The background solution comprised 0.01 mol/L CaCl2 , and 200 mg/L NaN3 to inhibit the biodegradation of the solution. The 8-mL vials were filled with a sorbate solution to minimize the headspace volumes of vials and sealed with aluminum foillined Teflon screw caps to avoid evaporation of sorbate, and then agitated in the dark for 3 days at 25 ± 0.5 ◦ C to reach apparent equilibrium. The solution was separated by centrifugation at 4000 rpm for 15 min. The equilibrium concentrations were measured using a high performance liquid chromatography (HPLC, Agilent 1200) with a 4.6 mm × 150 mm reverse phase XDB-C18 column and a fluorescence detector (FLD) using acetonitrile–water (v:v, 90/10) as the mobile phase at a flow rate of 1 mL/min. The individual excitation wavelengths of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene were 240, 240, 220, 244 and 237 nm, while the respective emission wavelengths were 360, 360, 315, 360 and 385 nm. Because of minimal sorption by the vials, and negligible losses by evaporation, biodegradation and photodegradation, the sorbed amount was calculated by mass difference between nominal concentration without sorbent and with sorbent in aqueous solutions. All competitive sorption experiments of phenanthrene and pyrene were conducted at 25 ± 0.5 ◦ C. Bi-solute sorption experiments included sorption of pyrene with 0.5 mg/L phenanthrene as a co-solute and sorption of phenanthrene with 0.05 mg/L pyrene as a co-solute. The remaining procedures of the competitive sorption experiment were performed the same way as described for single-solute sorption experiments. 2.4. Biosorption and biodegradation experiment A 4-day culture of white-rot fungi (CW-1) obtained from pure culture grown in about 200 mL of Martin broth was homogenized in the sterilized conical flask of 500 mL, and was used for inoculating. Five groups were designed: (i) Martin broth without the fungi was used as blank control; (ii) heat-killed fungi + Martin broth, was used as control to study the effect of Martin broth (in contrast to heat-killed fungi in aqueous solutions of the batch sorption experiment); (iii) live fungi + Martin broth (total nutrient solution); (iv) live fungi + sterilized water (without nutrient); and (v) live fungi + Martin broth without glucose. These experimental groups were used to investigate the contributions of biosorption and biodegradation to the removal of PAHs under different nutrient conditions. The 20-mL solution with the concentrations of 1.0 mg/L phenanthrene and 0.1 mg/L pyrene were added to the 40-mL vials. Replicate samples were prepared for each group, including three experimental groups and two controls. After the addition of solution, the 40-mL vials were sealed with aluminum foil-lined Teflon screw caps to avoid sorbate’s evaporation, and then agitated in the dark for 7 days at 25 ± 0.5 ◦ C with aeration for 2 min each day. After 7-day of culture, the vials were centrifuged at 4000 rpm for 15 min. The supernatant was removed and diluted with CH3 CN (1:1), and the residual PAHs in the solution was detected by HPLC–FLD. Then the solution in the vials was decanted as much as possible, and the residual PAHs in fungal biomass were extracted by ultrasonication for 30 min with a 10 mL mixture of acetone and hexane (1:1, v:v) for 3 successive extractions. The extracts were pooled and evaporated to almost dry using rotary evaporator and re-dissolved in 5 mL of hexane, followed by a clean-up procedure through a 2.5-g silica gel column with a 15 mL mixture of hexane and dichloromethane
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Table 1 The selected physico-chemical parameters of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr). Molecular weight (g/mol)
Cs (25 ◦ C)a (mg/L)
Kow b
C10 H8
128.2
31.02
1.95 × 103
Acen
C12 H8
154.2
3.47
8.4 × 103
Flu
C13 H10
116.2
1.69
1.5 × 104
Phen
C14 H10
178.2
1.00
2.8 × 104
Pyr
C16 H10
202.3
0.13
8.0 × 104
PAHs
Molecular formula
Naph
a b
Structural formula
Cs : aqueous solubility at 25 ◦ C. Kow : octanol–water partition coefficients.
(1:1, v:v). Samples were evaporated again and re-dissolved in acetonitrile to a final volume of 4 mL. After filtration through 0.22 mm filter units, PAHs concentrations were determined by the same HPLC method. The removal of PAHs was determined by mass difference in solution concentration between the initial and the final, which includes biosorption and biodegradation. The residual PAHs extracted from fungal biomass was assigned to biosorption amounts, and the biodegradation amounts were determined by mass difference in the total removal and biosorption amounts. The water content of live white-rot fungi measured by dry–wet method was 97.63% (n = 3, RSD <0.36%). 2.5. Data analysis The Freundlich parameters (Kf and N) were calculated using the logarithmic form of the equation Q = Kf CeN , where Q is the amount sorbed per unit weight of sorbent, mg/kg; Ce is the equilibrium concentration, mg/L; Kf [(mg/kg)/(mg/L)N ] is the Freundlich capacity coefficient, and N (dimensionless) describes the isotherm curvature. In biosorption and biodegradation experiment, the average recoveries obtained by spiked fungi samples with phenanthrene and pyrene were 87.1% (n = 6, RSD <1.6%) and 94.8% (n = 6, RSD <2.2%) for the entire procedure of extraction and determination, which applied to data reconciliation of extraction. The intrinsic sorption coefficients (Kd = Q/Ce ) derived from the batch sorption experiment and the apparent partition coefficient (Kd∗ ) derived from biosorption and biodegradation experiment were calculated. The Koc values were calculated by normalizing Kd to the carbon level (foc ) of white-rot fungi (Koc = Kd /foc ). The Kd∗ /Kd ratios were calculated and employed to evaluate relative contributions of biosorption and biodegradation. If Kd∗ /Kd ≈ 1, it may indicate that biosorption governs the bio-dissipation of PAHs; if Kd∗ /Kd < 1, it may suggest that the biodegradation occurs.
carbonyl, carboxyl, sulfhydryl, amide, phosphonate and phosphodiester groups) have been suggested for their contribution to biosorption [25]. These aliphatic and aromatic components and high carbon content may contribute to the biosorption of PAHs by white-rot fungi (CW-1). 3.2. Sorption of PAHs with white-rot fungi Isotherms of five PAHs by heat-killed white-rot fungal biomass (CW-1) are presented in Fig. 2, and the regression parameters are listed in Table 2. Sorption isotherms of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene to heat-killed fungi fit well with the Freundlich equation. Sorption of organic pollutant to biomass may involve simultaneous surface sorption, partitioning processes and chemical reactions. The Freundlich N values were approximately 1, indicating that the primary mechanism of sorption is partitioning into fungal biomass. This is consistent with the previous report about dead bacterial biomass [12,26]. But Stringfellow and Alvarez-Cohen [12] suggested that the biosorption of PAHs by live bacterial biomass involved surface sorption rather than nonspecific partition. Therefore, the physiology of the biomass itself may influence sorption processes. The calculated partition coefficients (Kd ) of PAHs were 361.0 mL/g (naphthalene), 1656 mL/g (acenaphthene), 2890 mL/g (fluorene), 6822 mL/g (phenanthrene) and 24,140 mL/g (pyrene), which show that white-rot fungi (CW-1) have a high affinity for PAHs. A linear correlation was observed between log Koc and
3. Results and discussion 3.1. Characterization of white-rot fungal biomass The elemental compositions of white-rot fungal biomass (CW1) obtained from pure culture were as follows: C 43.70%; N 6.44%; H 7.22%. The FTIR spectrum of the fungal biomass is shown in Fig. 1. The band at 3300 cm−1 represents the stretching vibration of hydroxyl groups. The bands at 2930 and 1400 cm−1 are assigned mainly to CH2 units in biopolymers. The band at 1710 cm−1 is assigned to C O stretching vibration of ester groups. The band at 1650 cm−1 is assigned to C C and C O stretching in the aromatic ring and 571 cm−1 is also assigned to aromatic components. The bands at 1240 and 1060 cm−1 are assigned to C–O stretching of polysaccharides. Numerous chemical groups (e.g., hydroxyl,
Fig. 1. FTIR spectrum of dry biomass of the consortium of white-rot fungi (CW-1).
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Table 2 Regression parameters of isotherms of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr) by heat-killed fungal biomass (CW-1). PAHs
Freundlich regression parametersa N
Naph Acen Flu Phen Pyr Phen (0.05 mg/L Pyr as co-solute) Pyr (0.5 mg/L Phen as co-solute)
Linear parametersb 2
Log Kf
1.119 1.020 0.942 1.062 1.093 1.011 1.155
± ± ± ± ± ± ±
0.027 0.009 0.009 0.016 0.044 0.007 0.070
2.458 3.223 3.449 3.870 4.573 3.848 4.703
± ± ± ± ± ± ±
0.020 0.013 0.010 0.020 0.020 0.008 0.028
Freundlich r
Kd
0.991 0.999 0.999 0.996 0.996 0.999 0.991
361.0 1656 2890 6822 24,140 6971 26,020
r2 ± ± ± ± ± ± ±
2.7 7 38 117 695 106 740
0.999 0.999 0.997 0.995 0.985 0.996 0.986
Koc 826.2 3789 6613 15,610 55,240 15,950 59,540
a The Freundlich parameters (Kf and N) were calculated using the logarithmic form of the equation Q = Kf CeN , where Q is the amount sorbed per unit weight of sorbent, mg/kg; Ce is the equilibrium concentration, mg/L; Kf [(mg/kg)/(mg/L)N ] is the Freundlich capacity coefficient, and N (dimensionless) describes the isotherm curvature. r2 is regression coefficient. b Linear parameters (Kd and Koc ) were regressed, and the intrinsic sorption coefficients (Kd = Q/Ce ) derived from batch sorption experiment, and Koc values were calculated by normalizing Kd to the carbon level (foc ) of white-rot fungi (Koc = Kd /foc ).
log Kow for PAHs to white-rot fungi (see Fig. 3): log Koc = 1.13 log Kow − 0.84
2
(n = 5, r = 0.996)
(1)
According to the linear equation of log Koc –log Kow , biosorption of PAHs to the fungal biomass could be predicted. By contrast, the relationship for PAHs and their derivatives over a comparable range of log Kow (2.11–6.34) on river sediments presented by Karickhoff et al. [27] was as follows: log Koc = 1.00 log Kow − 0.21
(n = 10, r 2 = 0.996)
(2)
The two equations are similar despite different kinds of sorbents (fungi vs. river sediment). Partition coefficient (Kd ) is an effective parameter to estimate the distribution of target chemicals between two phases and hence their fate and mobility in the environment. Based on Kd value, the ranking of sorption efficiency of these sorbents to aqueous PAHs can be estimated [14]. For naphthalene, the log Kd value of the heatkilled fungi–water system was higher than that of soil–water and sediment–water systems due to the higher carbon content of the former [28]. Biotic-origin sorbents have log Kd values of phenanthrene from 2.98 to 4.45 [29], and the determined log Kd value of phenanthrene by the white-rot fungi was 3.83, which was higher than log Kd of cellulose (2.98) and lower than that of humic acid (4.45) [29]. Therefore, biosorption of microorganisms may play an important role in governing the fate of PAHs in soils and sediments.
Fig. 2. Sorption of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr) to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
Competitive sorption of phenanthrene and pyrene to heat-killed white-rot fungi (CW-1) was investigated. Sorption isotherms of single phenanthrene and phenanthrene with co-solute 0.05 mg/L pyrene were nearly consistent (Fig. 4A) , suggesting that co-solute pyrene had no influence on the sorption of phenanthrene to fungal biomass. Sorption isotherm of single pyrene and pyrene with co-solute 0.5 mg/L phenanthrene showed that co-solute phenanthrene had little effect on the sorption of pyrene to fungal biomass (Fig. 4B). Therefore, simultaneous sorption of phenanthrene and pyrene were compatible and not competitive. However, this is inconsistent with the previous report which suggested that PAHs exhibit competition for surface binding sites on bacterial biomass [12]. 3.3. Relative contributions of biosorption and biodegradation to the removal of PAHs by white-rot fungi Microorganisms play an important role in the fate of PAHs in the environment, including biosorption and biodegradation [12]. The contributions of biosorption and biodegradation of phenanthrene and pyrene to live and heat-killed white-rot fungi (CW-1) in different nutrient solutions are listed in Table 3. For heat-killed fungi (Group II), the biosorption controlled the bio-dissipation of PAHs since biodegradation was not involved. For live fungi, the contributions of biosorption and biodegradation to the total removal of phenanthrene and pyrene under different nutrient con-
Fig. 3. Relationship between log Koc and log Kow of PAHs to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
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Table 3 The contributions of biosorption and biodegradation in removal of phenanthrene (Phen) and pyrene (Pyr) by white-rot fungi (CW-1) under different nutrient conditions. Group
Biomass
Solution
Average biomass (mg) Initial
Group I (blank control) Group II (control) Group III Group IV Group V a b
Phen
Final
–
Pyr a
Removal (%) 5.44 ± 0.51
–
b
Biosorption (%) 0
Removala (%) 19.21 ± 2.21
Biosorptionb (%)
None
Culture solution
0
Heat-killed fungi
Culture solution
10.07 ± 0.04
10.07 ± 0.04
82.07 ± 2.09
63.85 ± 11.03
93.87 ± 0.56
86.14 ± 22.77
Live fungi Live fungi Live fungi
Culture solution Sterilized water Culture solution without carbon
6.09 ± 0.60 6.09 ± 0.60 6.09 ± 0.60
53.03 ± 2.85 8.03 ± 0.34 12.52 ± 1.60
75.06 ± 1.14 66.01 ± 1.05 60.44 ± 3.66
53.11 ± 12.25 51.36 ± 2.36 39.01 ± 8.62
91.06 ± 0.33 87.91 ± 1.93 87.73 ± 3.09
79.50 ± 13.24 76.87 ± 5.97 61.93 ± 8.21
Removal: the removal PAHs was determined by PAH mass difference in solution concentration between the initial and the final. Biosorption: the residual PAHs extracted from fungi is assigned to biosorption amounts.
ditions were examined. The removal efficiency of PAHs by live fungi (Group III) approached that of heat-killed fungi (Group II). For all live groups, the removal efficiency of pyrene (90%) was higher than that of phenanthrene (60–80%) because of higher hydrophobicity of pyrene. Secondly, significant differences between the total removal and biosorption (see Table 3) could be used to elucidate the bio-dissipation mechanisms. For phenanthrene, the
Fig. 4. Competitive sorption of phenanthrene (A) and pyrene (B) to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
significant difference between the total removal and biosorption (10–20%) suggested that the degradation of phenanthrene occurred under different nutrient conditions, including total nutrient (Martin broth), glucose-free medium (low-carbon Martin broth) and non-nutrient solution (sterilized water). For live fungi in total nutrient solution (Group III) and non-nutrient solution (Group IV), there was no significant difference for pyrene between the total removal and biosorption (the amounts extracted from fungal biomass), indicating that biosorption dominates the bio-dissipation of PAHs in these two nutrient conditions. For live fungi in non-nutrient culture (Group IV), the biodegradation of phenanthrene suggested that white-rot fungi may use phenanthrene as sole source of carbon for growth and thus degrade it. In addition, in glucose-free culture condition (Group V) simultaneous biodegradation of the mixture of phenanthrene and pyrene was observed and the same phenomenon was also reported but the mechanism has not been known yet [14]. Another probable reason was that nutrient limitation in low carbon culture condition promoted release of degrading enzymes such as extracellular lignin-modifying enzymes to facilitate the degradation of PAHs [30]. The bio-dissipation mechanism of phenanthrene and pyrene from solution containing live and heat-killed fungi (CW-1) were further elucidated by the growth of fungal biomass (see Table 3). Previous studies believed that biosorption was facilitated by the exopolysaccharide sheath around the fungal mycelium [13]. For heat-killed fungi, the biomass was constant before and after culture in the total nutrient solution, indicating that there was no metabolization involved in the removal process and supported that biosorption governed the process. For live fungi in total nutrient solution (Group III), the dry fungal biomass increased from the initial mass of 6.09 mg to the final mass of 53.03 mg (i.e., 9 times), which approached 5 times that of heat-killed fungi in total nutrient solution (Group II). Interestingly, for the removal efficiency and biosorption of PAHs there was no significant difference in live and heat-killed fungal systems (Group II and Group III), suggesting that the new fungal biomass was inaccessible to PAHs in aqueous solutions. For live fungi in non-nutrient solution (Group IV), the fungal biomass increased from the initial mass of 6.09 mg to the final mass 8.03 mg. For live fungi in a culture solution with low carbon (Group V), the biomass increased from 6.09 to 12.52 mg. These observations proved that phenanthrene and pyrene can be used as carbon sources by white-rot fungi. Furthermore, in long-term experiments (56 days) for all live groups, although the total removal increased, the PAHs stored in fungal biomass were almost constant. This suggested that biosorption limits the availability of PAHs for biodegradation, which works extracellularly by released lignin degrading enzymes to degrade lignin-like PAHs [31]. The reduced bioavailability by biosorption may contribute to the reduced biodegradation by live fungi in total nutrient solution (Group III) with high biomass than that achieved in glucose-free culture medium (Group V) with low biomass. These
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Table 4 Apparent partition coefficients (Kd∗ ) in culture systems and partition coefficient (Kd ) in batch sorption experiment of phenanthrene (Phen) and pyrene (Pyr), and the ratio of Kd∗ /Kd . Parameter
Sorbate
Heat-killed fungi (Kd )
Kd∗
Phen Pyr Phen Pyr
6822 24,140 1.000 1.000
Kd∗ /Kd a b c d
Controla 7174 28,250 1.052 1.170
Group IIIb 809.1 3369 0.119 0.140
Group IVc 3777 16,070 0.554 0.666
Group Vd 1588 8283 0.233 0.343
Control: heat-killed fungi in culture medium. Group III: live fungi in culture solution. Group IV: live fungi in sterilized water. Group V: live fungi in culture solution without carbon.
Fig. 5. The schematics of the correlations of biosorption and biodegradation with sorption parameters in live and dead fungal systems (CW-1) according to Kd∗ /Kd ratios.
results supported that the activated sludge plant effluent solids may be a significant source of PAH release to the environment [12]. 3.4. Comparison of partition coefficients between live and heat-killed white-rot fungi The apparent partition coefficients (Kd∗ ) and Kd∗ /Kd ratios for phenanthrene and pyrene were calculated and presented in Table 4. The Kd∗ were calculated as follow: Kd∗ =
Q∗ mPAHs /mbiomass = Ce∗ Ce∗
(3)
where Q* and Ce∗ are the PAHs apparent sorbed-amount and aqueous equilibrium concentration, respectively, in the culture system. Q* = mPAHs /mbiomass , where mPAHs is the PAHs amount stored in biomass, and mbiomass is the final fungal biomass. Based on Eq. (3), the Kd∗ value will decrease due to the biodegradation of PAHs and increase in fungal biomass (CW-1). Firstly, the Kd∗ value and the Kd value for heat-killed fungi were comparable with and without the presence of Martin broth in Group II and in the batch sorption experiment, indicating that the Martin broth has no effect on the sorption of PAHs. Secondly, Kd∗ of PAHs by live fungi in total nutrient solution (Group III) was almost 8 times less than the Kd value of heat-killed fungi (Group II), which mainly attributes to growth dilution of fungal biomass, i.e., the final biomass of live fungi was about 8 times higher than the initial and 5 times that of heat-killed fungi. Thirdly, Kd∗ by live fungi in non-nutrient solution (Group IV) was half of Kd of heat-killed fungi, while the final fungal biomass was about one third higher than the initial and four fifth that of heatkilled fungi. Presumably, the difference of Kd∗ (or Kd ) between live fungi in non-nutrient solution and heat-killed fungi may ascribe to small growth of fungi and its biodegradation. Finally, though the final biomass was twice the original and one fifth more than that of heat-killed fungi, Kd∗ of phenanthrene and pyrene by live fungi in glucose-free culture solution (Group V) was about a quarter of Kd of heat-killed fungi, mainly due to biodegradation of PAHs.
The correlations of biosorption and biodegradation with sorption parameters in live and dead fungal systems (CW-1) are demonstrated in Fig. 5. If Kd∗ /Kd ≈ 1, it indicated that biosorption governs the fate of PAHs, while if Kd∗ /Kd < 1, it may suggest that the biodegradation occurs. For phenanthrene and pyrene with heat-killed fungi in total nutrient solution (Group II), Kd∗ /Kd ≈ 1 suggested that the biosorption plays a critical role in the transport and fate of PAHs. For phenanthrene with live fungi in different nutrient conditions, Kd∗ /Kd < 1 may attribute to growth dilution of fungal biomass and its biodegradation. Live fungi in the nonnutrient solution degraded phenanthrene by utilizing it as the sole source of carbon, while simultaneous biodegradation of pyrene and phenanthrene occurred in Martin broth without carbon (Group V). However, for pyrene with live fungi in the total nutrient solution (Group III), Kd∗ /Kd < 1 was mainly ascribed to the increase of mbiomass of the final fungal biomass (i.e., growth dilution). Similar results were shown by Raghukumar et al. [13]. Therefore, Kd∗ /Kd ratios and fungal biomass are important parameters to distinguish the relative contributions of biosorption and biodegradation to total bio-dissipation of PAHs from aqueous solutions. 4. Conclusions Biosorption of PAHs by white-rot fungi is very important process influencing the fate of PAHs in the environment. The partitioning of PAHs into fungal biomass (CW-1) is the primary mechanism of biosorption, and sorption capabilities (Koc ) are linearly related with Kow . Simultaneous sorption of phenanthrene and pyrene is not competitive. Both biosorption and biodegradation of PAHs play a crucial role in the removal of PAHs, whereas the extent of biosorption and biodegradation vary with nutrient conditions. Biodegradation of phenanthrene occurs in all of different nutrient conditions, while simultaneous biodegradation of phenanthrene and pyrene occurs only in the glucose-free culture solution. Whiterot fungi may use phenanthrene and pyrene as the source of carbon for growth under nutrient-limited conditions. Based on analysis of biosorption and biodegradation experiment, Kd∗ /Kd ratios can be
B. Chen et al. / Journal of Hazardous Materials 179 (2010) 845–851
utilized to illustrate the relative contributions of biosorption and biodegradation in different nutrient conditions. Acknowledgements We are highly grateful to the anonymous reviewers for their valuable comments. This project was supported by The National High Technology Research and Development Program of China (No. 2007AA061101), National Natural Science Foundation of China (Grant Nos. 40671168 and 20737002), and Foundation for the Author of National Excellent Doctoral Dissertation of China (No. 200765). References [1] Q. Cai, C. Mo, Q. Wu, A. Katsoyiannis, Q. Zeng, The status of soil contamination by semivolatile organic chemicals (SVOCs) in China: a review, Sci. Total Environ. 389 (2008) 209–224. [2] B. Chen, X. Xuan, L. Zhu, J. Wang, Y. Gao, K. Yang, X. Shen, B. Lou, Distributions of polycyclic aromatic hydrocarbons in surface waters, sediments and soils of Hangzhou city, China, Water Res. 38 (2004) 3558–3568. [3] S.M. Bamforth, L. Singleton, Bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions, Chem. Technol. Biotechnol. 80 (2005) 723–736. [4] A.R. Johnsen, L.Y. Wick, H. Harms, Principles of microbial PAH-degradation in soil, Environ. Pollut. 133 (2005) 71–84. [5] S.B. Pointing, Feasibility of bioremediation by white-rot fungi, Appl. Microbiol. Biotechnol. 57 (2001) 20–33. [6] S.K. Samanta, O.V. Singh, R.K. Jain, Polycyclic aromatic hydrocarbons: environmental pollution and bioremediation, Trend. Biotechnol. 20 (2002) 243–248. [7] C.E. Cerniglia, Biodegradation of polycyclic aromatic hydrocarbons, Biodegradation 3 (1992) 351–368. [8] D.P. Barr, S.D. Aust, Mechanisms white-rot fungi use to degrade pollutants, Environ. Sci. Technol. 28 (1994) A78–A87. [9] C.E. Cerniglia, Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation, J. Ind. Microbiol. Biotechnol. 19 (1997) 324–333. [10] A.L. Juhasz, R. Naidu, Bioremediation of high molecular polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene, Int. Biodeterior. Biodegrad. 45 (2000) 57–88. [11] S.S. Cameotra, J.M. Bollag, Biosurfactant-enhanced bioremediation of polycyclic aromatic hydrocarbons, Environ. Sci. Technol. 30 (2003) 112–126. [12] W.T. Stringfellow, L. Alvarez-Cohen, Evaluating the relationship between the sorption of PAHs to bacterial biomass and biodegradation, Water. Res. 33 (1999) 2535–2544.
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Biosorption and biodegradation of polycyclic aromatic hydrocarbons in aqueous solutions by a consortium of white-rot fungi Baoliang Chen a,∗ , Yinshan Wang a , Dingfei Hu b a b
Department of Environmental Sciences, Zhejiang University, Tianmushan Road 148, Hangzhou, Zhejiang 310028, China Department of Civil and Environmental Engineering, The University of Iowa, Iowa City, IA 52242, USA
a r t i c l e
i n f o
Article history: Received 23 April 2009 Received in revised form 14 March 2010 Accepted 16 March 2010 Available online 27 March 2010 Keywords: White-rot fungi Polycyclic aromatic hydrocarbons Biosorption Biodegradation Nutrient limitation
a b s t r a c t Bioremediation is a popular approach used to abate polycyclic aromatic hydrocarbons (PAHs) in the environment. A consortium of white-rot fungi (CW-1) isolated from wood pieces was used for studying their potential of bioremediation of PAHs. Biosorption and biodegradation of PAHs by live and heatkilled white-rot fungi (CW-1) were investigated to elucidate the bio-dissipation mechanisms of PAHs. Sorption isotherms of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene to heat-killed fungal biomass were linear and non-competitive, indicating the primary mechanism of biosorption to be by partition. The carbon-normalized partition coefficients (Koc ) were linearly correlated with octanol–water partition coefficients (Kow ), i.e., log Koc = 1.13 log Kow − 0.84 (n = 5, r2 = 0.996). Biosorption and biodegradation of phenanthrene and pyrene by live white-rot fungi were quantified. In 1 week, the removal efficiency of phenanthrene (70–80%) and pyrene (90%) by live fungi from aqueous solution were comparable to those by heat-killed fungi. However, approximately 40–65% of phenanthrene and 60–85% of pyrene were still stored in organismal bodies. Biosorption might restrict biodegradation while nutrient limitation and presence of a PAH mixture might stimulate biodegradation. The apparent partition coefficients (Kd∗ ) in live fungal systems and the Kd of heat-killed fungi without biodegradation were compared, and then the Kd∗ /Kd ratios were employed to illustrate the relative contributions of biosorption and biodegradation under different nutrient conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) resulting from the combustion of fossil fuels and biomass are potentially harmful to human health due to their high bioaccumulation, potential mutagenic, carcinogenic, and terategenic properties [1]. PAHs are prevailing in surface water, sediments and soils [2]. The removal of PAHs in water and soil has been extensively studied, and bioremediation is thought to be one of the most useful and advisable measures [3–6]. Usually, a few genera of bacteria are capable of degrading low molecular weight PAHs with three or less fused benzene rings, whereas, fungi are capable of mineralizing high molecular weight PAHs with different enzymes. In particular, white-rot fungi are capable of extensively degrading lignin from lignocelluosic substrates [3,7–10]. Commonly, biosorption and biodegradation are involved in the removal of PAHs by white-rot fungi. Recently, mechanisms of biodegradation of PAHs have been studied, especially fungal metabolism of PAHs. [3,7–11].
∗ Corresponding author. Tel.: +86 571 8827 3901; fax: +86 571 8827 3693. E-mail addresses: [email protected] (B. Chen), [email protected] (Y. Wang), [email protected] (D. Hu). 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.03.082
However, the biosorption of PAHs and the relative contributions of biosorption and biodegradation are still unclear [12,13]. Biosorption is a physico-chemical process involved in the sorption of a chemical substance in/on a biological matrix/surface [14]. As one of the processes in bioremediation, biosorption plays a significant role in the removal of PAHs. Many types of biosorbents (e.g., fungi, bacteria, algae and plant cuticle) were investigated for their ability to sequester heavy metals, dyes, pesticides, and organic pollutants [14–23], but the use of biosorption for removing PAHs in environment has received considerably less attention [12–14,22]. By far, studies on biosorption and biodegradation of PAHs have mainly concentrated on bacteria rather than fungi [12,13]. Biosorption of PAHs is ubiquitous in the environment. It is well-known that soil organic matter has a major influence on transport and fate of PAHs in the environment. As an important original source of soil organic matter [24], microbial biomass also plays a significant role through biosorption and biodegradation in abating PAHs contamination. Microorganisms have been widely introduced to remediate PAHs contaminated soils and to treat refinery wastewater by activated sludge treatment systems [10–12]. Generally, biodegradation is considered to account for the removal of PAHs from water by microorganisms, while the process of biosorption and storage in biomass is ignored.
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The objective of the current study is to elucidate biosorption of PAHs to fungal biomass and the relative contributions of biosorption and biodegradation to the total removal of PAHs by white-rot fungi. Biosorption of five PAHs (naphthalene, acenaphthene, fluorene, phenanthrene and pyrene) to heat-killed white-rot fungi and bi-solute sorption of phenanthrene and pyrene in aqueous solutions were performed using batch sorption experiment. Biosorption and biodegradation of phenanthrene and pyrene by live white-rot fungi in solution with different nutrient conditions were investigated. The partition coefficients (Kd ) derived from heatkilled fungal biomass and the apparent partition coefficients (Kd∗ ) derived from live fungal system were calculated and compared, and the Kd∗ /Kd ratios were employed to illustrate the relative contribution of biosorption and biodegradation in different nutrient conditions. 2. Materials and methods 2.1. Isolation of fungi and sample preparation Culture medium (Martin broth, a selective medium for fungi) was made by combining 1 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 10 g glucose, and 10 g peptone in 1 L of distilled–deionized water for liquid shake culture. For plate culture, 17 g/L agar was added to the Martin broth liquid culture solution. The two culture media were used for isolating fungi from wood pieces. Growths of bacteria and actinomyces were restricted in the two culture media, while fungi were influenced rarely. Several fungi were recovered, but specific genera from these have not been defined. The isolation of fungi in brief was as follows: all of the equipments and containers were sterilized, and the entire experiment procedures were carried out under aseptic conditions. Initially, the white colony on wood pieces was selected and streaked with a sterilized loop, and then selective incubation was performed by the plate culture at 28 ◦ C for 3 days to develop the new colony. Subsequently, the new white colony was selected for preparing suspension solution with sterilized water, which was pipetted for further culture by spread plate method. Several of these white colonies were prepared for morphological study. The colony with white mycelium with abundant mycelia and plenty of conidiophores in the culture solution was selected. The fungi thus obtained (CW-1) were inoculated in 500 mL conical flask containing 200 mL culture solutions. After 3 days, the culture solution with fungi was divided into three conical flasks, and 200 mL fresh culture solution was added to each flask. After three cycles, fungal biomass were collected from the culture solution, and washed with distilled–deionized water to remove the residue of the culture solution. The clean fungal biomass was oven-dried at 60 ◦ C for 48 h. The dry fungal biomass was pulverized and then passed through a 0.154 mm sieve for use. On the other hand, some live fungal biomass was used for further amplification culture, and prepared for biosorption and biodegradation experiment. 2.2. Characterization of white-rot fungi Elemental (C, H, N) analyses of the fungal biomass were conducted using an EA 112 CHN elemental analyzer (Thermo Finnigan). FTIR spectra were recorded in the 4000–400 cm−1 region for a KBrpellet by a Nicolet FTIR spectrophotometer (model 560) with a resolution of 1.0 cm−1 . 2.3. Batch sorption experiment Five PAHs (naphthalene, acenaphthene, fluorene, phenanthrene and pyrene) were chosen as model sorbates due to their prevailing presence in environments. The selected properties of the PAHs are presented in Table 1. All the single-solute sorption isotherms by
white-rot fungi (CW-1) were obtained using a batch equilibration technique described elsewhere [23]. In brief, initial concentrations ranged from 0.20 to 25 mg/L for naphthalene, from 0.002 to 2.5 mg/L for acenaphthene, from 0.008 to 1.7 mg/L for fluorene, from 0.001 to 1 mg/L for phenanthrene and from 0.0002 to 0.1 mg/L for pyrene. Each isotherm consisted of 10 concentration points; each point, including the control and calibration, was run in duplicate. The background solution comprised 0.01 mol/L CaCl2 , and 200 mg/L NaN3 to inhibit the biodegradation of the solution. The 8-mL vials were filled with a sorbate solution to minimize the headspace volumes of vials and sealed with aluminum foillined Teflon screw caps to avoid evaporation of sorbate, and then agitated in the dark for 3 days at 25 ± 0.5 ◦ C to reach apparent equilibrium. The solution was separated by centrifugation at 4000 rpm for 15 min. The equilibrium concentrations were measured using a high performance liquid chromatography (HPLC, Agilent 1200) with a 4.6 mm × 150 mm reverse phase XDB-C18 column and a fluorescence detector (FLD) using acetonitrile–water (v:v, 90/10) as the mobile phase at a flow rate of 1 mL/min. The individual excitation wavelengths of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene were 240, 240, 220, 244 and 237 nm, while the respective emission wavelengths were 360, 360, 315, 360 and 385 nm. Because of minimal sorption by the vials, and negligible losses by evaporation, biodegradation and photodegradation, the sorbed amount was calculated by mass difference between nominal concentration without sorbent and with sorbent in aqueous solutions. All competitive sorption experiments of phenanthrene and pyrene were conducted at 25 ± 0.5 ◦ C. Bi-solute sorption experiments included sorption of pyrene with 0.5 mg/L phenanthrene as a co-solute and sorption of phenanthrene with 0.05 mg/L pyrene as a co-solute. The remaining procedures of the competitive sorption experiment were performed the same way as described for single-solute sorption experiments. 2.4. Biosorption and biodegradation experiment A 4-day culture of white-rot fungi (CW-1) obtained from pure culture grown in about 200 mL of Martin broth was homogenized in the sterilized conical flask of 500 mL, and was used for inoculating. Five groups were designed: (i) Martin broth without the fungi was used as blank control; (ii) heat-killed fungi + Martin broth, was used as control to study the effect of Martin broth (in contrast to heat-killed fungi in aqueous solutions of the batch sorption experiment); (iii) live fungi + Martin broth (total nutrient solution); (iv) live fungi + sterilized water (without nutrient); and (v) live fungi + Martin broth without glucose. These experimental groups were used to investigate the contributions of biosorption and biodegradation to the removal of PAHs under different nutrient conditions. The 20-mL solution with the concentrations of 1.0 mg/L phenanthrene and 0.1 mg/L pyrene were added to the 40-mL vials. Replicate samples were prepared for each group, including three experimental groups and two controls. After the addition of solution, the 40-mL vials were sealed with aluminum foil-lined Teflon screw caps to avoid sorbate’s evaporation, and then agitated in the dark for 7 days at 25 ± 0.5 ◦ C with aeration for 2 min each day. After 7-day of culture, the vials were centrifuged at 4000 rpm for 15 min. The supernatant was removed and diluted with CH3 CN (1:1), and the residual PAHs in the solution was detected by HPLC–FLD. Then the solution in the vials was decanted as much as possible, and the residual PAHs in fungal biomass were extracted by ultrasonication for 30 min with a 10 mL mixture of acetone and hexane (1:1, v:v) for 3 successive extractions. The extracts were pooled and evaporated to almost dry using rotary evaporator and re-dissolved in 5 mL of hexane, followed by a clean-up procedure through a 2.5-g silica gel column with a 15 mL mixture of hexane and dichloromethane
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Table 1 The selected physico-chemical parameters of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr). Molecular weight (g/mol)
Cs (25 ◦ C)a (mg/L)
Kow b
C10 H8
128.2
31.02
1.95 × 103
Acen
C12 H8
154.2
3.47
8.4 × 103
Flu
C13 H10
116.2
1.69
1.5 × 104
Phen
C14 H10
178.2
1.00
2.8 × 104
Pyr
C16 H10
202.3
0.13
8.0 × 104
PAHs
Molecular formula
Naph
a b
Structural formula
Cs : aqueous solubility at 25 ◦ C. Kow : octanol–water partition coefficients.
(1:1, v:v). Samples were evaporated again and re-dissolved in acetonitrile to a final volume of 4 mL. After filtration through 0.22 mm filter units, PAHs concentrations were determined by the same HPLC method. The removal of PAHs was determined by mass difference in solution concentration between the initial and the final, which includes biosorption and biodegradation. The residual PAHs extracted from fungal biomass was assigned to biosorption amounts, and the biodegradation amounts were determined by mass difference in the total removal and biosorption amounts. The water content of live white-rot fungi measured by dry–wet method was 97.63% (n = 3, RSD <0.36%). 2.5. Data analysis The Freundlich parameters (Kf and N) were calculated using the logarithmic form of the equation Q = Kf CeN , where Q is the amount sorbed per unit weight of sorbent, mg/kg; Ce is the equilibrium concentration, mg/L; Kf [(mg/kg)/(mg/L)N ] is the Freundlich capacity coefficient, and N (dimensionless) describes the isotherm curvature. In biosorption and biodegradation experiment, the average recoveries obtained by spiked fungi samples with phenanthrene and pyrene were 87.1% (n = 6, RSD <1.6%) and 94.8% (n = 6, RSD <2.2%) for the entire procedure of extraction and determination, which applied to data reconciliation of extraction. The intrinsic sorption coefficients (Kd = Q/Ce ) derived from the batch sorption experiment and the apparent partition coefficient (Kd∗ ) derived from biosorption and biodegradation experiment were calculated. The Koc values were calculated by normalizing Kd to the carbon level (foc ) of white-rot fungi (Koc = Kd /foc ). The Kd∗ /Kd ratios were calculated and employed to evaluate relative contributions of biosorption and biodegradation. If Kd∗ /Kd ≈ 1, it may indicate that biosorption governs the bio-dissipation of PAHs; if Kd∗ /Kd < 1, it may suggest that the biodegradation occurs.
carbonyl, carboxyl, sulfhydryl, amide, phosphonate and phosphodiester groups) have been suggested for their contribution to biosorption [25]. These aliphatic and aromatic components and high carbon content may contribute to the biosorption of PAHs by white-rot fungi (CW-1). 3.2. Sorption of PAHs with white-rot fungi Isotherms of five PAHs by heat-killed white-rot fungal biomass (CW-1) are presented in Fig. 2, and the regression parameters are listed in Table 2. Sorption isotherms of naphthalene, acenaphthene, fluorene, phenanthrene and pyrene to heat-killed fungi fit well with the Freundlich equation. Sorption of organic pollutant to biomass may involve simultaneous surface sorption, partitioning processes and chemical reactions. The Freundlich N values were approximately 1, indicating that the primary mechanism of sorption is partitioning into fungal biomass. This is consistent with the previous report about dead bacterial biomass [12,26]. But Stringfellow and Alvarez-Cohen [12] suggested that the biosorption of PAHs by live bacterial biomass involved surface sorption rather than nonspecific partition. Therefore, the physiology of the biomass itself may influence sorption processes. The calculated partition coefficients (Kd ) of PAHs were 361.0 mL/g (naphthalene), 1656 mL/g (acenaphthene), 2890 mL/g (fluorene), 6822 mL/g (phenanthrene) and 24,140 mL/g (pyrene), which show that white-rot fungi (CW-1) have a high affinity for PAHs. A linear correlation was observed between log Koc and
3. Results and discussion 3.1. Characterization of white-rot fungal biomass The elemental compositions of white-rot fungal biomass (CW1) obtained from pure culture were as follows: C 43.70%; N 6.44%; H 7.22%. The FTIR spectrum of the fungal biomass is shown in Fig. 1. The band at 3300 cm−1 represents the stretching vibration of hydroxyl groups. The bands at 2930 and 1400 cm−1 are assigned mainly to CH2 units in biopolymers. The band at 1710 cm−1 is assigned to C O stretching vibration of ester groups. The band at 1650 cm−1 is assigned to C C and C O stretching in the aromatic ring and 571 cm−1 is also assigned to aromatic components. The bands at 1240 and 1060 cm−1 are assigned to C–O stretching of polysaccharides. Numerous chemical groups (e.g., hydroxyl,
Fig. 1. FTIR spectrum of dry biomass of the consortium of white-rot fungi (CW-1).
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Table 2 Regression parameters of isotherms of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr) by heat-killed fungal biomass (CW-1). PAHs
Freundlich regression parametersa N
Naph Acen Flu Phen Pyr Phen (0.05 mg/L Pyr as co-solute) Pyr (0.5 mg/L Phen as co-solute)
Linear parametersb 2
Log Kf
1.119 1.020 0.942 1.062 1.093 1.011 1.155
± ± ± ± ± ± ±
0.027 0.009 0.009 0.016 0.044 0.007 0.070
2.458 3.223 3.449 3.870 4.573 3.848 4.703
± ± ± ± ± ± ±
0.020 0.013 0.010 0.020 0.020 0.008 0.028
Freundlich r
Kd
0.991 0.999 0.999 0.996 0.996 0.999 0.991
361.0 1656 2890 6822 24,140 6971 26,020
r2 ± ± ± ± ± ± ±
2.7 7 38 117 695 106 740
0.999 0.999 0.997 0.995 0.985 0.996 0.986
Koc 826.2 3789 6613 15,610 55,240 15,950 59,540
a The Freundlich parameters (Kf and N) were calculated using the logarithmic form of the equation Q = Kf CeN , where Q is the amount sorbed per unit weight of sorbent, mg/kg; Ce is the equilibrium concentration, mg/L; Kf [(mg/kg)/(mg/L)N ] is the Freundlich capacity coefficient, and N (dimensionless) describes the isotherm curvature. r2 is regression coefficient. b Linear parameters (Kd and Koc ) were regressed, and the intrinsic sorption coefficients (Kd = Q/Ce ) derived from batch sorption experiment, and Koc values were calculated by normalizing Kd to the carbon level (foc ) of white-rot fungi (Koc = Kd /foc ).
log Kow for PAHs to white-rot fungi (see Fig. 3): log Koc = 1.13 log Kow − 0.84
2
(n = 5, r = 0.996)
(1)
According to the linear equation of log Koc –log Kow , biosorption of PAHs to the fungal biomass could be predicted. By contrast, the relationship for PAHs and their derivatives over a comparable range of log Kow (2.11–6.34) on river sediments presented by Karickhoff et al. [27] was as follows: log Koc = 1.00 log Kow − 0.21
(n = 10, r 2 = 0.996)
(2)
The two equations are similar despite different kinds of sorbents (fungi vs. river sediment). Partition coefficient (Kd ) is an effective parameter to estimate the distribution of target chemicals between two phases and hence their fate and mobility in the environment. Based on Kd value, the ranking of sorption efficiency of these sorbents to aqueous PAHs can be estimated [14]. For naphthalene, the log Kd value of the heatkilled fungi–water system was higher than that of soil–water and sediment–water systems due to the higher carbon content of the former [28]. Biotic-origin sorbents have log Kd values of phenanthrene from 2.98 to 4.45 [29], and the determined log Kd value of phenanthrene by the white-rot fungi was 3.83, which was higher than log Kd of cellulose (2.98) and lower than that of humic acid (4.45) [29]. Therefore, biosorption of microorganisms may play an important role in governing the fate of PAHs in soils and sediments.
Fig. 2. Sorption of naphthalene (Naph), acenaphthene (Acen), fluorene (Flu), phenanthrene (Phen) and pyrene (Pyr) to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
Competitive sorption of phenanthrene and pyrene to heat-killed white-rot fungi (CW-1) was investigated. Sorption isotherms of single phenanthrene and phenanthrene with co-solute 0.05 mg/L pyrene were nearly consistent (Fig. 4A) , suggesting that co-solute pyrene had no influence on the sorption of phenanthrene to fungal biomass. Sorption isotherm of single pyrene and pyrene with co-solute 0.5 mg/L phenanthrene showed that co-solute phenanthrene had little effect on the sorption of pyrene to fungal biomass (Fig. 4B). Therefore, simultaneous sorption of phenanthrene and pyrene were compatible and not competitive. However, this is inconsistent with the previous report which suggested that PAHs exhibit competition for surface binding sites on bacterial biomass [12]. 3.3. Relative contributions of biosorption and biodegradation to the removal of PAHs by white-rot fungi Microorganisms play an important role in the fate of PAHs in the environment, including biosorption and biodegradation [12]. The contributions of biosorption and biodegradation of phenanthrene and pyrene to live and heat-killed white-rot fungi (CW-1) in different nutrient solutions are listed in Table 3. For heat-killed fungi (Group II), the biosorption controlled the bio-dissipation of PAHs since biodegradation was not involved. For live fungi, the contributions of biosorption and biodegradation to the total removal of phenanthrene and pyrene under different nutrient con-
Fig. 3. Relationship between log Koc and log Kow of PAHs to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
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849
Table 3 The contributions of biosorption and biodegradation in removal of phenanthrene (Phen) and pyrene (Pyr) by white-rot fungi (CW-1) under different nutrient conditions. Group
Biomass
Solution
Average biomass (mg) Initial
Group I (blank control) Group II (control) Group III Group IV Group V a b
Phen
Final
–
Pyr a
Removal (%) 5.44 ± 0.51
–
b
Biosorption (%) 0
Removala (%) 19.21 ± 2.21
Biosorptionb (%)
None
Culture solution
0
Heat-killed fungi
Culture solution
10.07 ± 0.04
10.07 ± 0.04
82.07 ± 2.09
63.85 ± 11.03
93.87 ± 0.56
86.14 ± 22.77
Live fungi Live fungi Live fungi
Culture solution Sterilized water Culture solution without carbon
6.09 ± 0.60 6.09 ± 0.60 6.09 ± 0.60
53.03 ± 2.85 8.03 ± 0.34 12.52 ± 1.60
75.06 ± 1.14 66.01 ± 1.05 60.44 ± 3.66
53.11 ± 12.25 51.36 ± 2.36 39.01 ± 8.62
91.06 ± 0.33 87.91 ± 1.93 87.73 ± 3.09
79.50 ± 13.24 76.87 ± 5.97 61.93 ± 8.21
Removal: the removal PAHs was determined by PAH mass difference in solution concentration between the initial and the final. Biosorption: the residual PAHs extracted from fungi is assigned to biosorption amounts.
ditions were examined. The removal efficiency of PAHs by live fungi (Group III) approached that of heat-killed fungi (Group II). For all live groups, the removal efficiency of pyrene (90%) was higher than that of phenanthrene (60–80%) because of higher hydrophobicity of pyrene. Secondly, significant differences between the total removal and biosorption (see Table 3) could be used to elucidate the bio-dissipation mechanisms. For phenanthrene, the
Fig. 4. Competitive sorption of phenanthrene (A) and pyrene (B) to the heat-killed white-rot fungal biomass (CW-1) in aqueous solution.
significant difference between the total removal and biosorption (10–20%) suggested that the degradation of phenanthrene occurred under different nutrient conditions, including total nutrient (Martin broth), glucose-free medium (low-carbon Martin broth) and non-nutrient solution (sterilized water). For live fungi in total nutrient solution (Group III) and non-nutrient solution (Group IV), there was no significant difference for pyrene between the total removal and biosorption (the amounts extracted from fungal biomass), indicating that biosorption dominates the bio-dissipation of PAHs in these two nutrient conditions. For live fungi in non-nutrient culture (Group IV), the biodegradation of phenanthrene suggested that white-rot fungi may use phenanthrene as sole source of carbon for growth and thus degrade it. In addition, in glucose-free culture condition (Group V) simultaneous biodegradation of the mixture of phenanthrene and pyrene was observed and the same phenomenon was also reported but the mechanism has not been known yet [14]. Another probable reason was that nutrient limitation in low carbon culture condition promoted release of degrading enzymes such as extracellular lignin-modifying enzymes to facilitate the degradation of PAHs [30]. The bio-dissipation mechanism of phenanthrene and pyrene from solution containing live and heat-killed fungi (CW-1) were further elucidated by the growth of fungal biomass (see Table 3). Previous studies believed that biosorption was facilitated by the exopolysaccharide sheath around the fungal mycelium [13]. For heat-killed fungi, the biomass was constant before and after culture in the total nutrient solution, indicating that there was no metabolization involved in the removal process and supported that biosorption governed the process. For live fungi in total nutrient solution (Group III), the dry fungal biomass increased from the initial mass of 6.09 mg to the final mass of 53.03 mg (i.e., 9 times), which approached 5 times that of heat-killed fungi in total nutrient solution (Group II). Interestingly, for the removal efficiency and biosorption of PAHs there was no significant difference in live and heat-killed fungal systems (Group II and Group III), suggesting that the new fungal biomass was inaccessible to PAHs in aqueous solutions. For live fungi in non-nutrient solution (Group IV), the fungal biomass increased from the initial mass of 6.09 mg to the final mass 8.03 mg. For live fungi in a culture solution with low carbon (Group V), the biomass increased from 6.09 to 12.52 mg. These observations proved that phenanthrene and pyrene can be used as carbon sources by white-rot fungi. Furthermore, in long-term experiments (56 days) for all live groups, although the total removal increased, the PAHs stored in fungal biomass were almost constant. This suggested that biosorption limits the availability of PAHs for biodegradation, which works extracellularly by released lignin degrading enzymes to degrade lignin-like PAHs [31]. The reduced bioavailability by biosorption may contribute to the reduced biodegradation by live fungi in total nutrient solution (Group III) with high biomass than that achieved in glucose-free culture medium (Group V) with low biomass. These
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Table 4 Apparent partition coefficients (Kd∗ ) in culture systems and partition coefficient (Kd ) in batch sorption experiment of phenanthrene (Phen) and pyrene (Pyr), and the ratio of Kd∗ /Kd . Parameter
Sorbate
Heat-killed fungi (Kd )
Kd∗
Phen Pyr Phen Pyr
6822 24,140 1.000 1.000
Kd∗ /Kd a b c d
Controla 7174 28,250 1.052 1.170
Group IIIb 809.1 3369 0.119 0.140
Group IVc 3777 16,070 0.554 0.666
Group Vd 1588 8283 0.233 0.343
Control: heat-killed fungi in culture medium. Group III: live fungi in culture solution. Group IV: live fungi in sterilized water. Group V: live fungi in culture solution without carbon.
Fig. 5. The schematics of the correlations of biosorption and biodegradation with sorption parameters in live and dead fungal systems (CW-1) according to Kd∗ /Kd ratios.
results supported that the activated sludge plant effluent solids may be a significant source of PAH release to the environment [12]. 3.4. Comparison of partition coefficients between live and heat-killed white-rot fungi The apparent partition coefficients (Kd∗ ) and Kd∗ /Kd ratios for phenanthrene and pyrene were calculated and presented in Table 4. The Kd∗ were calculated as follow: Kd∗ =
Q∗ mPAHs /mbiomass = Ce∗ Ce∗
(3)
where Q* and Ce∗ are the PAHs apparent sorbed-amount and aqueous equilibrium concentration, respectively, in the culture system. Q* = mPAHs /mbiomass , where mPAHs is the PAHs amount stored in biomass, and mbiomass is the final fungal biomass. Based on Eq. (3), the Kd∗ value will decrease due to the biodegradation of PAHs and increase in fungal biomass (CW-1). Firstly, the Kd∗ value and the Kd value for heat-killed fungi were comparable with and without the presence of Martin broth in Group II and in the batch sorption experiment, indicating that the Martin broth has no effect on the sorption of PAHs. Secondly, Kd∗ of PAHs by live fungi in total nutrient solution (Group III) was almost 8 times less than the Kd value of heat-killed fungi (Group II), which mainly attributes to growth dilution of fungal biomass, i.e., the final biomass of live fungi was about 8 times higher than the initial and 5 times that of heat-killed fungi. Thirdly, Kd∗ by live fungi in non-nutrient solution (Group IV) was half of Kd of heat-killed fungi, while the final fungal biomass was about one third higher than the initial and four fifth that of heatkilled fungi. Presumably, the difference of Kd∗ (or Kd ) between live fungi in non-nutrient solution and heat-killed fungi may ascribe to small growth of fungi and its biodegradation. Finally, though the final biomass was twice the original and one fifth more than that of heat-killed fungi, Kd∗ of phenanthrene and pyrene by live fungi in glucose-free culture solution (Group V) was about a quarter of Kd of heat-killed fungi, mainly due to biodegradation of PAHs.
The correlations of biosorption and biodegradation with sorption parameters in live and dead fungal systems (CW-1) are demonstrated in Fig. 5. If Kd∗ /Kd ≈ 1, it indicated that biosorption governs the fate of PAHs, while if Kd∗ /Kd < 1, it may suggest that the biodegradation occurs. For phenanthrene and pyrene with heat-killed fungi in total nutrient solution (Group II), Kd∗ /Kd ≈ 1 suggested that the biosorption plays a critical role in the transport and fate of PAHs. For phenanthrene with live fungi in different nutrient conditions, Kd∗ /Kd < 1 may attribute to growth dilution of fungal biomass and its biodegradation. Live fungi in the nonnutrient solution degraded phenanthrene by utilizing it as the sole source of carbon, while simultaneous biodegradation of pyrene and phenanthrene occurred in Martin broth without carbon (Group V). However, for pyrene with live fungi in the total nutrient solution (Group III), Kd∗ /Kd < 1 was mainly ascribed to the increase of mbiomass of the final fungal biomass (i.e., growth dilution). Similar results were shown by Raghukumar et al. [13]. Therefore, Kd∗ /Kd ratios and fungal biomass are important parameters to distinguish the relative contributions of biosorption and biodegradation to total bio-dissipation of PAHs from aqueous solutions. 4. Conclusions Biosorption of PAHs by white-rot fungi is very important process influencing the fate of PAHs in the environment. The partitioning of PAHs into fungal biomass (CW-1) is the primary mechanism of biosorption, and sorption capabilities (Koc ) are linearly related with Kow . Simultaneous sorption of phenanthrene and pyrene is not competitive. Both biosorption and biodegradation of PAHs play a crucial role in the removal of PAHs, whereas the extent of biosorption and biodegradation vary with nutrient conditions. Biodegradation of phenanthrene occurs in all of different nutrient conditions, while simultaneous biodegradation of phenanthrene and pyrene occurs only in the glucose-free culture solution. Whiterot fungi may use phenanthrene and pyrene as the source of carbon for growth under nutrient-limited conditions. Based on analysis of biosorption and biodegradation experiment, Kd∗ /Kd ratios can be
B. Chen et al. / Journal of Hazardous Materials 179 (2010) 845–851
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