Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite

Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite

ISSN 1001-0742 J o d of Environmental Sciences Vol. 18, No. 6, pp. 1204-1209,2006 Article ID: 1001-0742(2006)06-1204-06 CN 11-2629iX CLC nnmber:X1...

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ISSN 1001-0742

J o d of Environmental Sciences Vol. 18, No. 6, pp. 1204-1209,2006

Article ID: 1001-0742(2006)06-1204-06

CN 11-2629iX

CLC nnmber:X172; X144

Document code: A

Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite SU Dan'J, LI Pei-jun'?, FRANK Stagnitti3,XIONG Xian-he' (1. Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. E-mail: [email protected]; 2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China; 3. School of Life and Environmental Science, Faculty of Science and Technology, P. 0, Box

423, WarrnamboolVictoria 3280, Australia)

Abstract: Two indigenous microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, capable of degradmg polycyclic aromatic hydrocarbons (PAHs) were co-immobilized on vermiculite by physical adsorption and used to degrade benzo[a] pyrene (BaP). The characteristics of BaP degradation by both h e and co-immobilized microorganism were then investigated and compared. The removal rate using the immobilized bacterial-fungal mixed consortium was higher than that of the fieely mobile mixed consortium. 95.3% of BaP was degraded using the co-immobilized system within 42 d, which was remarkably higher than the removal rate of that by the t7ee strains. The optimal amount of inoculated co-immobilized system for BaP degradation was 2%. The immobilized bacterial-bgal mixed consortium also showed better water stability than the fiee strains. Kinetics of BaP biodegradation by co-immobilized SF06 and SB02 were also studied. The results demonstrated that BaP degradation could be well described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10-200 mgkg. The scanning electronic microscope (SEM)analysis showed that the co-immobilized microstructure was suitable for the growth of SF06 and SB02. The mass transmission process of co-immobilized system in soil is discussed. The results demonstrate the potential for employing the bacterial-fungal mixed consortium, co-immobilized on vermiculite, for in sku bioremediation of BaP. Keywords: biodegradation; Bacillus sp. SB02; Mucor sp. SF06;benzo[a]pyrene; immobilization; soil pollution

Introduction Polycyclic aromatic hydrocarbons (PAHs) consist of two or more fused aromatic rings in linear, angular or clustered arrangements (Sims and Overcash, 1983). They are common, persistent and recalcitrant environmental contaminants with a tendency to bioaccumulate. Many PAHs are toxic, carcinogenic and/or mutagenic (Crawford and Crawford, 1996). Sixteen PAHs are listed as priority pollutants by the US EPA including benzo[a]pyrene (BaP) (Juhasz and Naidu, 2000). Soil and sediment are the major sinks for their accumulation (Kotterman et d.,1998) with PAHs contamination ranges from 5 ng/g soil in an undeveloped areas to 1.7 X lo6ng/g at a spillage in an oil refinery. The clean-up criteria for PAHs in soil vary widely within and between nations. For example, the Dutch standard for threshold levels are 1 to 10 mg/kg and from 0.2 to 7 Fg/kg for different PAHs (Wilson and Jones, 1993). Although PAHs may undergo adsorption, volatilization, photolysis and chemical oxidation, microbial degradation is the major decomposition mechanism (Bogan and Lamar, 1995; Kilbane, 1998; Yuan et d., 2001). Bacteria may possess PAH catabolic pathways presumably capable of degrading BaP, but degradation of this compound may not occur due to the inability of the compound to pass through bacteria cell walls (Hammel et d.,1986). However, these limitations do not apply to fungi due to their

ability to produce extracellular enzymes (lignindegrading enzymes). Fungal degradation of BaP does not result in significant mineralization of the compound, however, ligninases convert BaP to water soluble products which are more bioavailable (Meulenberg et d.,1997) and potentially more toxic (Cerniglia et d.,1980) than the parent compound. As the ability of the organisms to hydroxylate the compound, the initial transformation of polar metabolites could lead to an effective strategy for BaP degradation. Co-culture (e.g. bacterial and fimgal cultures) studies have demonstrated the potential for the mineralization and detoxification of BaP. For example, Kottermann et d. (1998) investigated BaP degradation by successively incubating BaP with the mixed cultures of micro-organisms from soil, sediment and sludge. Despite the abundknce of promising experimental data, a number of limitations still restrict the use of free microorganisms to remove PAHs in terrestrial systems. Firstly, organisms having the potential to degrade PAHs, may not be prevalent in soil where PAHs/BaP remediation is required (Juhasz and Naidu, 2000). Secondly, the die-off of laboratory-adapted strains and the inability of inocula to compete with the indigenous microflora are also reasons for limited success (Mueller et d.,1989). Thirdly, the extent of PAHs degradation by these organisms may be limited to the production of polar metabolites, which often may pose even a greater toxicological threat due to

Foundation item: The National Basic Research Program (973) of China (No. 2004CB418506); the National Natural Science Foundation of China (No. 20337010) and the Hi-Tech Research and Development Program (863) of China (No. 2004AA649060); *Correspondingauthor


Biodegradation ofbenzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized ......

their increased toxicity, solubility and mobility (Juhasz and Naidu, 2000). Fourthly, the degradative performance of any inocula will depend on soil types and environmental conditions which may not be easily controlled in the field (Kanaly and Harayama, 2000). Delivering such bacterial-fungal co-cultures in an immobilized form may offer more complete and/or more rapid degradation. Immobilization is known to reduce competition with indigenous microorganisms (Lin and Wang, 1991), offer protection fiom predation, extremes of pH and toxic compounds in the contaminated soil (Pritchard, 1992). There is also evidence of increased biological stability, including plasmid stability, in immobilized cells (Cassidy et al., 1996). In this application, the immobilized matrix may also act as a bulking agent in contaminated soil, facilitating the transfer of oxygen crucial for rapid hydrocarbon mineralization. Immobilization has been achieved in a number of ways either by physical adsorption, ion-coagulation, cross-linking and/or entrapment methods in a variety of matrices. Of those four immobilization methods, physical adsorption on vermiculite was found to be the optimal method for bacterial-hgal mixed consortium co-immobilization in our experiments. The use of natural supports, such as vermiculite or clay represents an attractive option for the treatment of PAHs contamination. In a previous study, bacterial-fungal mixed consortium consisting of two indigenous strains, Mucor sp. SF06, and Bacillus sp. SB02, were screened with the purpose to biodegrade PAHs in contaminated soils. For the present work, mixed bacterial-fungal consortia were immobilized on vermiculite with the objective to determine the correct mixture of bacteria and fungi to optimize the remediation of soil polluted with BaP. We also evaluated the performance of degradation by the co-immobilized system compared with its fiee form counterpart. Subsequently, the morphology and the mass transmission process of the co-immobilized system were observed using scanning electron microscopy (SEM).

1 Materials and methods 1.1 Microorganism cuttivation Both SF06 (Mucor sp.) and SF02 (Bacillus sp.) were isolated fiom soil fiom the Shenfu Irrigation Area (Liaoning Province of China). The seed medium used for supply of abundant mixed consortium of bacteria and hngi for co-immobilization contained ( g L )potato extract 200, saccharose 20, beef extracts 2.0, NaCl 5.0, and was sterilized by autoclave at 115°C for 30 min. The proliferation medium used for multiplication of co-immobilized mixed consortium contained (gL) saccharose 40, yeast extract 3.0, 2.0, MgS04 HzO 0.25, and m2Po4 0.5, (N&)2mo4


was sterilized by autoclave at 115°C for 30 min. The pH of the two media was adjusted to fall fkom 6.0 to 6.5. A constant temperature incubator (DHP-SOOBS, China) was used for cultivation and the biodegradation experiments. 1.2 Carrier materials and immobilization method Vermiculite with a diameter of 0.5-1.0 cm was chosen as the carrier material for immobilization, and was purchased fiom the Shenyang Ecological Station. An FT-IR analysis was carried out to reveal the functional groups in vermiculite (Guibal et al., 1995). Twenty grams vermiculite was weighed and placed into a 50-ml culture jar spiked with 20 ml of nutrient medium and autoclaved at 121T for 30 min. The mixed consortium consisting of 3 d old SB02 and SF06 were scraped from the agar slant and inoculated into the culture jar with sterilized vermiculite. This was then incubated in a constant temperature incubator at 28°C. A 5-ml proliferation medium was added into the co-immobilized system every day and inoculated for 3 d under identical cultivation conditions. 1.3 Soil The soil used in this study was collected fiom the top 10 cm from the Shenyang Ecological Station in the Liaoning Province of China. The brown soil consisted of: TOC, 1.78%; TN,0.1 1%; TP, 0.035%; TK 0.604% and pH 6.8. Prior to testing, soils without detectable PAHs were air-dried, passed through a 2-mm sieve, and stored at 4°C. 1.4 Degradation of BaP in soil The target PAHs was BaP dissolved in HPLC grade acetone and kept at -20% until required. 30.00 g sterilized soil was spiked with 5 ml of acetone containing BaP. The acetone was alIowed to evaporate for 2 h at room temperature. Then co-immobilized SB02 and SF06 or an equal quantity of free microorganisms were added to the soil samples and the moisture content of soil was adjusted to different levels (30% , 55% , or 100% of maximum water-holding capacity (WHC) which was 380 g/kg of soil). The sample was mixed and incubated in a darkened drawer at 25°C for the duration of the experiment. As a control media without microorganism (sterile vermiculite) was also prepared and used to exclude the possibility that a decrease of the compound concentration was due to an adsorption of the compounds and not to a biodegradation by the mixed consortium being investigated (designated as control). All the treatments were mixed manually once per week to enhance oxygenation, and kept moist. Samples were taken at zero-time and at intervals to 42 d. Stabilities of the BaP in the soil were monitored on the spiked soil samples without vermiculite. Over 90% extraction efficiencies were obtained with the BaP. Five replicates were performed for each treatment.


SU Dan et al.

1.5 High performance liquid chromatography (HPLC)analysis of degradation BaP concentrations of all samples in this work were analyzed by HPLC (HP 1090 II, USA). Five volumes of dichloromethane were added to one volume of the BaP sample. Extraction was performed in a Branson ultrasonic cleaner at 50 Hz for 2 h. After centrifugation at 3000 r/min for 15 min at room temperature, the organic phase was collected. The sample was extracted twice with dichloromethane. Two dichloromethane extracts were pooled together and evaporated dry using nitrogen gas. The residue was then re-dissolved in 1 ml of HPLC grade methanol and filtered by 0.45 km PTFE membrane into a borosilicate GC vial before storage in darkness at -2OT until further analysis. The column temperature was 40T and the liquid phase flow rate was 0.80 pVmin, the volume of the injected samples was 10.0 p1, and the detection limit was 1 ng. 1.6 SEM examination of co-immobilized system SB02 and SF06 co-immobilized on vermiculite were fixed in 2.5% glutaraldehyde in 0.1 m o w phosphate buffer for 3 h, rinsed twice for 10- 15 min each in phosphate buffer and post-fixed in 1% osmium tetraoxide buffer for 1-2 h at 4°C. The vermiculite was then washed twice in phosphate buffer for 10-15 min each at room temperature and dehydrated with a series of ethanol (30%, 50%, 70%, go%, 90% and 100% ) at room temperature. The vermiculite was dried using the C02 critical point technique (Hitachi Critical Point Dryer-HCP 2, Japan) and sputter-coated with gold (Eiko E3 5 Ion Coater, Japan). Observations were done using a SEM (JSM-300, Japan).

2 Results and discussion 2.1 Structure of the vermiculite Fig. 1 is an infrared spectrum (between 4000 and 400 cm-') of the vermiculite without Mwor sp. or Bacillus sp. A large and broad absorption band was 'observed between 3800 and 3000 cm-' due to the presence of -OH groups. The absorption band at 1646 cm'l probably indicated C=O bonds (Lau et al., 2003). The peak located at around 1001 cm-' corresponded to -NH groups (Guibal et al., 1995). These fimctional groups in the vermiculite served as biosorption sites for bacteria and fungi. Vermiculite had excellent absorbent properties and a good cell-loading capacity. In order to assess the immobilization of the two microorganisms on vermiculite, the enumeration of immobilized viable cells on vermiculite were observed. There were 10I2 CFU (colony forming units)/g and lo6 CFU/g of immobilized SB02 and SF06 cells, respectively, on vermiculite. The number of immobilized cells remained fairly constant throughout the period of immobilization for these microbes. The results

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showed that the mixed consortium was efficiently co-immobilized on vermiculite in terms of the number of immobilized cells. Hence, vermiculite was used in the immobilization of mixed consortium in subsequent experiments in this work. Chellapandian (1998) also chose vermiculite as carrier to immobilized alkaline protease by covalent linkage. Although the exact mechanism may be complex and varied, the natural ability of microorganism to attach to vermiculite is nevertheless an advantage if co-immobilization is to be scaled up.


-20 4000


W a v e l e n ~0m-l ,

Fig.1 An idinred spectnun of vermiculiteWithout the microorganisms

2.2 Comparison of BaP degradation in soil by free and co-immobilized system A comparison of the biodegradation of BaP in control samples (without introduced microorganisms) and in samples with both free and co-immobilized SB02 and SF06 is shown in Table 1, which shows that BaP was more quickly degraded by the co-immobilized system than that by the free strains. Under the experimental conditions of 2% co-immobilized mixed consortium treating 50 mflg BaP at 2 5 T , biodegradative removal reached 95.3% after 42 d. In contrast the highest sorption removal was only 17.2% BaP (Garrigues and Lamotte, 1991). There are some possible reasons for this, such as both biotic e.g. predation by protozoa and abiotic (e.g. influence of local chemistry or UV irradiation). This subject has been previously considered and reviewed (Veen et al., 1997). One of the possible advantages of vermiculite encapsulation is that mixed consortium are protected Table 1

Removal effidency of BaP in soil by free or co-

immobilized of SB02 and SFM' Removal efficiency Time, d



Control %










































Notes: *Initial BaP concentrationwas 50 mgkg, pH 6.8, and 55% of the soil water-holding capacity, inoculation and growth conditions are described in the text

Biodegradation ofbenzo[a]pyrene in soil by Mutor sp. SF06 and Bacillus sp. SB02 co-immobilized


from such deleterious effects. There are studies reporting that immobilized cells showed a faster degradation rate than ftee cells (Wang and Qian, 1999; Yamaguchi et d.,1999; Na et d., 2000; Manohar et d.,2001). This was most likely due to the high immobilization efficiency of the cells onto the immobilization material and high affinity between the hydrophobic immobilization material and substrates (Wilson and Bradley, 1996). These lead to an increased availability of the substrates for the cells and a better interaction between the substrates and immobilized cells, synergistically enhancing the degradation rate (Diaz et d.,2002). Co-immobilized system could degrade about 95% of BaP after 6 weeks (Table 1). No significant increase in degradation was observed after this time(data not shown). These results suggested that co-immobilized system could degrade ex situ BaP. It also suggests the possibility of using co-immobilized bacteria and h g i for in situ degradation of PAHs 2.3 Effect of initial inoculation amount on biodegradation The inoculation amount of co-immobilized SB02 and SF06 on the BaP degradation was also investigated and the results are summarized in Fig.2. It shows the most efficient level of inoculation occurred at approximately 2% . Below this, the removal efficiency dropped markedly and above 2% incoluation, the removal efficiency did not statistically improve. Thus, the optimal amount of inoculated co-immobilized mixed consortium was 2%. 100 I













co-immobilized system (Fig.3). During incubation

with co-immobilized SB02 and SF06, the soil moisture had little effect on BaP disappearance. Under 30% WHC the disappearance of BaP was somewhat enhanced in the presence of co-immobilized system, and the co-immobilized microorganism could function at a wide range of moisture conditions. 100





40 20 n "



14 t, d





4Cu-immobilized microorganism at 30% WHC -A- Free microorganism at 30% WHC f Co-immobilizcd microorganiemat 55% WHC -8Free microorganism at 55% WHC tCo-immobilhd microorganism at 100% WHC Free rnicroorgadsm at 100%W C

Fig.3 BaP disappearanceby fkee and co-immobilized of Mucor sp. and

Bacillus sp. under different moisture conditions (30, 55, or 100% of maximum water-holdingcapacity, WHC) Initial BaP concentration was 50 [email protected], pH 6.8, the control was BaP-polluted soils with vermiculite and without microorganisms, and inoculation and growth conditions are described in the text

2.5 Effect of different initial BaP concentrations on degradation rate The biodegradation of BaP by fiee or co-immobilized SB02 and SF06 was investigated using various initial concentrations of BaP. The results in Fig.4 show that when the initial concentrations of BaP were between 10 and 400 mgkg, the BaP could degraded by either the free or the co-immobilized microorganism. However, a higher concentration than 400 [email protected] of BaP could not be degraded within 42 d





1 2 3 Initial amount, %


F i g 2 Removal efficiency of BaP by various amounts of inoculated co-immobilized SB02 and SF06 within 42 d Values represent the mean of three replicates; bars indicate standard deviation of the mean

2.4 Effect of moisture conditions on biodegradation Increasing the soil moisture was expected to: (1) enhance the BaP transformation by facilitating microorganism contact with the substrate, and (2) reduce the inhibitory effect of soil organic matter following its dilution with water (Ahn et d.,2002). Increasing water content in soil had a significant effect on the disappearance of BaP in the presence of fiee mixed consortium consisting of SB02 and SF06, but it did not have any effect in the presence of




8 60





c4 0







1, d

Fig.4 Removal of free and co-immobilized microorganisms for different initial concentrations of BaP under 55% of maximum water-holdingcapacity (WHC) Symbols:(o) free microorganism for BaP 10 mgkg; ( m ) co-immobilized for BaP 10 mgkg; (A) free microorganism for BaP 50 mgkg; (A)co-immobilizedfor 50 mgkg; ( 0 )t h e microorganism for BaP 100 m a g ; ( 0 )co-immobilized for BaP 100 mgikg;(o) free microorganism for BaP 200 mgkg; (+) co-immobilized for BaP 200 m a g ; (-) free microorganism for BaP 400 gmnCg; (+) co-immobilized for BaP 400 mag


SU Dan et al.

by fiee system (data not shown), moreover, the co-immobilized system also needed a longer lag time to accommodate the high concentration of BaP before starting to degrade it. According to kinetic analysis (Wang et d., 2002), the concentration (C) verses time ( t ) (C-t) curve and the In (C-t) curve were calculated to fit the data in Fig.4. The biodegradation kinetics of BaP by SB02 and SF06 co-immobilized on the vermiculite can be described well with a zero-order kinetic model when initial BaP concentrations were 10, 50, 100, and 200 mg/kg, respectively. The zero-order realtionship breaks down at higher initial concentrations of 400 mgkg. The kinetic results of BaP biodegradation in Table 2 indicate that BaP was utilized by the


Table 2 Kinetic results of BeP biodegradation by SB02 and SF06 co-immobilized on vermiculite Biodegradation rate constant (k3, mg/(kg-4

Correlation coefficient

C=-0.2 1t+7.73















Initial conc.,

Kinetic equation





co-immobilized SB02 and SF06 at a constant rate when the initial concentration of BaP was less than 400 mg/kg.

Fig.5 The scanning electronicmicroscope(SEM) micrograph and mass transmissionprocess of co-immobilizedSB02 and SF06 on vermiculite (a)X750; (b)X 1500; (c)X 1500; (d)X 10000; (e)X 10000; (OX 1500

2.6 Microenvironment and mass transmission of co-immobilized system Co-immobilization of SB02 and SF06 were examined under the SEM shown in Fig.5. FigSa shows the relationship between mycelium of SF06 and vermiculites. FigSb shows fungal spores spiked into the surface of vermiculite resulted in the mass transfer

among vermiculites and/or soil. Figs% and 5d show the adsorption of SF06 h g a l spore and the SB02 cells on the vermiculite surface at the same time, some of SB02 cells were also absorbed by fungal spores. FigsSe and 5f show SB02 absorbed on the mycelium of SF06 and moved fkeely with the growth of h g a l mycelium of SF06. The SEM analysis clearly


Biodegradation ofbenzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized ..-...

demonstrates that SB02 and SF06 were readily co-immobilized on the vermiculite by adsorption and entrapment permitting excellent mass diffusion of substrate and oxygen. It can also be seen that SB02 and SF06 were well distributed in the inner and outer surface of the matrix. Numbers of microorganisms added to the vermiculite have to be such that growth can subsequently occur. Growth and replication within the vermiculite matrix may release some bacteria and fungi to the surrounding environment. The effect on bioremediation would thus be two-fold: an increase in rate within the protected, vermiculite environment, and an increased rate within the contaminated soil due to slow release of bacterial and fungal. The growth of Mucor sp. and BaciZZzu sp. during incubation provides an obvious explanation for the increase in the degradation of BaP with successive utilization of co-immobilized system.

3 Conclusions Co-immobilized SB02 and SF06 with vermiculite as a carrier showed significant advantages over free strains. The BaP degradation rate was 95.3% within 42 d for co-immobilized system but only 79.6% for fiee strains. The optimal amount of inoculated co-immobilized microorganism was 2% in batch experiments. The co-immobilized system had excellent tolerance to water changes, and could function in a wide range of moisture conditions. BaP degradation could be described by a zero-order reaction rate equation when the initial BaP concentration was in the range of 10 to 200 m a g . The SEM analysis demonstrated advantages of the immobilized microstructure and the process of mass transmission. In a conclusion, the use of co-immobilized bacterial-fungal strains for the remediation of PAHs-contaminated soil in situ is a promising technology. Acknowledgements: The authors would like to thank the reviewers for their comments and ,JU Jing-li for technical laboratory support.

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