R ESEARCH ARTICLE

doi: 10.2306/scienceasia1513-1874.2012.38.147

ScienceAsia 38 (2012): 147–156

Degradation of polycyclic aromatic hydrocarbons bynewly isolated Curvularia sp. F18, Lentinus sp. S5, andPhanerochaete sp. T20Kanokpan Juckpecha, Onruthai Pinyakonga,b, Panan Rerngsamrana,b,∗

a Bioremediation Research Unit, Department of Microbiology, Faculty of Science, Chulalongkorn University,Bangkok 10330 Thailand

b National Centre of Excellence for Environmental and Hazardous Waste Management (NCE-EHWM),Chulalongkorn University, Bangkok 10330 Thailand

∗Corresponding author, e-mail: panan.r@chula.ac.thReceived 9 Nov 2011

Accepted 19 Apr 2012

ABSTRACT: Three chromogenic substances with structures resembling those of polycyclic aromatic hydrocarbons (PAHs)were incorporated in culture medium in order to screen for fungi capable of degrading PAHs. Curvularia sp. F18,Lentinus sp. S5, and Phanerochaete sp. T20 were isolated and shown to have the ability to degrade both low- and high-molecular weight PAHs, with the most prominent degradation being observed with Phanerochaete sp. T20. Preliminarymetabolite analysis of fluorene degradation by Phanerochaete sp. T20 using HPLC and GC-MS revealed that one of theearly metabolites was 9-fluorenol, which is a less toxic substance. This fungus survived in 500 mg/l of PAH for at least30 days. The fungus could degrade a mixture of four PAHs (25 mg/l each), resulting in the reduction of 97, 59, 39, and47% of fluorene, phenanthrene, fluoranthene, and pyrene, respectively. This work demonstrates that Phanerochaete sp. T20could be used to bioremediate environments contaminated with high concentrations and/or mixtures of PAHs.

KEYWORDS: biodegradation, bioremediation, fungi, mixed PAHs

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are a groupof environmental pollutants that are composed ofcarbon and hydrogen with fused benzene rings inlinear, angular, and clustered arrangements. Basedon the molecular weight of these hydrocarbons, PAHscan be classified into two broad groups: (i) the low-molecular weight PAHs that contain 2–3 benzenerings, such as naphthalene, fluorene, and phenan-threne, and (ii) the high-molecular weight PAHs, suchas fluoranthene, pyrene, and chrysene1. PAHs aregenerated as byproducts of incomplete combustionof organic substances, which are found in burnt fos-sil fuels, forest fires, volcano eruptions, and motorvehicle emissions, as well as in grilled and smokedfoods2. PAHs can also be found as contaminantsat industrial sites, especially those associated withpetroleum or gas production and wood preservingprocesses3, 4. PAHs and their metabolites are reportedto possess mutagenic and carcinogenic properties forhumans and other animals5, 6. Consequently, theUS Environmental Protection Agency has listed somePAHs as priority pollutants1. Generally, the high-molecular-weight PAHs are less water-soluble and

more recalcitrant to degradation than low-molecular-weight PAHs7. Investigations of the content of PAHsfound in several contaminated areas reveal that con-tamination is the result of a mixture of PAHs ratherthan a single type of contaminant4, 8–10. Due to thelong half life of PAHs and the human activities thatcause the emissions of these contaminants into theenvironment every day, PAHs continuously increaseand accumulate in the soil, water, and sediments andthus appropriate treatment is required to reduce theconcentration and toxicity of these substances.

Chemical methods, such as chemical oxidationand liquid solvent extraction, and physical meth-ods, such as incineration and microwave energytreatments, have been shown to have high levelsof efficiency in remediating sites contaminated withPAHs11. However, these methods require complextechnologies, have high treatment cost, tend to useexcessive amounts of organic solvents, and may harmliving organisms12. Bioremediation, a safe, envi-ronmentally friendly, and effective method, uses theability of organisms, such as bacteria, fungi, algae,or plants, to reduce the concentrations of PAHs toan acceptable level by transforming them into lesstoxic forms or to completely mineralize them into

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CO213. Fungi have advantages over other organisms

in that they produce classes of enzymes, such as ligninperoxidase, manganese peroxidase, and laccase, thatcan interact with several types of PAHs with a fairlyhigh degree of non-specific activity14. They also haveother enzymatic systems, such as cytochrome P450monooxygenase and epoxide hydrolase that oxidizePAHs15, 16. Fungi are also tolerant to high concen-trations of recalcitrant compounds and are able toflourish in extreme conditions, such as at high temper-atures and under low pH conditions. In addition, thefact that fungi form large, branching mycelia makesit possible for them to grow and distribute througha solid matrix to degrade PAHs within contaminatedareas (in situ) by virtue of secreting extracellularenzymes or by sequestration of PAHs17–19. Fungi canalso degrade PAHs under microaerobic and very-low-oxygen conditions20. In addition to biodegradationand mineralization of the PAHs, fungi adsorb PAHsonto their hydrophobic cell wall21 and/or store themin vacuoles or other organelles inside the cells22, 23.In combination, all of these mechanisms lead to thereduction of PAHs in the environment. Several reportshave demonstrated that fungi, such as Phanerochaetechrysosporium, Cunninghamella elegans, Trametesversicolor, Bjerkandera adusta, and Pleurotus ostrea-tus, play an important role in the degradation of a widevariety of xenobiotic compounds, including PAHs18.Most of the current research in the field has studiedthe ability of a specific fungus to degrade a particularPAH compound24–27. However, contaminated sitesare commonly contaminated with a mixture of PAHs.Therefore, the objective of this study was to screenfor fungi that could degrade a mixture of four PAHs,including fluorene and phenanthrene, and fluoran-thene and pyrene, as representatives of low- and high-molecular weight PAHs, respectively. These fungihave the potential to be used for in situ bioremediationat environmental sites where contamination is causedby several types of PAHs, a more common scenario.

MATERIALS AND METHODS

Polycyclic aromatic hydrocarbons (PAHs)

Fluorene was obtained from the Wako Pure ChemicalIndustries Co. (Japan). Fluoranthene was obtainedfrom the Kanto Chemical (Japan). Phenanthrene,pyrene, benomyl, and the three chromogenic PAH-like substances (guaiacol, azureB, and phenol red)were obtained from the Sigma-Aldrich Co. (USA),9-fluorenone was obtained from Nacalai Tesque Co.(Japan), and 9-fluorenol was obtained from the TCICo. (Japan). All chemicals were of analytical grade.

Media

Two percent malt extract agar (MEA), used for thepreliminary isolation of fungi, contained (per litre):20 g malt extract, 5 g peptone, 20 g glucose, 15 g agar,3 mg benomyl, and 50 mg chloramphenicol28.

Minimal medium (MM), used for peroxidase en-zyme screening, contained (per litre): 0.5 g KH2PO4,0.5 g MnSO4, 0.1 g NH4NO3, 18 g agar, and 200 mlof trace elements solution containing the followingreagents (per litre): 5 g Na2EDTA, 0.5 g FeCl3, 0.05 gZnCl2, 0.01 g CuCl2, 0.01 g CoCl2 · 6 H2O, 0.01 gH3BO3, and 1.6 g MnCl2 25.

Modified GPY (mGPY), used for the preparationof fungal inocula, contained (per litre): 10 g glucose,3 g peptone, 2 g yeast extract, 1 g KH2PO4, 1 gMgSO4 · 7 H2O, and 0.4 g Na-tartrate25.

N-limited medium, used for the biodegradationexperiments, contained (per litre): 10 g glucose,0.1 g NH4NO3, 1 g KH2PO4, 1 g MgSO4 · 7 H2O,0.01 g FeSO4 · 7 H2O, 0.01 g ZnSO4 · 7 H2O, 0.001 gMnSO4, and 0.001 g CuSO4 · 5 H2O29.

Fungal isolation

Wood-rot fungi or mushrooms growing on rottenwood and soil contaminated with petroleum oil werecollected from five provinces in Thailand (Bangkok,Chonburi, Nakhon Pathom, Phatthalung, and Ra-yong). Pieces of the inner tissue of the mushroomor rotten wood fungi were placed on MEA mediacontaining 3 mg/l benomyl and 50 mg/l chloram-phenicol to inhibit fast-growing fungi and bacteria,respectively. Fungi from soil samples were isolatedusing the soil dilution plate technique on MEA mediabearing benomyl and chloramphenicol. All plateswere incubated at room temperature for 5–7 days.The fungi were isolated as pure cultures using thesame media. The purified isolates were kept as stockcultures at 4 °C on MEA slants until used.

Screening for potential fungi using chromogenicsubstances

0.1% Guaiacol, 0.1% azureB, and 0.0025% phenolred were used for the screening of potential fungi thatcould produce peroxidase and laccase enzymes30–33.Three 7-mm agar plugs containing fungal myceliafrom 7-day-old cultures on MEA plates were placedon an MM plate that contained each chromogenicsubstance. All MM plates were incubated in thedark at room temperature for 3 days. The fungi thatwere able to change these chromogenic substances,as determined by visual appearance of a differentcoloured halo around the fungal colony, were selected

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for further study.Fungal identification was performed by ITS1 - 5.8

RNA - ITS2 DNA sequence identity using standardconditions and primers ITS1 and ITS4 or ITS1-Fand ITS434. Direct sequencing of both strands ofeach purified amplicon was commercially performedby 1st BASE DNA Sequencing Service (Malaysia).The consensus nucleotide sequences were comparedto those available in the GenBank database using theBLASTn algorithm.

Degradation of PAHs in liquid medium andmetabolite analysis

Fungi that exhibited positive results from the screen-ing step were prepared for inocula in 100 ml of mGPYliquid medium at 30 °C with shaking at 120 rpm for5 days. Mycelia were harvested by centrifugation at1120g for 15 min and washed twice with 0.85% (w/v)NaCl. Three grams of fresh mycelia were added into30 ml of N-limited medium in 125-ml Erlenmeyerflasks containing three glass marbles. Each PAH(100 mg/l) was added to these cultures and shakenat 120 rpm in the dark at 30 °C. For the controlexperiment, the flasks containing fungal mycelia wereautoclaved at 121 °C for 15 min prior to adding thePAH. Samples were collected 15 days after incuba-tion.

PAHs and their metabolites were extracted from5 ml of samples using ethyl acetate, as previously de-scribed24, and analysed by HPLC. HPLC analysis wasperformed with a liquid chromatograph system (Shi-madzu) equipped with an LC-3A pump, an SPD-2AUV-Vis detector and a C-RIA recorder. The separationcolumn was 4.6× 150 mm (Inersil ODS-3) and themobile phase was methanol:water (80:30 (v/v)) at aflow rate of 1 ml/min. The reduction of each PAHwas calculated as (1−AT/AC), where AC is the areaunder the peak of the substrate from the control set andAT is the area under the peak of the substrate from thetest set. Prior to extraction step of some experiments,100 mg/l of pyrene was added as an internal standardto monitor the extraction efficiency.

For metabolite analysis, the metabolites werecollected at the optimum production time. Thesemetabolites were acidified with hydrochloric acid topH 2–3 and extracted by ethyl acetate, evaporated,resuspended in ethanol, and evaporated a secondtime24. The precipitate was resuspended in methanoland these were analysed for the presence of PAHs andmetabolites by HPLC and GC-MS. HPLC analyseswere performed as described above. Samples forGC-MS analyses were analysed using gas chromatog-raphy with time of flight mass spectrometry (Pegasus

III, LECO). The GC used a 50 m long HP-5 columnof 320 µm in diameter and was coated to a 0.25-µmfilm thickness with 5% phenyl-methyl-syloxane.

Survival of selected fungi in differentconcentration of PAHs

Three grams of fresh mycelia of each selected funguswere inoculated into N-limited medium supplementedwith each single PAH at 25, 50, 100, 300, and500 mg/l and was incubated in the dark at 30 °C for30 days. To test for the survival of the fungus, 100 µlof fungal culture was dropped on to MEA medium.The plate was incubated for 7 days and fungal growthwas observed.

Ability of fungi to grow on solid mediumcontaining mixed PAHs

The four PAHs, including fluorene, phenanthrene,fluoranthene, and pyrene, were incorporated into MMagar plates at a concentration of 25 mg/l each. Three7-mm agar plugs containing fungal mycelia from 7-day-old cultures grown on MEA plates were placedon each of the MM plates. All plates were incubatedin the dark at room temperature for 10 days.

Degradation of mixed PAHs in liquid medium

Each fungal inoculum was prepared and 3 mg of freshmycelia were added into 30 ml of N-limited mediumin 125 ml Erlenmeyer flasks containing three glassmarbles. A mixture of the four PAHs (25 mg/l each)was added to each flask. All flasks were shakenat 120 rpm in the dark at 30 °C. Flasks containingfungal mycelia that were autoclaved at 121 °C for15 min prior to the addition of the PAHs were usedas controls. Samples were collected at 15 days afterincubation, whereupon PAHs were extracted and anal-ysed as previously described24. All data are presentedas the mean value derived from duplicate samples.

RESULTS

Fungal isolation and screening

From the initial 55 fungal isolates, 47 isolates (85.5%)were unable to change the colour of any of thethree indicator compounds. However, eight isolates(14.5%) were able to change at least one of the threechromogenic substances. Among the latter, threeisolates consisting of F18, S5, and T20 gave thewidest zone of colour changes and, therefore, were se-lected for further characterization because the colourchanges of these substances have been reported to berelated to the production of peroxidase and laccaseenzymes, which are responsible for the degradation ofPAHs30–33.

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Nucleotide sequencing of the ITS regions of therRNA genes (ITS1 - 5.8S RNA - ITS2) was per-formed for the F18, S5, and T20 isolates followedby BLASTn searches to identify close relatives (highsequence identity) in the GenBank database. The F18isolate had 100% nucleotide sequence identity to thesequence designated as Curvularia sp. F SMR-2011(HQ909079). Isolates S5 and T20 showed 98% and93% sequence identity to those designated as Lentinussquarrosulus strain 7-4-2 (GU001951) and Phane-rochaete sp. ATT215 (HQ607891), respectively. Inthis study, we refer to these isolates as Curvulariasp. F18, Lentinus sp. S5 and Phanerochaete sp. T20.The consensus ITS nucleotide sequences for isolatesF18, S5, and T20 have been submitted to the Gen-Bank database under accession numbers JN253597,JN253598, and JN253599, respectively.

Degradation of single PAH in liquid medium

These three fungi were tested for their ability todegrade the four representative PAHs of fluorene,phenanthrene, fluoranthene, and pyrene in nitrogen-limited liquid media (100 mg/l). After analysis of themetabolites by HPLC, the areas under the peaks werecompared to that of the control set using the killedfungus. The reduction for each PAH was calculatedusing the aforementioned formula. Curvularia sp. F18was found to be unable to degrade fluoranthene,phenanthrene, and pyrene, as the substrate peaks werestill present (data not shown). However, Curvulariasp. F18 was able to degrade fluorene, as it showed a90% reduction in the initial level added after 15 daysof incubation. In addition, the analysis of Curvulariasp. F18 showed the appearance of four major newpeaks (Fig. 1) that are assumed to be intermediatemetabolites. Lentinus sp. S5 was unable to degradefluoranthene under these conditions (data not shown)but degraded fluorene, phenanthrene, and pyrene at60, 86, and 85% relative to the control cultures,respectively, (Fig. 2). Finally, Phanerochaete sp. T20was able to degrade 83% of fluorene, 87% of phenan-threne, and 31% of fluoranthene (Fig. 3) but wasunable to degrade pyrene (data not shown).

Metabolite analysis

Although the results indicate that Lentinus sp. S5 andPhanerochaete sp. T20 are potentially good candi-dates for PAHs degradation, isolate S5 exhibited aslow growth rate. Thus we selected Phanerochaetesp. T20 for further characterization. This funguswas also closely related to P. chrysosporium, whichis the most reported PAH-degrading fungus35–38. Inorder to determine the preliminary PAH degradation

Area

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Fig. 1 HPLC chromatograms of fluorene degradation byCurvularia sp. F18. Representative HPLC chromatogramsfrom a 15-day-old culture of (a) autoclaved and (b) livemycelia of Curvularia sp. F18 grown in N-limited mediacontaining fluorene at 100 mg/l. Arrows indicate the peaksof fluorene. A circle specifies the intermediate peaks.

pathway used by Phanerochaete sp. T20, we de-termined the early metabolites that arose from thedegradation of fluorene. The Phanerochaete sp. T20fungus was grown in N-limited medium supplementedwith 100 mg/l of fluorene. The first intermediatepeak was observed on the third day of incubation andwas extracted, purified, and analysed by HPLC andGC-MS. In each case, 9-fluorenol and 9-fluorenonewere used as external standards because these prod-ucts have been reported to be the first metabolitesof fluorene degradation in most white rot fungi39.The HPLC analysis also revealed that the purifiedintermediate had the same retention time (RT =0.71) as 9-fluorenol (Fig. 4), while GC-MS analysisrevealed that the purified intermediate gave m/z ratiovalues of 63, 76, 91, 126, 152, and 181, which werethe same as those for 9-fluorenol. Taken together,the results from HPLC and GC-MS methods stronglysuggested that the first observed intermediate fromfluorene degradation by Phanerochaete sp. T20 waslikely to be 9-fluorenol.

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(f)

Fig. 2 HPLC chromatograms of fluorene, phenanthrene, and pyrene degradation by Lentinus sp. S5. Representative HPLCchromatograms from a 15-day-old culture of (a, c, e) autoclaved and (b, d, f) live mycelia of Lentinus sp. S5 grown inN-limited media containing (a, b) fluorene, (c, d) phenanthrene, and (e, f) pyrene at 100 mg/l. Arrows indicate the peaks offluorene. Circles specify the intermediate peaks. The internal loading standard of pyrene is found to the right of the substratepeaks in the fluorene and phenanthrene degradations.

Survival of Phanerochaete sp. T20 at variousconcentrations of PAHs

To investigate the ability of this fungus to surviveunder different concentrations of PAHs, fresh myceliafrom mGPY medium were cultured in media con-taining different concentrations (25, 50, 100, 300,or 500 mg/l) of fluorene, phenanthrene, fluoranthene,or pyrene. After 30 days, survival of the funguswas investigated by dropping the fungal culture ontoan MEA plate. Growth of Phanerochaete sp. T20was observed on MEA plates from all five testedconcentrations of all four evaluated PAHs (data not

shown). The fungus was able to survive, at least tosome extent, in all four tested PAHs at concentrationof up to 500 mg/l for at least 30 days.

Testing the ability of Phanerochaete sp. T20 togrow on solid medium containing mixed PAHsand to degrade mixed PAHs in liquid medium

To further test for the potential of Phanerochaetesp. T20 to degrade a mixture of PAHs, this isolate wasevaluated for its ability to grow on MM agar mediumcontaining an equal concentration of fluorene, phenan-threne, fluoranthene, and pyrene as the sole carbon

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74 6

5174 3

2.3

08 8

2940 4

2.6

58 1

36437 5

3.2

54 2

57776 6

3.8

78 3

4508 7

4.2

51 4

08461 8

4.8

18 1

44984 9

5.7

83 1

4461 10

6.6

83 5

07 11

7.9

20 8

37 12

9.6

75 8

713597 13

11.5

77 1

74780 14

Retention Time

Area

Time: 13.0112 Minutes - Amplitude: -- Volts

Detector A (275 nm) (a)

0.5

04

7

37

1

1.6

43

1

65

68

57

2

1.9

41

9

45

4 3

2.2

27

9

20

2 4

2.4

77

2

09

87

5

2.6

56

2

65

08

6

3.8

86

8

70

21

8

4.2

53

5

31

51

2 9

4.8

20

1

98

96

5 1

0

5.7

15

1

43

56

1

1

6.1

45

1

23

50

1

2

6.5

16

5

47

3 1

3

8.6

42

1

42

8 1

4

9.1

67

7

22

1

5

9.6

78

2

49

44

2 1

6

11

.56

9 4

32

2 1

7

3.2

57

3

93

90

75

7

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Minutes

Retention Time

Area

Time: 13.0112 Minutes - Amplitude: -- Volts

Detector A (275 nm) (b)2

.02

4 2

55

40

4

2.5

17

1

05

73

65

2.9

33

8

22

46

1

4.0

63

1

82

13

7

4.4

59

1

57

20

84

.67

7 2

88

14

7

5.3

09

3

02

89

6

6.1

59

3

83

85

7

6.7

16

1

95

93

3

7.5

41

1

06

14

81

3

9.3

91

2

08

73

10

.26

2 1

96

37

48

7

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Minutes

Time: 13.0114 Minutes - Amplitude: -- Volts

Detector A (275 nm)

Retention Time

Area

(c)

1.9

19

2

37

77

4

2.4

51

3

33

03

03

2.9

44

5

84

45

9

3.2

96

1

43

60

26

3.8

91

1

18

33

74

.05

8 2

14

91

5

4.5

12

4

03

74

12

5.3

42

2

50

87

1

5.7

24

1

18

74

8

6.4

18

5

81

07

2

7.5

54

1

41

02

30

9.4

05

2

00

94

10

.27

9 1

95

20

19

8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13Minutes

Retention Time

Area

Time: 12.9929 Minutes - Amplitude: -0.001647 Volts

Detector A (275 nm) (d)

Retention Time

Area

Detector A (275 nm)

1.4

92 1

6

1.9

30

8

90

55

2.3

58

4

79

30

6

2.6

88

6

28

01

2.9

39

1

82

27

4

3.3

05

2

84

07

1

4.5

18

3

79

12

2

5.3

21

1

47

58

3

6.6

08

1

83

45

7.2

75

8

56

13

8.3

27

1

05

4

8.7

29

1

83

9

9.4

04

1

76

99

00

4

10

.29

5 2

05

85

66

3

12

.11

1 3

48

95

0.8

0.6

0.4

0.2

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Minutes

Time: 13.0114 Minutes - Amplitude: -- Volts

(e)

2.0

10

1

09

33

3

2.5

72

1

01

69

89

2.7

67

2

25

54

42

.96

2 1

76

42

14

3.3

04

3

77

56

51

3.8

65

3

78

79

74

.05

2 3

60

72

8

4.5

12

1

04

95

97

5

5.3

47

1

20

59

63

5.7

51

7

69

68

3

6.3

81

1

56

86

05

7.2

44

7

85

45

5

8.0

83

2

58

99

8

8.4

22

5

49

39

7

9.4

05

1

36

71

56

9

10

.29

5 2

11

16

92

3

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7 8 9 10 11 12 13Minutes

Retention Time

Area

Detector A (275 nm)

Time: 13.0114 Minutes - Amplitude: -- Volts

(f)

Fig. 3 HPLC chromatograms of fluorene, phenanthrene, and fluoranthene degradations by Phanerochaete sp. T20.Representative HPLC chromatograms from a 15-day-old culture of (a, c, e) autoclaved and (b, d, f) live mycelia ofPhanerochaete sp. T20 grown in N-limited media containing (a, b) fluorene, (c, d) phenanthrene, and (e, f) fluorantheneat 100 mg/l. Arrows indicate the peaks of fluorene. Circles specify the intermediate peaks. The internal loading standard ofpyrene is located to the right of the substrate peak.

sources. Clear growth was observed on this medium,although with a slower growth rate compared to itsgrowth on MEA medium alone (data not shown). Thisobservation serves as a preliminary indication that thisfungus should be able to use or degrade these mixedPAHs, therefore, we evaluated its ability to degrademixed PAHs (25 mg/l each) in liquid medium. After15 days in culture, PAHs and their metabolites wereextracted from the culture media, analysed by HPLCand the results were compared with those obtainedfrom the control cultures (autoclaved mycelia). A

decrease in the area of the substrate peaks and theappearance of new peaks (assumed metabolites) as-sociated with the live mycelia cultures, but not in theautoclaved controls, were indicators of the ability ofthe fungus to degrade multiple PAHs (Fig. 5). Theseresults indicated that Phanerochaete sp. T20 coulddegrade a mixture of four different PAHs with initialconcentrations of 25 mg/l each. Next, we calculatedthe reduction in the level of each PAH relative tothe level in the corresponding control (autoclavedmycelia), and the results revealed that Phanerochaete

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ScienceAsia 38 (2012) 153

Retention Time

Area0.20

0.15

0.10

0.05

0.00

0 1 2 3 4 5 6 7Minutes

0.1

25 7

20.2

50 4

20.3

23 1

60

0.4

94 1

67

0.8

28 1

30

1.0

07 1

31

1.1

29 8

0

1.3

52

2

70

6

1.7

66

8

07

1

1.9

35

1

87

82

2.2

95

6

88

7

2.6

70

1

87

06

3.2

57

1

24

53

68

4.0

97

1

25

38

4.5

92 6

34.6

92 2

7

4.8

59

1

76

8

5.1

42 3

2

5.3

18 2

80

5.5

08 3

3

5.7

12 2

92

5.8

75 8

56.0

17 3

98

6.2

00 5

67

6.3

08 3

78

6.4

82 2

20

6.6

12 7

14

6.7

75 2

61

6.9

07 1

89

Time: 7.00605 Minutes - Amplitude: -- Volts

Detector A (275 nm) (a)

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5 6 7Minutes

0.1

58

1

33

0.2

17

7

40

.30

3 7

3

0.5

55

1

66

0.8

33

3

22

1.0

26

2

58

1.0

94

3

67

1.2

81

1

31

8

1.7

65

4

06

2

2.0

00

1

11

2.1

30

9

2

2.6

18

3

50

3.2

58

2

89

33

46

4.1

04

4

04

78

4.7

31

2

55

4.8

77

3

50

4.9

49

1

30

5.1

41

2

88

5.3

30

1

39

5.5

92

8

35

.65

3 6

35

.82

5 8

35

.95

8 9

9

6.1

70

3

46

.30

8 1

23

6.4

58

4

46

.53

7 5

76

.66

4 1

49

6.7

99

2

56

Retention Time

Area

Time: 0.0801945 Minutes - Amplitude: 2.5e-005 Volts

Detector A (275 nm) (b)

Fig. 4 HPLC chromatograms of purified intermediate and 9-fluorenol. Representative HPLC chromatograms illustrating(a) the purity of the purified intermediate compound froma three-day-old culture of Phanerochaete sp. T20 grown inN-limited media containing fluorene at 100 mg/l and (b) 9-fluorenol.

sp. T20 was able to remove fluorene, phenanthrene,pyrene, and fluoranthene by 97, 59, 47, and 39%, re-spectively, (Fig. 5). Thus this fungus has the potentialto degrade and remove a mixture of at least four PAHs(fluorene, phenanthrene, fluoranthene, and pyrene).

DISCUSSION

The white-rot fungi are a group of organisms ca-pable of degrading a wide variety of environmentalpollutants, including PAHs, due to the productionof extracellular and relatively nonspecific (for thesubstrate) ligninolytic enzymes, including lignin per-oxidase, manganese peroxidase, and laccase13. In thisstudy, we initially isolated fungi that had the potentialto degrade PAHs by screening for the ability of theseisolates to degrade the chromogenic substrates phenolred, guaiacol, and azureB, which have structures thatresemble PAHs. We used these substrates as indicatorsfor the potential production of extracellular peroxidaseand/or laccase enzymes. Two white-rot fungi, whichwere identified as Lentinus sp. S5 and Phanerochaetesp. T20, and one Ascomycetes fungus, Curvulariasp. F18, were isolated using this criterion. These fungi

1.6

46

1

32

18

41

.90

6 3

31

69

22

.25

8 4

32

06

62

.67

7 1

47

84

9

3.2

76

3

41

91

7

3.9

06

1

03

04

24

.28

5 4

72

49

14

.62

2 8

27

66

4.9

58

5

55

66

5.5

47

3

09

21

5.8

36

2

52

29

6.2

24

7

37

10

7.2

89

7

17

4

8.0

10

2

28

2

8.8

98

8

07

8 9.7

83

1

55

96

61

10

.40

7 3

88

60

08

11

.31

8 1

26

32

12

.11

7 5

67

99

14

.00

1 5

06

54

12

15

.40

7 6

00

41

47

0.4

0.3

0.2

0.1

0.0

0 2 4 6 8 10 12 14 16 18 20Minutes

Time: 20.0173 Minutes - Amplitude: -- Volts

Detector A (275 nm)

Retention Time

Area

(a)

0.25

0.20

0.15

0.10

0.05

0.00

0 2 4 6 8 10 12 14 16 18 20

Minutes

Retention Time

Area

Time: 20.0173 Minutes - Amplitude: -- Volts

Detector A (275 nm)

1.6

56

1

08

29

41

.98

9 1

24

83

32

.33

1 1

44

93

82

.72

1 3

20

92

8 3.2

79

1

13

22

26

3.8

21

1

51

24

4.2

40

1

24

50

42

4.8

64

4

15

88

5

5.9

82

7

67

27

6.1

92

3

51

51

6.6

08

1

74

33

1

8.5

88 9

68.8

82 8

40

9.2

75 7

9

9.7

88

4

61

66

10

.40

6 1

60

58

13

12

.15

7 8

56

66

13

.99

8 3

11

49

17

15

.40

7 3

20

31

19

(b)

Fig. 5 HPLC chromatograms of mixed-PAHs degradationby Phanerochaete sp. T20. Representative HPLC chro-matograms from a 15-day-old culture of (a) autoclavedand (b) live mycelia of Phanerochaete sp. T20 grown inN-limited media containing fluorene, phenanthrene, fluoran-thene, and pyrene at 25 mg/l each. The retention time offluorene, phenanthrene, fluoranthene, and pyrene were 9.6,10.3, 13.9, and 15.3 min, respectively. Arrows indicate thepeaks of these four substrates.

were then tested for the ability to degrade two low-molecular weight PAHs (phenanthrene and fluorene)and two high-molecular weight PAHs (fluorantheneand pyrene), both alone and together. All three fungalisolates were found to be good candidates for PAHbioremediation, showing good degradation of the low-molecular weight PAHs. In addition, S5 and T20also showed good and moderate degradation of thehigh-molecular weight pyrene and fluoranthene, re-spectively. Further characterization of Phanerochaetesp. T20 revealed that it can withstand and survive inhigh concentrations of PAHs (at least up to 500 mg/lfor 30 days) and that the degradation of fluoreneproduced a less toxic compound, 9-fluorenol, as themajor early intermediate. In addition, this strainshowed the potential ability to degrade a mixture ofall four of these PAHs.

Although fungi in the genus Curvularia have beenreported to be able to degrade tricyclic PAHs, such

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154 ScienceAsia 38 (2012)

as phenanthrene and anthracene40, 41, the degradationof fluorene (which has a more complex structure) byfungi in this genus had never been demonstrated priorto this study. Our results indicate that, among thefour PAHs tested, Curvularia sp. F18 could only de-grade fluorene, a tricyclic PAH with a five-memberedring, at high efficiency. Although the intermediatemetabolites of fluorene were not investigated in thisstudy, several previous reports have shown that fornon-white-rot fungi fluorene and a number of otherPAHs are generally metabolized by cytochrome P450monooxygenase and epoxide hydrolase to form trans-dihydrodiols39. Due to its robust ability to degradefluorene, Curvularia sp. F18 should be further investi-gated for its potential use in situ bioremediation wherehigh levels of fluorene contamination are present.

Lentinus sp. S5 is a member of the white-rotBasidiomycetes fungi. The fungi in this genus havebeen reported to degrade several types of PAHs usingboth extracellular ligninolytic enzymes and intracellu-lar P450 monooxygenase systems42, 43. In this study,Lentinus sp. S5 was capable of degrading phenan-threne, fluorene, and pyrene. However, it could notdegrade fluoranthene. The chromatograms from thephenanthrene, fluorene, and pyrene degradations byLentinus sp. S5 (Fig. 2) revealed new peaks with thesame retention times for all three PAHs, indicatingthat Lentinus sp. S5 used the same pathway or thesame mechanism to degrade these PAHs, giving riseto the same intermediate metabolites. This possibilityremains to be confirmed. However, if this hypothesisis correct, these metabolites would probably be thePAH-quinone compounds, which, in general, are theproducts of PAHs degradation by white rot fungiincluding Lentinus via the ligninolytic and laccaseenzymatic reactions42. In the case of complete miner-alization, this PAH-quinone will pass the ring fissionprocess and produce CO2 as the final product44.

Phanerochaete sp. T20 is a white-rot Basid-iomycetes fungus that belongs to the same genusas the well-known PAHs degrader, P. chrysospo-rium35, 45. In the degradation of each PAH alone,Phanerochaete sp. T20 appeared to be capable ofdegrading fluorene, phenanthrene, and fluoranthene;and several intermediate peaks were seen in the chro-matograms of the degradation. However, it could notdegrade pyrene, which is a fused tetracyclic aromatichigh-molecular weight hydrocarbon. In contrast,when Phanerochaete sp. T20 was grown in N-limitingmedium containing all four PAHs, it was able todegrade pyrene, as well as fluorene, phenanthrene, andfluoranthene, and at a higher level than fluoranthenealone. The apparent degradation of pyrene in the

presence of other easily degradable substrates wouldprobably result from the interactions between sub-strates that subsequently enhance the degradation ofcompounds that are more difficult to degrade, such aspyrene46. Synergistic effect between PAHs and co-metabolism were also proposed for this feature47. Inaddition to its ability to degrade a mixture of low- andhigh-molecular weight PAHs, Phanerochaete sp. T20was able to grow and survive (at least to some extent)at relatively high concentrations of PAHs (500 mg/l)for at least 30 days. These results indicate a notablePAH degradation efficiency of Phanerochaete sp. T20and suggest that this fungus might be useful for insitu bioremediation at sites where mixed and/or highconcentrations of PAHs are a problem.

The early metabolites of fluorene degradationwere further investigated to preliminarily determinethe degradation pathway of Phanerochaete sp. T20.The results from HPLC and GC-MS indicated thatthis metabolite was most probably 9-fluorenol, whichis a less mutagenic compound than fluorene39. Thisobservation is considered to be an indicator for thefirst step of fluorene detoxification11. Thus Phane-rochaete sp. T20 likely degrades fluorene under non-ligninolytic conditions such that it degrades fluorenevia cytochrome P450 monooxygenase, as is frequentlyfound with P. chrysosporium and other white-rotfungi39, 48, 49.

The three PAH-degrading fungi that were isolatedin this study can potentially be used for bioremedi-ation. Curvularia sp. F18 may be suitable for condi-tions where the contamination is fluorene, while Lenti-nus sp. S5 and Phanerochaete sp. T20 may be suitablefor contamination sites where more than one type ofPAHs is the main concern. This study reports on thepreliminary isolation and investigation of three PAH-degrading fungi. However, further detailed studies arerequired to investigate the degradation pathways andthe degradation products of these useful fungi.

Acknowledgements: This study was financially sup-ported by a grant for the development of new faculty atChulalongkorn University; the Thai Government StimulusPackage 2 (TKK2555), under the Project for Establishmentof Comprehensive Centre for Innovative food, Health Prod-ucts and Agriculture; and a CU graduate school thesis grant.

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