Bio Degradation Of F By Soil Fungi

Chemosphere, Vol.

38, No. 13, pp. 3031-3039, 1999 © 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter

Pergamon PII: S0045-6535(98)00504-9

Biodegradation of Fluoranthene by Soil Fungi. F. SALICIS ( 1), S. KRIVOBOK (1) M. JACK & J.-L BENOIT-GUYOD (2)

Groupe pour l'Etude du Devenir des Xdnobiotiques dans l'Environnement (GEDEXE) (I) - Laboratoire de Botanique, Cryptogamie, Biologie Cellulaire ct G6ndtique (2) - Laboratoire de Toxicologie et t~cotoxicologie UFR de Pharmacie de Grenoble, Universit6 J. Fourier, BP 138, 38243 Meylan ccdex. France (Received in Germany 27 July 1998; accepted 23 September 1998) A b s t r a c t . A selection of 39 strains of micromycetes k n o w n as good degraders of polychlorinated aromatic compounds, mostly isolated from soil and belonging to various taxonomic groups, have been investigated lor fluoranthene degradation. Toxicity assays, first evaluated on solid m e d i u m MEA, have not shown any toxicity of fluoranthene (1-100 mg.L -1) towards fungi. Whereas, c o n s u m p t i o n assays on a solid synthetic m e d i u m , h o w e d a toxicity at 100 mg.L -1. The degradation of fluoranthene (10 mg.L -1) was then investigated in a liquid synthetic m e d i u m for 4 days and evaluated by HPLC. A m o n g the 39 strains tested. 18 degraded fluoranthene at 60% or more. Zygomycetes appeared to be the most efficient group (mean degradation : 9091 ). A m o n g 18 p e r f o r m a n t strains, 10 had not yet been reported in the literature : Sporormiella

australi.v,

Cryptococcus albidus, Cicinobolus cesatii, Pestalotia pahnarum, Beauveria alba, Ast~ergilhts terreu,~. Cmminghamella blakesleeana, C. echinulata, Mortierella ramanniana and Rhi.-rOl)USarrhizus. Fluoranthene adsorption on fungi was very low for the strains which degraded well 11uoranthene (mean adsorption : 4~7~). Whereas. some strains adsorbed it m u c h more such as Colletotrichum dematium (47%) and Penicil/ium

ila/icum (43%). ©1999 Elsevier Science Ltd. All rights reserved K e y w o r d s . Fluoranthene • Biodegradation • Adsorption • Micromycetes • Fungi.

INTRODUCTION

Polycyclic aromatic h y d r o c a r b o n s (PAHs), a c o m p l e x group of chemical c o m p o u n d s , are present ill petroleum and mainly formed as byproducts of any incomplete combustion (Guerin, 1978 : National Academy ~ Sciences, 1983). The latter is mainly responsible for their ubiquitous distribution in the enxironlncnt ~IARC. 1983 : Karcher, 1988 ; Fernandez et al., 1992). Some P A H s are considered hazardous becausc of their mutagenic and carcinogenic activities (IARC, 1983 ; Dipple et al., 1990 : Hansen et al., 1994/, and are nn the list of priority pollutants established by the US Environmental Protection A g e n c y ( Keith & Teillarcl,

1979). Fluoranthene, a tetracyclic aromatic hydrocarbon, is naturally present in fossil fuels and also formed during Iheir c o m b u s t i o n (Schuetzle et al., 1981 ; Jones et al., 1989). Fluoranthene is consistently the most abundant PAH in e n v i r o n m e n t a l samples (Mc Elroy et al., 1989 ; W e s t et al., 1986) and has been reported to be %'totoxic, mutagenic and potentially carcinogenic (Kaden et al., 1979 : Thilly et al.. 1980 : Rice et al.. 1982 • Busby et al., 1984 ; K a g a n et al., 1985 ; Bos, 1987 ; Irvin & Martin. 1987 ; T u v e s o n et al.. 1987). It is c~msidered more of a potential health hazard, by virtue of its abundance, than the widely studied, but 1ess3031

3032 a b u n d a n t carcinogen b e n z o [ a ] p y r e n e (Rastetter et al., 1982 : IARC,

1983 : Sakai et al.. 1985 :

l~abson et al., 1986 : Bos et al., 1987 ; Mersch-Sundermann et al., 1992). Because its structure is found in carcinogenic PAHs such as benzo[a]pyrene and benzo[a]fluoranthene, fluoranthene is used as a model compound (Pothuluri & Cemiglia, 1994). Biodegradation of PAHs by bacteria is well documented (Gibson & Subramanian, 1984 : Cerniglia, 1993 ; Pothuluri & Cerniglia, 1994) but such knowledge on fungi is limited / Sutherland. 1992 : Mtincnerova & Augustin, 1994). Only a detailed investigation on fungal metabolism of fluoranthene was realized with Cunninghamella elegans (Pothuluri et al., 1990 ; 1992). The aim of this study was first to evaluate the toxicity of fluoranthene on solid media towards 39 microlnycetes at levels far exceeding environmental concentrations, then to assess its depletion from liquid mediuln.

MATERIALS

AND METHODS

M i c r o o r g a n i s m s . Preliminary studies included 39 strains of our cuhure collection (CMPG : Collection Mycology Pharmacy Grenoble), distributed between the main taxonomic groups (Table I). They were isolated from soil samples (Lacharme et al., 1978 : Seigle-Murandi et al., 1980 : 1981) or from w m o u s other ,ubstrates (De Hoog et al., 1985 ; Gains et al., 1990). Stocks cultures were maintained at 4"~C on solid malt extract medium (MEA) or potato dextrose agar medium (PDA). Media. Malt extract agar (MEA) used for stock cultures contained : malt extract. 15 g.L-I. Potato dextrose a~ar (PDA), was preparated with : potato extract, 200 g.L -1 ; dextrose, 20 g.L -1 : agar. 15 g.L -I . Galzy & ~lonimski i 1957) liquid synthetic medium (GS) was slightly modified by adding glucose at a concentration of 5 g.L-I. Before use, the media were sterilized by autoclaving (20 rain. i21 °C). Chemicals. Fluoranthene was purchased from Sigma-Aldrich (Sigma Chemical Co., SL Louis. MO}. Agar ,rod malt extract were respectively from Coop6rative Pharmaceutique Fran~aise (Melun. France l and Difal Villefianche-sur-Sa6ne, France). Other products were from Prolabo (Paris, France). t ' u l t u r e s conditions. The strains were reactivated on MEA medium for 8 days without fluoranthene. For the first experilnents, the final concentrations of fluoranthene were 0.001. 0.01. 0. l and 1 g.L I. MEA ~ c d i u m were used to check the toxicity of fluoranthene against microorganisms. Cultivation on Galzy and !';Ionimski synthetic solid medium (GS) (1957) at pH 4.5 without glucose was a test for consumption. GS medium was also used without glucose and without fluoranthene to estimate any nutrient effect of agarose. which is not an inert substrate (Seigle-Murandi et al., 1980). Cultivation was made for a 9-days period at 23°C ~m~d 37°C for Phanerochaete cho,sosporium). The results allowed to select a final concentration of 0.01 g.L I f,~v fu,ther cultivation of all the strains. D e g r a d a t i o n assays. To obtain sufficient inoculum for liquid medium cultures, strains were grown l~r 1-2 a'eeks on MEA medium without any adaptation to fluoranthene. They were inoculated (mycelmm and spores I. l~cptically, in GS liquid medium with glucose (5 g.L -1) at pH 4.5. which had been sterilized by autoclaving '~t,r 20 rain at 121°C. The 125 mL Erlenmeyer flasks containing 25 mL of medium were incubated with ,baking (180 rpm, orbital shaker), for 2 days, in order to allow the biomass to grow and reach 140 mg +_ 10'~ cdr~ weight). At this stage, no glucose remained in the medium.

3O33 Fluoranthene in dimethylsulfoxide stock solution was sterilized by filtration through 0.2 p.m Millipore membranes, and added to the 2-day-old cultures to a final concentration of 0.01 g.L-I. The depletion of fluoranthene was evaluated after 2 more days of cultivation. Temperature was 24°C (and 37°C for

Plumerochaete chrysosporium). Light was 1200 lux with a photoperiod of 12 hours per day. Each series of experiments was run at least in triplicate and included cell-free f a s k s for abiotic degradation assessment. M e t h o d s of analysis. The disappearance of fluoranthene was monitored by HPLC. Liquid media with mycelia containing fluoranthene were extracted with one volume of bidistilled ethyl acetate (25 ml) by rotary shaking for 30 min. Mycelia were filtered off and rinsed with ethyl acetate (5 ml). The media and the rinse ~ere transferred in a separating funnel. The mixture was shaken for 2 rain and allowed to stand for 15 min. The organic phase was collected and saved. The extraction was then twice repeated. The combined crude extracts were dried over anhydrous Na2SO4, and evaporated to dryness at 40°C under reduced pressure. The residue was dissolved in acetonitrile (1.5 ml) from which, after gently vortexing and filtration through a 0.2 ttm membrane filter, an aliquot of 20 ~tl, was removed for HPLC analysis. Mvcelia were separated from the media by filtration and rinsed with bidistilled ethyl acetate (5 ml) as above mentioned. They were then oven-dried (60°C, 2 days) and further extracted with 10 ml of methanol by rotary shaking during 1 hour. Volumes were carefully readjusted to 10 nil, filtered through a 0.2 ~tm membrane filter and an aliquot of 20 ILtlwas removed for HPLC analysis. HPLC was performed with a liquid chromatograph I Shimadzu) equipped with a LC 6A pump, a SIL-9A automatic injector and a RF- 10AXL spectrofluorescence detector. The separation column, Supelcosil TM LC-PAH 5 ~m, was 4.6 m m inside diameter x 150 mm I Supelco Inc., Bellefonte, PA). The mobile phase was acetonitrile : water (70 : 30, v : v). The flow rate was I mL.min -1 and detection was made at 280 nm (~, excitation) and 450 nm (~, emission). Each sample was injected at least 3 times and the mean was calculated. Results were within a 5% range.

RESULTS AND DISCUSSION Toxicity and consumption of fluoranthene. Toxicity assays on malt extract medium showed that O. 1 g.L 1 fluoranthene was not toxic to most of thefungal strains (Table I). 35 strains among the 39 tested I90%) showed a good (++) or a very good (+++) growth at this concentration. The best growth was observed with Rhizoctonia solani, Cryptococcus albidus, Phoma herbarum and Rhodotorula rubrcl. Various performances of micromycetes were observed when the concentration of fluoranthene increased : decrease of growth, apparition of sterile mycelia and discoloration of the colonies (especially Aspergillus terreus.

A.~pergillus versicolor, Colletotrichum dematium, Penicillium italicum, Rhizoctonia solani. Sporormiella mtstralis ). Only four strains showed a weak growth at 0.I g.L -1, particularly Cr~phonectria parasitica and Coniot~hora arida. By comparaison with others PAHs, fluoranthene showed a toxicity similar to anthracene on the same (ungal strains ; 95% of strains showed a good or a very good growth at 0.1 g,L-I of anthracene (Krivobok et al.. 1998). On the other hand, fluorene was more toxic than fluoranthene and anthracene, with only 53c/+ performant strains (publication submitted by Garon et al.).

3034 T a b l e I. Toxicity (MEA medium) and consumption (GS medium) of fluoranthene t o w a r d s A s c o m y c e t e s ,

Basidiomycetes, Deuteromycetes and Zygomycetes on solid media were evaluated by measure of the growth diameter expressed as a percentage of the growth diameter of reference strains : +++ over 80%, ++ between 50 and 80%, + under 50%. Concentration of fluoranthene (g,L -1) between brackets. Class / Genus

MEA

GS

Nomenclature

Ascomycetes Clyphonectriaparasitica Ikichotomonlyces cejpii Sporormiella australis

++ (0.01) +++ (0.1) +++ (0.1)

++ (0.1) ++ (0.001) ++ (0.1)

( M u m ) M . Baar (Milko) Scott (Speg.) Ahmed & Cain

++ (0.1) ++ (0.1)

++ (0.1) + (0.001)

(Wildd.:Fr.) Karst. (Pilat) Gilbertson Ryv

Basidiomycetes 131ed~anderacuhtsta ('criporiopsis subvermispora Ceriporiopsis subverntispora ConiophoraaHda O.kvsl?ortts sp. Phauerochaete cho'sosporium - 23°C - 37°C Phanetwehaete chrysosporium - 23°C - 37°C Phanerochaete cho'sosporium - 23~'C - 37°C

++ (0.1)

+ (0.001 )

++ (0.01) +++ (0.01) ++ (0.1) ++ (0.1) ++ (0.1) ++ (0.1) ++ (0.1) ++ (0.1)

++ (0,01) ++ (O.l) ++ (0.001) ++ (0.001) +.+- (0.01) ++ (0.01) + (0.001) ++ (0.01)

(Fr.:Fr.) Karst.

++ (1) +++ (0.1) +++ (0.1) ++ (0.1) +++ (0.1) +++ (0.1) +++ (0.1) +++ (1) +++ (0.1) +++ (0,01) +++ (0.01) +++ (0.1) ++ (1) +++ (0.1) +++ (0.1) +++ (0.1) +++ (0.1) +++ (1) +++ (I) + + + (1) +++ (0.1) +++ (O.l)

+ (0.001) + (0.00l) ++ (0.1) ++ (0.1) +++ (0.01) ++ (0.1) +++ (0.01) +++ ( 1) +++ (0.1) + (0.001) ++ (0.1) + (0.001) ++ (0.1) ++ (0.1) + + (0.1) ++ (0.1) ++ (0.1) + + (I) +-~ (0.001) + + + (1) ++(0. I ) ++ (O.OOI)

Thorn (Vuill.) Tiraboschi (Limber) Saccas de Baly (Pels.:Fr.) Link (Pers.:Fr.) Grove (W. Gams & Domsch) van der Aa (Saito) Skinner (Zins.) Scholten (Sacc.) Morton & G. Smith (Bain.) v. Arx de Hoog Sheldon/Wollenw. & Reinking (Mart.) Sacc. Thorn Wehmer Cooke Weslend. KiJhn (Demme) Lodder de Hoog & de Vries (Zimm.) Viegas

++ (0.l) +++ (0.1) ++ (0. l ) +++ (0. I ) ++ (l) + + (1)

++ (I) ++ (0.1) + (0.001) + (0.001) ++ (1) ++ (0.001)

Lendner (Thaxter) Thaxter Lendner

BurdsaII

Deuteromycetes .~wergillus terreus 4 ~l~ergillus versicolor Beauvecia alba ('ic'inobolus cesatii (huhw~oriltnl herbarum Colletotrichum dematium U~,niothyrium sporulosum ('~37)toeoccus albidus Q~lindrocaq~on destrucmns I)oraromvces stemonitis l)rechsleraspicifelzl Embelli~ia amlulata I-,sarium monilifi~rme vat. subglutinans f-,~ariumsolani Penicillium cho'sogenum Pcnicillium italicum Pcstalotia l)ahuarum I)homa herbarum Rhizoctonia solani Rhodotornh~ rubra gporothrir cvanescens 'crticillimn lecanii

Zylaomycetes (umnfinghctmella blakesleeana ('wlninghamella echinulata (mminghamella elegans ("mminghamella elegans ,tlortierella ramanniana Rhizolms arrhizns

Consumption

a s s a y s o n G S s o l i d m e d i u m w i t h fluoranthene as s o l e c a r b o n s o u r c e s h o w e d t h a t it w a s t o x i c

f o r 5 0 % o f t e s t e d s t r a i n s at 0.1 g . L - I

w h i l e 9 0 % o f fungal strains g r e w w e l l o n M E A

represented groups were Deuteromycetes ,bowed

(M6ller) Linnemann Fischer

(66% of performant strains) and Zygomycetes

lnedmm. The best (50%). These results

t h a t t h e s e strains are able to use fluoranthene as sole carbon source. Howewer, in B a s i d i o m y c e t e s

g r o u p . 7 5 % o f the strains were inhibited by flu0ranthene. P h a n e r o c h a e t e chr),sosporium

is a n e x a m p l e o f this

i n h i b i t i o n " a s i m i l a r g r o w t h inhibition was observed rather at 2 3 ° C t h a n at 3 7 ° C . W h i l e . in p r e s e n c e o f a n t h r a c e n e . K r i v o b o k et al. ( 1 9 9 8 ) c o n f i r m e d t h a t P h a n e r o c h a e t e chr)'sosporittm a~ p r e v i o u s l y o b s e r v e d b y K i r k e t al. ( 1 9 7 8 ) .

h a d a b e t t e r g r o w t h at 3 7 ° C

3035 Thus, comparison of results on M E A and GS media showed that various micromycetes better degraded fluoranthene in cometabolism (MEA medium) than as sole carbon source (GS medium) as previously reported by Cerniglia (1993). D e g r a d a t i o n o f fluoranthene. Results are given according to the taxonomic distribution. The values in Table II are expressed as percentage of degradation of fluoranthene, and we choose to qualify as efficient any strain that degraded it at least at 60%. Abiotic degradation, mainly photodegradation, was negligible after 4 days of cultivation.

Table II. Degradation and adsorption of fluoranthene (FA) (%) by A s c o m y c e t e s , Deuteromycetes and Zygomycetes. Values for efficient strains are given in bold.

Class / Genus

FA degradation

FA adsorption

Basidiomycetes,

Nomenclature

Ascomycetes Ctyphoneetria parasitica Diehotmnomyces cejpii 3"lnnvrmiella australis

25 38 58

25 0 5

(Murr.) M. Baar (Milko) Scott (Speg.) Ahmed & Cain

64 87

1I 2

(Wildd.: El. ) Karst. (Pilat) Gilbertson Ryv

71

6

58 79 0 0 30 45 36 7

3 12 22 5 10 9 31 1

(Fr.:Fr.) Karst.

89 5 80 86 77 39 33 93 49 54 66 I1 33 0 56 24 78 53 47 1 23 76

0 23 6 0 8 47 7 0 2 4 15 0 0 I 7 43 7 I 35 0 1 0

Thorn (Vuill.) Tiraboschi (Limber) Saccas de Bary (Pers.:Fr.I Link (Pers. :Fr. ) Grove (W. Gains & Domsch) van der Aa (Saito) Skinner (Zins.) Scholten (Sate.) Morton & G. Sinith (B ai n. ) v. Arx de Hoog Sheldon/Wollenw. & Reinking (Mart.) Sacc. Thorn Wehmer Cooke Westend. Ktihn (Demme) Lodder de Hoog & de Vries (Zimm.) Viegas

86 94 94

0 1 0

98

1

84 82

0 0

Basidiomycetes Bjerl~tndetzt adusta Ceriporiopsis subvermispora Ceriporiopsis subvermi~7~ora ('oniophora arida O~3'.worus sp. Phanetvchaete chtTsosporium - 23°C

- 37°C Phanetwehctete cht3sosporium - 23°C

- 37°C P/lanetwehaete cht3'so~porium - 23°C

- 37°C

Burdsall

Deuteromycetes .4~t~ergilhts terreus ,4spel\~ilhts versicolor Becmveria alba Cicim)hohts eesatii CladospoHum herbarton Colletotriehttm dem~ltium Coniothyrium sporulosum CJ3pmcoceus alhidus C~limh'twatpon destruetans /)oratom~ces stemonitis I)re~ hsk'ra ~v~ie(fem Emhellisia anmdata Fu~arittm moniliforme var. subglutinans ]'-IIS~IIJItlll solani Penicillittm eho'sogenum Penicillium italicum Pestalotia pahnarum Phoma herbarunl Rhizoctonia so[ani Rhock~unTt[a rnbtzt ,S'potv)tllrix e)'anescens Verticillium leeanii

Zygomycetes Cmmuinghamelkl blakesleeana Cunninghcmtella echinttlam Ctmningllantelkt elegans Cmtnin,~,,hamella elegans Mentie~ella ramanniana Rlli~olmS atv'llizto"

Lendner (Thaxter) Thaxter

Lendner (M~ller) Linnemann Fischer

3036 Among the 39 strains tested, 18 strains were selected for their high capacity to degrade fluoranthenc. Zygomycetes with the genus Cunninghamella was the most efficient group (mean degradation : 90% ). Among I)cuteromycetes, Cr3'ptoccocus albidus strongly degraded fluoranthene at 93%. Aspergillus terretts (89~/~).

('icinobulus cesatii (86%) and Beauveria alba (80%) showed also a good degradation capacity oi this PAH. Among Basidiomycetes, only 3 strains had a noticeable activity : Ceriporiopsis subvermispora (87%). O.xysporus sp. (79%) and Bjerkandera adusta (64%). These 3 micromycetes were previously reported for anthracene degradation (Krivobok et al., 1998) and fluorene degradation (publication submitted by Garon et al. ). A good growth on GS solid medium did not always imply a good biotransformation in liquid medium. This phenomenom has been previously reported with biphenyl-oxyde (Seigle-Murandi et al., 19911 and 2~tcctyhhiophene biotransformation (Seigle-Murandi et al., 1991). These observations confirm the first cometabolism studies (Cerniglia, 1993). The consumption of fluoranthene by micromycetes as sole carbon ~,ource used much energy, consequently the strains tested showed only weak growth. The white-rot fungi Phanerochaete chrysosporium was not efficient in fluoranthene degradation. These results confirmed the weak performance of this genus observed with anthracene (Krivobok et al., 1998) and fluorene (publication submitted by Garon et al.). But this fungus is well known for its large capability of bi~degradation of organopollutants PAHs (Bogan et al., 1996). This low level of degradation could be explained by the time of metabolisation which was only 2 days. Indeed, studies on anthracene have reported a $ood degradation by P. chr),sosporium after 28 days of cultivation (Hammel et al., 1991). Among 18 performant strains upon fluoranthene (degradation >60%), 10 have never been cited in the literature : Sporormiella australis, Cr3'ptococcus albidus, Cicinobolus cesatii. Pestalotia palmarum, Beam'eria

(llha. Asl)ergillus terreus, Cunninghamella blakesleeana, C. echinulata. Mortierella ramamliamt and Rhiz~q~u.~ :trrhizus. Others were previously known for their actions on others HAPs : B/erkandera adusta, Ceril~()ri()psis ~td)vermispora, Oxysporus sp., Cladosporium herbarum, Drechslera spictlfera, Verticillium h, ctmii. P~,llicillium chr3,sogenum, Cunninghamella elegans and Rhizopus arrhi~us (Cerniglia, 1993 : Krivobok et al., ',t~']8). Only Ctmninghamella elegans was cited for its capacity to degrade the fluoranthene (Pothuluri et al., 1'390

:

1992).

~,dsorption of fluoranthene. To degrade a chemical pollutant is not the only way to remove it since the tL,o of microbial biomass as biosorbents may offer a potential alternative or complementary mechanism. An another way is adsorption of toxic organics which is a complicated process driven by various parameters. Our ~tudies on adsorption of fluoranthene on the fungal biomass showed that this adsorption is depending of ,pecies (Table II). In most cases, we observed thad adsorption was inversely proportional ~o ftmgal degradation. Performant strains (fluoranthene degradation >60%) showed a weak adsorption (mean : 4'51 with ~t maximum with Drechslera spic~[era 15%) ; for Zygomycetes and Ci3'ptococcus albidtls which highly degraded fluoranthene (>80%), adsorption is nearly nil (mean : 0.3%). On the other hand. several particular ~:~ses were noted, concerning strains which showed an important fluoranthene disparition in liquid medium : but tiffs disparition was mostly due to adsorption : Rhizoctonia solani (82% disparition with 35% adsorption ),

CJ3t~lumectria parasitica (50%-25%), Phanerochaete chrysosporium (67%-31%), Collett)trichum dematiiml ~86~7c-47%) and Penicillium italicum (67%-43%).

3037 In conclusion, consumption and toxicity assays on solid media have showed slight toxicity of fluoranthene l~lO0 mg.L -I) towards the 39 selected micromycetes. 18 of them degraded fluoranthene (10 mg.L I) in ~ynthetic liquid medium at 60% or over and 10 of these 18 have never been cited in the literature : Sporormiella australis, Cryptococcus albidus, Cicinobolus cesatii, Pestalotia palmarum, Beauveria alba, A.vwrgillus terreus, Cunninghamella blakesleeana, C. echinulata, Mortierella ramanniana and Rhizop.s arrhiztts.

Acknowledgments We acknowledge L. Soubiran for his technical assistance.

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