Research Paper Removal of Polycyclic Aromatic Hydrocarbons by Pleurotus Ostreatus Sp. ATCC38540 in Liquid Medium

Research Paper

Removal of Polycyclic Aromatic Hydrocarbons by Pleurotus ostreatus sp. ATCC38540 in Liquid Medium

Accepted 3nd October 2016

David Tirado-Torres1, Martha Gayosso- Canales2, Yolanda Marmolejo-Santillán1, ABSTRACT Claudia Romo-Gómez1 and Otilio Acevedo-Sandoval2* Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in crude oil, by a white rot fungus, Pleurotus ostreatus sp., in broth culture was investigated. It was 1Instituto de Ciencias Básicas e Ingeniería, Universidad Autónoma del found that the biomass of the organism increased with the increase of PAHs Estado de Hidalgo (UAEH), Carr. concentration in the cultures. In the cultures with 25.99, 49.01 and 80.05 mg.kg-1 Pachuca-Tulancingo, Km. 4.5, Col. PAHs, the degradation increased with the PAHs of low molecular weight Carboneras, Mineral de la Reforma, concentration, whereas, the degradation was high and constant for high 42184, México. 2Instituto de Ciencias Agropecuarias, molecular weight. The addition of Tween 80 improved the ability to degrade UAEH. Av. Universitaria Km. 1, Ex-had. PAHs high molecular weight. In conclusion, P. ostreatus is a promising white rot deAquetzalpa, Tulancingo, Hgo. 43600. fungus to used degrade PAHs of low and high molecular weight in the México. environment. *Corresponding autor. E-mail: [email protected]. Key words: Pleurotus ostreatus, PAHs, biodegradation, White rot fungi.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are a group of fungi, such as Phanerochaete chrysosporium, Pleurotus organic lipophilic pollutants generated during incomplete ostreatus, Trametes versicolor and Bjerkandera sp., were combustion of organic carbons such as burning fossil fuels, reported as being capable of degrading naturally occurring wood and municipal solid waste. They are of great polymer, lignin. Lignin possesses similar structure to that environmental concern due to their ubiquitous presence of PAHs and thus, these fungi are plausible candidates for and persistence in air, water and soil, as well as, their PAH degradation (Hammel, 1995; Hestbjerg et al., 2003). toxicity to humans and other living organisms (Keith and The world of fungi provides a fascinating and almost Telliard, 1979). Humans may be exposed to these endless source of biological diversity, which is a rich compounds from a wide variety of sources, such as source of exploitation (Manoharachary et al., 2005). through occupation, natural environment and diets etc Although fungal flora mainly species Western fungi have (Doyle et al., 2008). Although PAHs may fade away from been well documented (Steffen, 2003), there is very little the environment through adsorption, chemical research on fungal species in relation to their use as degradation, photolysis and volatilization, microbial potential remediation (Manoharachary et al., 2005). degradation is the major process to remove PAH In addition, previous studies showed that various fungi contamination in the environment (Yuan et al., 2002). are able to mineralize PAHs and PAHs removal rates with Microbial diversity offers an immense field of friendly P. ostreatus at different concentrations and can be environment for mineralization of pollutants or compared with other investigations conducted (Run and transformation into non-hazardous compounds with less Eyini, 2011; Covino et al., 2010). Previous research damaging options. There is general interest in the study of showed that the enzymes secreted by fungi of white rot to the diversity of indigenous micro-organisms capable of degrade lignin (peroxidase, MNP and laccase) have been degrading different pollutants due to their varying effects recognized to be especially important in the degradation of on the environment (Watanabe et al., 2002). White rot PAHs (Chang et al., 2001; Wariishi et al., 1992). In this Academia Journal of Scientific Research; Torres et al. 377

study, PAHs biodegradation was evaluated by P. ostreatus Analytical methods in liquid culture at different concentrations of the pollutant. After the incubation, the culture broth was filtered and residue (fungal body) separated by filtration. Briefly, PAHs extract was prepared by mixing 10 ml culture sample and MATERIALS AND METHODS 30 ml hexane in a separating funnel and each sample vigorously shaken for 3 min. The hexane extract HAPs was Chemicals collected in vials amber and the extraction procedure repeated. The final extract was concentrated in a rotary The crude oil used in the study was managed from the evaporator and each concentrated sample was Lázaro Cardenas refinery in Minatitlán Ver. México. resuspended in cyclohexane and cleaned C-18 cartridge Standard PAHs were purchased from Sigma-Aldrich (98 to packed with silica gel SUPELCO mark. Each sample was 99% purity), containing Benzo [b] fluoranthene, Benzo eluted with 30 ml of the solvent mixture dichloromethane: [g,h,i] perylene, Benzo [k] fluoranthene, Indeno [1,2,3-cd] hexane (2:3). The eluate was concentrated on Rotavapor pyrene, acenaphthene, fluoranthere, naphthalene, Benzo and resuspended in acetonitrile. Samples were stored in [a] anthracene, Benzo [a] pyrene, chrysene, amber vials at -4°C until analysis. acenaphthylene, anthracene, fluorene, phenanthrene, The mycelium of each culture incubated at 15 days was pyrene and dibenzo [a,h] anthracene; that according the cleaned with acetone and dried at 55°C for 24 h. The dry EPA are the most dangerous pollutants. Solvents used in weight of each mycelium was measured. Approximately 1 this study were purchased from Sigma-Aldrich (USA) of an g of mycelium (previously milled in a mortar) dry weight analytical and HPLC grade. was mixed in 15 ml of acetone: dichloromethane (1:5) in a test tube for 1 min using a vortex mixer. The sample was incubated at 15°C for 40 min in an ultrasonic cleaner Organism and culture conditions Branson 2510 and centrifuged for 15 min at 8000 rpm. The supernatant was transferred to a vial and the P. ostreatus ATCC38540 was obtained from Difco, Detroit, extraction procedure repeated for the same sample earlier MI, USA. Was stored in potato dextrose agar (PDA) plate at mentioned. The two sample extractions were combined 5°C and was then activated at 27°C for 3 days darkness. and concentrated in a rotary evaporator; the concentrate When the entire plate was covered by mycelia, 10 agar also was resuspended in cyclohexane and cleaned in a C- plugs (5 mm in diameter) cut from the PDA culture plate 18 cartridge. The resuspended sample in cyclohexane was were transferred into 100 ml of a sterilized 3% malt eluted with 15 ml of dichloromethane: hexane (2:3, V/V). extract liquid culture medium in a 250 ml Erlenmeyer flask Finally, the eluate was concentrated again on a rotary and incubated at 27°C on a rotary shaker (150 rpm) in the evaporator and resuspended in acetonitrile. Each sample dark for 5 d before use. was analyzed in a chromatograph high performance liquid (HPLC) 1260 equipped with a fluorescence detector, quaternary pump, autosampler and Chemstation software Experimental design (all from Agilent technologies, Germany). Separation of the PAHs was Carried out on a ZORBAX Eclipse PAH (4.6 mm × Experiments were performed in 250-ml Erlenmeyer flasks 50 mm, 1.8 um) column. The mobile phase was containing 100 ml of fungal culture in minimum medium acetonitrile: water mixture (60:40), flow rate 0.8 ml.min-1, for basidiomycetes plus 1 ml tween 80. The experimental the sample volume of 20 µl and a run time of 16 min. design was a randomized completely block design (RCBD) Fluorescence levels excitation and emission were set at where three treatments at concentrations of 17400, 32700 260/352, 260/420 and 260/460 respectively. and 53400 mg.L-1 of crude oil were handled to study their effects on the degradation of PAHs. Mycelia plugs of selected fungi were cut from the outer edge of an actively Data analysis growing culture on an inoculums plate. Ten 5-mm disks obtained by punching out with a cork-borer from the outer The data was recorded as means ± standard deviations. edge of an actively growing culture of a particular fungus One-way analysis of variance (ANOVA) and Tukey´s were inoculated into a flask containing 100 ml of liquid multiple comparisons were carried out to find the best medium. The flasks were incubated at 27°C on a rotary treatment using the software SAS 9.0. Differences between shaker (150 rpm) in the dark. Growth and substrate means at 5% (P <0.05) level were considered significant. consumption were determined at 15 days. All media were sterilized by autoclaving at 121°C for 20 min. Control experiments were performed by incubating crude oil in RESULTS AND DISCUSSION autoclaved liquid medium (121°C for 20 min) without inoculums. All assays were conducted in triplicate. The growth of the organism in cultures with different PAH Academia Journal of Scientific Research; Torres et al. 378

Table 1. Effect of the initial PAH concentration on biomass and PAH degradation in the culture of P. ostreatus ATCC38540 at 27°C.

% Degradation of PAHs PAHs conc. (mg.L-1) Biomass (g) LMW HMW Total PAHs 25.99 ± 1.28 1.253 ± 0.052 77.7c ± 1.59 97.3b ± 1.09 85.8c ± 1.35 49.01 ± 2.18 1.421 ± 0.061 88.5b ± 1.33 97b ± 1.05 91.9b ± 1.2 80.05 ± 3.85 1.671 ± 0.073 97.7a ± 1.13 98.9a ± 1.6 98.2a ± 1.34

Data are presented as means ± SD. Means in the same column with a letter in common are not significantly different among treatments (P < 0.05).

Table 2. Degradation and bioaccumulation of PAHs at a concentration of 80.051 mg.l-1, P. ostreatus as degrading agent.

Control Biodegradation Intracellular Total removal PAH Rings (mg.L-1) (mg.L-1) accumulation (mg.L-1) (mg.L-1) Naphthalene 2 15.712 ± 0.07 14.661 ± 0.02(93.3) 1.051 ± 0.00(6.7) 15.712 ± 0.83(100) Acenaphthene 3 9.459 ± 0.09 7.954 ± 0.07(84.1) 0.547 ± 0.01(5.8) 8.501 ± 0.27(89.9) Phenantjrene 3 3.694 ± 0.04 3.594 ± 0.01(97.3) 0.037 ± 0.01(1.0) 3.631 ± 0.10(98.3) Fluoranthene 3 16.680 ± 0.10 16.140 ± 0.01(96.8) 0.527 ± 0.01(3.2) 16.667 ± 1.00(99.9) Total LMW PAHs 2 y 3 45.545 ± 0.15 42.349 ± 0.07 (93.0) 2.162 ± 0.01(4.7) 44.511 ± 1.13(97.7) Chrysene 4 6.783 ± 0.05 6.645 ± 0.01(98.0) 0.055 ± 0.00(0.8) 6.700 ± 0.21(98.8) Pyrene 4 2.853 ± 0.06 2.795 ± 0.00(98.0) 0.000 ± 0.00(0.0) 2.795 ± 0.10(98.0) Benzo[a]anthracene 4 3.060 ± 0.01 2.990 ± 0.01(97.7) 0.025 ± 0.00(0.8) 3.015 ± 0.21(98.5) Benzo[b]fluoranthene 4 2.358 ± 0.01 2.338 ± 0.00(99.2) 0.000 ± 0.00(0.0) 2.338 ± 0.00(99.2) Benzo[k]fluoranthene 5 0.742 ± 0.01 0.737 ± 0.00(99.3) 0.000 ± 0.00(0.0) 0.737 ± 0.00(99.3) Benzo[a]pyrene 5 4.269 ± 0.02 4.248 ± 0.00(99.5) 0.000 ± 0.00(0.0) 4.248 ± 0.01(99.5) Benzo[g,h,i]perylene 6 7.400 ± 0.08 6.588 ± 0.02(89.0) 0.660 ± 0.03(8.9) 7.248 ± 0.13(97.9) Indene[1,2,3-cd] pyrene 6 7.041 ± 0.04 6.567 ± 0.02(93.3) 0.455 ± 0.02(6.5) 7.022 ± 0.22(99.8) Total HMW PAHs 4 a 6 34.506 ± 0.05 32.908 ± 0.04(95.4) 1.195 ± 0.01(3.5) 34.103 ± 1.6(98.9) Total PAHs 80.051 ± 0.55 75.257 ± 0.02(94.0) 3.357 ± 0.02(4.2) 78.614 ± 1.34(98.2)

Numbers in () are % degradation defined by the following equation: (PAH removed*100)/control). Data are presented as means ± SD.

concentrations were compared at the end of 15 days was capable of removing 98.2% (Table 2; high rates of incubation (Table 1). It was found that the higher the PAH degradation were observed in each HAP) in these concentration, the higher the biomass. These data are concentrations. The HAP presented a complete removal of contrary to those reported by Ting et al. (2011) where naphthalene. PAHs achieved low molecular weight (LMW) with an incubation time of 10 days it was observed that the of 97.7%, while 98.9% of high molecular weight (HMW) micellar biomass decreased with increasing concentration PAHs. In the case of P. ostreatus at concentrations of of PAHs using G. lucidum. This result suggested that PAH 80.051 mg.L-1 high rates of degradation of individuals did not inhibit the growth of P. ostreatus. When the PAHs were observed and this may be due to the concentrations of PAHs in the cultures were elevated to concentration of tween 80 added to them. In cultures of P. 49.01 and 80.05 mg.L-1, the degradation activities were ostreatus, the fenanthrene, pyrene and benzo[a]anthracene enhanced to 91.9 and 98.2% respectively. The increment degraded evenly over a period of 15 days culture. In of degradation activities in the cultures with high addition to the stimulating effects caused by the type of concentrations of PAHs was probably due to no growth inoculums, support could be derived from its ability to inhibition. This is because basidiomycetes are able to act slowly release phenolic compounds capable of acting as through mechanisms based on degradation unspecific mediators in the degradation of PAHs (Cañas et al., 2007) radicals produced in the extracellular environment (Chung and stimulating of ligninolytic fungal system (Crestini et et al., 2000). Previous studies showed that several fungi al., 1996). are able to mineralize PAHs and removal rates of PAHs A study with P. ostreatus and addition of tween 80 with P. ostreatus at different concentrations can be facilitated degradation of fenanthrene, pyrene and compared with studies already reported and varying benzo[a]pyrene in a reactor rotating biological contact degradations of 150 to 1500 mg.kg-1 PAHs (Run and Eyini, where the results showed that the tween 80 increased the 2011; Covino et al, 2010). solubility of PAHs so as to make available to ligninolytic It was also shown that enzymes secreted by fungi of enzyme system of P. ostreatus, (Akdogan and Pazarlioglu, white rot degraded lignin (peroxidase MnP and laccase) 2011). Moreover, the high removal bap at different and are responsible for the degradation of PAHs (Chang et concentrations is because it proved to be more susceptible al., 2001). At concentrations of 80.051 mg.L-1, P. ostreatus to oxidative attack by fungi of white rot (Novotny et al., Academia Journal of Scientific Research; Torres et al. 379

1999) and purified preparations of degradative enzymes Chung NH, Lee IS, Song HS, Bang WG (2000). Mechanisms used by white lignin (Johannes and Majcherczyk, 2000). Bioavailability of rot fungus to degrade lignin and toxic chemicals. J. Microbiol. Biotechnol. 10: 737-752. the compound increases by the effect of the surfactant Covino S, Svobodová K, Cvancarova M, D´Annibale A, Petruccioli M, used to emulsify the medium. Fluoranthene high removal Federici F, Kresinová Z, Galli E, Cajthaml T (2010). Inoculum carrier is comparable to that of other fungi such as Trametes and contaminant bioavailability affect fungal degradation versicolor, Trametes trogii, Ganoderma carnasum and P. performances of PAH contaminated solid matrices from a wood preservation plant. Chemosphere 79: 855-864. ostreatus, while T. versicolor, G. carnasum and T. trogii Crestini C, D'Annibale A, Giovannozzi-Sermanni G (1996). Aqueous plant degraded the fluoranthene 30% and P. ostreatus and extracts as stimulators of laccase production in liquid cultures of metabolized 85% approximately a concentration of 30 Lentinus edodes. Biotechnol. Tech. 10: 243-248. mg.L-1 within six weeks (Akdogan and Pazarlioglu, 2011). Díaz De Rienzo MA, Kamalanathan ID, Martin PJ (2016). Comparative study of the production of rhamnolipid biosurfactants by B. Degradation of chrysene was more efficient in other thailandensis E264 and P. aeruginosa ATCC 9027 using foam studies with fungi such as Nematoloma frowardii, fractionation. Process. Biochem. 51: 820-827. Bjerkandera, Irpex lacteus and Lentinus tigrinus where it Doyle E, Muckian L, Hickey AM, Clipson N (2008). Microbial PAH degrades up to 35% of 10 mg.L-1 in four weeks (Valentin et degradation: Advances. Appl. Microbiol. 65: 27-66. Hammel KE, (1995). Organopollutant degradation by ligninolytic fungi. al., 2006; Sack et al., 2006; Leonardi et al., 2008). The In: Young LY, Cerniglia CE, editors. Microbial transformation and enzymes released by P. ostreatus were oxidized by this degradation of toxicorganic chemicals. New York: Wiley-Liss Inc. 331- compound bioavailability tween 80 that produced the 346. observed increased intracellular degradation. In cultures Hestbjerg HP, Willumsen A, Christensen M, Andersen O, Jacobsen CS (2003). Bioaugmentation of tar-contaminated soils under field of P. ostreatus, fenanthrene, pyrene and conditions using Pleurotus ostreatus refuse from commercial benzo[a]anthracene its degradation percentages were mushroom production. Environ. Toxicol. Chem. 22: 692-698. similar though each PAH concentrations are different. In Johannes C, Majcherczyk A (2000). Natural mediators in the oxidation of addition, P. ostreatus can produce an emulsifying agent polycyclic aromatic hydrocarbons by laccase mediator systems. Appl. Environ. Microbiol. 66: 524-528. Pseudomonas capable of solubilizing as PAHs (Diaz De Keith LH, Telliard WA (1979). Priority pollutants I-a perspective view. Rienzo et al., 2016). Environ. Sci. Technol. 13: 416-423. Leonardi V, Giubilei MA, Federici E, Spaccapelo R, Sasek V, Novotny C, Petruccioli M, D'Annibale A (2008). Mobilizing agents enhance fungal degradation of polycyclic aromatic hydrocarbons and affect diversity Conclusion of indigenous bacteria in soil. Biotechnol. Bioeng. 101: 273-285. Manoharachary C, Sridhar K, Reena S, Adholeya A, Suryanarayanan TS, The results showed that PAHs were degraded by P. Seema-Rawat JBN (2005). Fungal biodiversity: distribution, ostreatus ATCC38540 in liquid culture. As the conservation and prospecting of fungi from India. Curr. Sci. 89: 58-71. Novotný C, Erbanová P, Sasek V, Kubátová A, Cajthaml T, Lang E, Krahl J, concentration of PAHs increased it was observed that P. Zadrazil F (1999). Extracellular oxidative enzyme production and PAH ostreatus was able to increase activity removal and removal in soil by exploratory mycelium of white-rot fungi. increase their biomass. Furthermore, addition of tween 80 Biodegradation 10: 159-168. achieved a better bioavailability HMW PAHs which led to a Run A, Eyini M (2011). Comparative studies on lignin and polycyclic aromatic hydrocarbons degradation by basidiomycetes fungi. Bio. high degradation of the compounds. Technol. 102: 8063-70. Sack U, Hofrichter M, Fritsche W (2006). Degradation of polycyclic aromatic hydrocarbons by manganese peroxidase of Nematoloma ACKNOWLEDGMENT frowardii. FEMS Microbiol. Lett. 152: 227-234. Steffen KT (2003). Degradation of recalcitrant biopolymers and polycyclic aromatic hydrocarbons by litter decomposing basidiomycetous fungi. The authors wish to thank CONACyT (Consejo Nacional de Academic Dissertation in Microbiology. Finland, University of Helsinki. Ciencia y Tecnología) from Mexico for PhD scholarship Ting W, Yuan S, Wu S, Chang B (2011). Biodegradation of phenanthrene granted. and pyrene by Ganoderma lucidum. Inter. Biodeter. biodeg. 65: 238- 242. Valentín L, Feijoo G, Moreira MT, Lema JM (2006). Biodegradation of polycyclic aromatic hydrocarbons in forest and salt marsh soils by REFERENCES white-rot fungi. Int. Biodeter. Biodegr. 58: 15-21. Wariishi H, Valli K, Gold MH (1992). Manganese (II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete Akdogan HA, Pazarlioglu NK (2011). Fluoreno biodegradation by P. chrysosporium. J. of Biological. Chem. 267(2): 2388–95. ostreatus Part I: Biodegradation by free cells. Process. Biochem. 46: 834- Watanabe K, Futamata H, Harayama S (2002). Understanding the 839. diversity in catabolic potential of microorganisms for the development Akdogan HA, Pazarlioglu NK (2011). Fluoreno biodegradatión by P. of bioremediation strategies. Anton. Van. Leeuwen. 81: 655-663. ostreatus Part II: Biodegradation by immobilized cells in a recycled Yuan SY, Shiung LC, Chang BV (2002). Biodegradation of polycyclic packed bed reactor. Process. Biochem. 46: 840-846. aromatic hydrocarbons bya mixed culture. C. Cañas AI, Alcalde M, Plou F, Martínez MJ, Martínez AT, Camareno S (2007). Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil. Environ. Sci. Technol. 41. 2964-71. Chang BV, Chang JS, Yuan SY (2001). Anaerobic degradation of phenanthrene in river sediment under nitrate reducing conditions. Bulletin of Environ. Contamin. Toxicol. 67: 898-905.