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Screening and Molecular Characterization of Polycyclic Aromatic Hydrocarbons Degrading Yeasts

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Screening and Molecular Characterization of Polycyclic Aromatic Hydrocarbons Degrading Yeasts Türk Biyokimya Dergisi [Turkish Journal of Biochemistry–Turk J Biochem] 2015; 40(2):105–110 doi: 10.5505/tjb.2015.16023 Research Article [Araştırma Makalesi] Yayın tarihi 5 Mart 2015 © TurkJBiochem.com [Published online March 5, 2015] Screening and molecular characterization of polycyclic aromatic hydrocarbons degrading yeasts [Polisiklik aromatik hidrokarbonları parçalayan mayaların taranması ve moleküler karakterizasyonu] Diğdem Tunalı Boz, ABSTRACT Hüsniye Tansel Yalçın, Objective: Disasters such as leakages and accidental falls are the main causes of environmental pollution by the petroleum industry product. Since no commercial yeast strain with bio- Cengiz Çorbacı, degradation capacity is available, we aimed to isolate and characterize hydrocarbon degrading Füsun Bahriye Uçar yeasts. Methods: Yeast isolates used in the study were isolated from samples of wastewater, active sludge and crude oil, which was obtained from a petroleum refinery as well as from soil samples, which were contaminated with crude oil. Yeast isolates used in the study were isolated from wastewater, active sludge and petroleum samples obtained from petroleum refinery and soil Ege University, Faculty of Science, Biology samples contaminated with petroleum. Degradation of naphthalene, phenanthrene, pyrene and Department, Basic and Industrial Microbiology crude petroleum by yeasts were determined using a microtiter plate-based method. Molecular Section, İzmir characterization was achieved by performing a sequence analysis of the ITS1-5.8S rRNA-ITS2 and 26S rRNA regions. Results: In total, 100 yeast isolates were obtained from four different samples. Following the incubation in media containing different polycyclic aromatic hydrocarbon compounds (naphthalene, phenanthrene, pyrene) and crude petroleum, 12 yeast isolates were detected to degrade more than one polyaromatic hydrocarbon. Sequence analyses of rRNA regions revealed that the identified yeasts represented 10 species belonging to 6 genera. The isolates were identified as Candida parasilopsis, Candida sinolaborantium, Cryptococcus albidus, Cryptococcus diffluens, Cryptococcus uzbekistanensis, Pichia kudriavzevii, Rhodosporidium diobovatum, Rhodotorula glutinis, Rhodotorula muciloginosa and Saccharomyces cerevisiae. Conclusion: Yeast strains that are capable of degrading more than one polycyclic aromatic hydrocarbon compound have the potential of being utilized in future research. Key Words: Identification, yeasts, polycyclic aromatic hydrocarbon, 26S rRNA, ITS1-5.8S rRNA-ITS2 Conflict of Interest: The authors have no conflict of interest. Correspondence Address ÖZET [Yazışma Adresi] Amaç: Petrol endüstrisi ürünleri ile oluşturulan sızıntılar ve kazalar gibi felaketler, çevresel kir- liliğe neden olan ana faktörlerdir. Mevcut durumda, biyo-parçalama kapasitesine sahip ticari bir Füsun Bahriye Uçar, MD. maya suşu bulunmamaktadır. Bu yüzden, geniş hidrokarbon parçalama aktivitesine sahip maya suşlarının saptanması, bu kirliliklerin azaltılmasında önemli bir rol oynamaktadır. Belirtilen Ege Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, bu sebeple, çalışmamızda, hidrokarbon parçalayan mayaların izolasyonu ve karakterizasyonu Temel ve Endüstriyel Mikrobiyoloji Anabilim Dalı, amaçlanmıştır. 35100, Bornova 35100, İzmir, Türkiye Phone: +90 232 3111799 Metod: Çalışmada kullanılan maya izolatları, petrol rafinerisinden alınmış atık su, aktif çamur E-mail: [email protected] ve petrol örnekleri ve petrol ile kontamine olmuş toprak örneklerinden izole edilmiştir. Nafta- len, fenantren, piren ve ham petrolün mayalar tarafından parçalanması, mikrotitre plaka yönte- mi kullanılarak saptanmıştır. Mayaların moleküler karakterizasyonu, ITS1-5.8S rRNA-ITS2 ve 26S rRNA bölgelerinin dizi analizi ile gerçekleştirilmiştir. Bulgular: Toplamda, dört farklı örnekten 100 maya izolatı elde edilmiştir. Farklı polisiklik aromatik hidrokarbon bileşikleri (naftalen, fenantren, piren) ve ham petrol içeren ortamlarda inkübasyondan sonra, 12 maya izolatının birden fazla poliaromatik hidrokarbonu parçalayabil- diği bulunmuştur. rRNA bölgelerinin dizi analizi, tanılanan mayaların 6 genusa dahil olan 10 türü temsil ettiklerini göstermiştir. İzolatlar, Candida parasilopsis, Candida sinolaborantium, Cryptococcus albidus, Cryptococcus diffluens, Cryptococcus uzbekistanensis, Pichia kudriav- zevii, Rhodosporidium diobovatum, Rhodotorula glutinis, Rhodotorula muciloginosa ve Sacc- haromyces cerevisiae olarak tanılanmıştır. Sonuç: Birden fazla polisiklik aromatik hidrokarbon bileşiğini parçalayan maya suşları, ileriki araştırmalarda kullanım potansiyeline sahiptir. Anahtar Kelimeler: Tanılama, mayalar, poliaromatik hidrokarbonlar, 26S rRNA, ITS1-5.8S Registered: 12 March 2014; Accepted: 30 September 2014 rRNA-ITS2 [Kayıt Tarihi: 12 Mart 2014; Kabul Tarihi: 30 Eylül 2014] Çıkar Çatışması: Yazarların çıkar çatışması yoktur. http://www.TurkJBiochem.com 105 ISSN 1303-829X (electronic) 0250-4685 (printed) Introduction In the present study, we aimed to isolate and characterize hydrocarbon degrading yeasts. Petroleum-based products are the main source of energy for industry and daily life [1]. Release of these com- Materials and Methods pounds into the environment is a main cause of water and soil pollution [1,2]. Leakages and accidental spills Sampling and isolation of yeasts occur frequently during surveying, production, refining, In the study, the active sludge (1) and petroleum samples transport, and storage of petroleum and petroleum-based (1) were obtained from İzmir Tüpraş petroleum refinery products [1]. and the two soil samples contaminated with petroleum Petroleum is a complex mixture of numerous compounds, were obtained from Mersin harbor from Turkey. Yeast which can be separated into four main groups: alkanes, isolation was performed on Yeast Malt (YM) agar com- aromatics, resins, and asphaltenes. The alkane fraction posed of (g/L); 3 g yeast extract, 3 g malt extract, 5 g pep- is the most biodegradable, whereas the resins and as- tone, 10 g glucose and 15 g agar. Inoculated plates were phaltenes are resistant to biological degradation. The incubated at 27°C for 5-7 days [19]. aromatic compounds, particularly the polycyclic aromatic Determination of degradation capabilities of yeasts by hydrocarbons (PAHs), are compounds composed of two microtiter plate technique or more bonded benzene rings [3]. Since some PAHs The screening of yeasts for their capability of utilizing are harmful, mutagenic and carcinogenic, they impose hydrocarbons was performed in microtiter plates (flat a potential risk to human health. The US Environmental bottom, 96 wells), using 200 µl Bushnell-Haas medium Protection Agency (USEPA) has recognized sixteen PAH (pH 7.2), per well. Each well received 10 µl of each stock compounds as important pollutants, whose levels in in- solution of phenanthrene, pyrene or naphthalene (>96% dustrial wastes require monitoring [4,5]. purity, Fluka) dissolved in acetone to give a final concen- Reducing PAHs in a contaminated environment is a con- tration of 10 mg PAH/l. The acetone was allowed to evapo- siderable challenge. Mechanical and chemical techniques rate at room temperature. Afterwards, the PAH-containing have limited efficiency and can be expensive [6]. Biodeg- medium was inoculated with 10 µl of a dense suspension radation via microorganisms is generally accepted as the of microbial cells. Each yeast isolate was inoculated in ideal and principal way of PAH removal from the polluted four different microtiter plates containing phenanthrene, environment owing to some characteristic advantages in- pyrene, naphthalene and crude petroleum. Plates were cluding cost effectiveness and comparatively high clean- incubated for 21 days at 27°C. At the end of the period, up efficiency [6-10]. Microorganisms, mainly bacteria 50 μl of p-iodonitrotetrazolium violet (INT) indicator was and fungi, were detected to reduce PAHs via either me- added to each well. Plates were re-incubated for 24 hours tabolism or co-metabolism [8,11,12]. More than 80% of and each microtiter plate was scored for positive results. the studies conducted on microbial biodegradation of oil- INT is one of the growth indicator reagents and thus, mi- related contaminants are devoted to bacterial biodegrada- crobial growth was observed easily as colored precipitate. tion. Bacteria are the most studied microorganisms and For medium to low chain alkanes a positive result was in- the contribution of bacteria during hydrocarbon mineral- dicated by red precipitate; crude oil samples were scored ization in soil has been widely studied [13-15], however, for brown precipitate [20,21]. a small number of fungal species capable of degrading Phenotypic characterization of the yeast isolates hydrocarbons were reported [8,10-12,14,16]. Individual yeast isolates with degradation capacity were Hydrocarbon pollutants have similar or analogous mo- characterized based on their physiological and morpho- lecular structures, which allow the fungi to act on them logical properties according to Kurtzman et al. [22]. as well. Fungi possess degrading capabilities to handle Sequencing of ribosomal RNA regions a series of hydrocarbon compounds that serve as poten- In our study, yeast isolates showing PAH-degrading ca- tial carbon sources. Also, fungi have numerous stress pabilities were also characterized by sequencing two responses that generate further phenotypic and genetic different rRNA genes. DNA isolation was performed as variety and thus, they can adapt to a new environmen- described
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