Petrol ve hidrokarbon parçalayan bakterilerin izolasyonu ve biyoteknolojik potansiyellerinin belirlenmesi
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Tarih
2022
Yazarlar
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Cilt Başlığı
Yayıncı
Dicle Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Petrol dünyadaki en yaygın çevresel kirleticilerdir. Petrol kirleticileri balıkçılık, deniz habitatları, halobiyolar ve insan sağlığı için ciddi bir tehdit oluşturmakta ve yıllarca hatta on yıllarca sürebilen ekolojik dengeyi tahrip etmektedir. Bu çalışmada, Batman bölgesinde petrolle kirletilmiş alanlardan bakteri izole ederek, petrol parçalayan bu bakteri varyeteler üzerinde morfolojik, fizyolojik, biyokimyasal ve filogenetik analizler yapılarak tanımlanmıştır. Ayrıca, bu bakterilerin ham petrolü ve çeşitli hidrokarbonları enerji kaynağı olarak kullanabilme kapasiteleri, hidrokarbonları parçalama özellikleri ve biyoparçalamada görev alan genlerin tespit edilmesi üzerinde çalışmalar gerçekleştirildi. Batman petrol sahalarından petrol ile kirlenmiş bölgelerden Pseudomonas, Enterobacter, Delftia, Acinetobacter cinslerine ait 8 bakteri izole edilmiştir. Bu izole edilen bakterilerin fenotipik analizleri yapılmış ve 16S rRNA gen dizi analizine göre, sırasıyla D7, D8, D9, D10, D11, D12, E1 ve E2 olarak adlandırılan bakterilerin Pseudomonas aeruginosa'ya %100, Enterobacter ludwigii'ye %100, Delftia tsuruhatensis'e %100, Pseudomonas putida'ya %100, Acinetobacter pittii'ye %99,93, Pseudomonas rhodesiae'ye %100, Acinetobacter pittii'ye %100, Acinetobacter calcoaceticus'a %100 benzer oldukları belirlenmiştir. Çalışılan tüm bakteri suşlarının ham petrolü karbon ve enerji kaynağı olarak kullandıkları belirlenmiştir. Bakterilerin farklı uzunluklara sahip alifatik hidrokarbonlardan, sadece uzun zincirli n-alkanlarda (pentadekan, hekzadekan) ve dekanda iyi ürediği görülmüştür. Test edilen uzun zincirli alkanlar arasında, genel olarak bakteriler tarafından en çok tercih edilen hekzadekan olmuştur. Beş günlük inkübasyon sonucunda GC-MS analizi yapılarak, ham petrolü parçalama potansiyeli açısından yüksekten düşüğe doğru sıralayacak olursak; D11'in %43, D8'in % 27, D12'nin % 21, D10'nun %18, D9'un %12, E2 'nin ise %12 ve E1'in %9 oranında degradasyona uğrattıkları tespit edildi. Üç günlük inkübasyon sonucunda bakterilerin %1'lik alkan hidrokarbonlarını parçalama oranları GC-MS analizi ile belirlenmiştir. Hekzadekanı, D9'un yaklaşık olarak %65, D11'in %70, D12'nin %38, E1'in %76 oranında parçaladığı tespit edilmiştir. Pentadekanı, D7'in %3, D8'in %29, E2'in ise %48 oranında parçaladığı tespit edilmiştir. Ayrıca, üç günlük inkübasyon sonucunda, bakterilerin tek veya karışım halinde hekzadekan ve pentadekandan oluşan hidrokarbon karışımlarını (toplam %1) parçalama oranları belirlenmiştir. Bireysel olarak D8 %31, D11 %43, E2 %53 oranında etkili olurken, karışım halinde D8+D11 %60, D11+E2 %49, D8+E2 %61 ve D8+D11+E2 ise %72 gibi yüksek oranda biyodegradasyon yaptıkları tespit edildi. D11 ve E2 suşları için tasarlanan primerlerden ilki (primer 1) kullanılarak elde edilen PCR ürünlerinin alkM geninin dizi analizi sonuçlarına göre sırasıyla %93,06 ve %92,72 oranında Acinetobacter sp. BUU8 (A6N7F9) alkM gen dizisine benzerlik gösterdiği, ayrıca D11 ve E2 suşları için tasarlanan diğer primer (primer 3) kullanıldığında elde edilen PCR ürünlerinin alkB geni dizi analizi sonuçlarına göre, sırasıyla %94,48 oranında Psudomonas citronellolis (A0A024A3Z2) ve %95,52 oranında Pseudomonas mendocina (A4XPE8) alkB gen dizilerine benzerlik gösterdiği belirlenmiştir.
Petroleum is the most common environmental pollutant in the world. Oil pollutants pose a serious threat to fisheries, marine habitats, halobionts and human health, and destroy the ecological balance that can last for years or even decades. In this study, by isolating bacteria from oil-contaminated areas in the Batman region, these oil-degrading bacteria were identified by performing morphological, physiological, biochemical and phylogenetic analyzes on the varieties. In addition, studies were carried out on the capacity of these bacteria to use crude oil and various hydrocarbons as energy sources, their ability to break down hydrocarbons, and the determination of genes involved in biodegradation. Eight bacteria belonging to Pseudomonas, Enterobacter, Delftia, Acinetobacter genera were isolated from oil-contaminated areas of Batman oil fields. Phenotypic analyzes of these isolated bacteria were carried out and according to the 16S rRNA gene sequence analysis, the bacteria designated as D7, D8, D9, D10, D11, D12, E1 and E2 were found to be similar as 100% to Pseudomonas aeruginosa, 100% to Enterobacter ludwigii, 100% to Delftia tsuruhatensis, 100% to Pseudomonas putida, 99,93% to Acinetobacter pittii, 100% to Pseudomonas rhodesiae, 100% to Acinetobacter pittii and 100% to Acinetobacter calcoaceticus, respectively. It has been determined that all bacterial strains studied use crude oil as a carbon and energy source. It has been observed that bacteria grow well from aliphatic hydrocarbons of different lengths, only in long chain n-alkanes (pentadecane, hexadecane) and decane. Among the long chain alkanes tested, hexadecane was the most preferred by bacteria overall. After five days of incubation, GC-MS analysis is performed and the order of crude oil degradation capacity by bacteria from high to low were as follows; D11, D8, D12, D10, D9, E2 and E1 degraded 43%, 27%, 21%, 18%, 12%, 12% and 9%, respectively. After three days of incubation, the rate of degradation of 1% alkane hydrocarbons by bacteria was determined by GC-MS analysis. It has been determined that 1% hexadecane decomposes approximately 65% by D9, 70% by D11, 38% by D12 and 76% by E1. Pentadecane was found to be decomposed 3% by D7, 29% by D8, and 48% by E2. In addition, as a result of three days of incubation, the rate of bacterial degradation of hydrocarbon mixtures (total 1%) consisting of hexadecane and pentadecane, singly or as a mixture, was determined. While individually D8, D11 and E2 degraded 31%, 43% and 53% respectively, in mixtures biodegradation of hydrocarbon mixtures was as high as 60% by D8+D11, 49% by D11+E2, 61% by D8+E2 and 72% by D8+D11+E2. The results of the sequence analysis of the alkM gene of PCR products obtained using the first primer (primer 1) designed for D11 and E2 strains showed 93,06% and 92,72% alkM gene similarity to that of Acinetobacter sp. BUU8 (A6N7F9), respectively. The sequence analysis of the alkb gene PCR products using the other primer (primer 3) designed for D11and E2 strains showed 94,48% and %95,52% alkB gene similarity to that of Psudomonas citronellolis (A0A024A3Z2) and Pseudomonas mendocina (A4XPE8), respectively.
Petroleum is the most common environmental pollutant in the world. Oil pollutants pose a serious threat to fisheries, marine habitats, halobionts and human health, and destroy the ecological balance that can last for years or even decades. In this study, by isolating bacteria from oil-contaminated areas in the Batman region, these oil-degrading bacteria were identified by performing morphological, physiological, biochemical and phylogenetic analyzes on the varieties. In addition, studies were carried out on the capacity of these bacteria to use crude oil and various hydrocarbons as energy sources, their ability to break down hydrocarbons, and the determination of genes involved in biodegradation. Eight bacteria belonging to Pseudomonas, Enterobacter, Delftia, Acinetobacter genera were isolated from oil-contaminated areas of Batman oil fields. Phenotypic analyzes of these isolated bacteria were carried out and according to the 16S rRNA gene sequence analysis, the bacteria designated as D7, D8, D9, D10, D11, D12, E1 and E2 were found to be similar as 100% to Pseudomonas aeruginosa, 100% to Enterobacter ludwigii, 100% to Delftia tsuruhatensis, 100% to Pseudomonas putida, 99,93% to Acinetobacter pittii, 100% to Pseudomonas rhodesiae, 100% to Acinetobacter pittii and 100% to Acinetobacter calcoaceticus, respectively. It has been determined that all bacterial strains studied use crude oil as a carbon and energy source. It has been observed that bacteria grow well from aliphatic hydrocarbons of different lengths, only in long chain n-alkanes (pentadecane, hexadecane) and decane. Among the long chain alkanes tested, hexadecane was the most preferred by bacteria overall. After five days of incubation, GC-MS analysis is performed and the order of crude oil degradation capacity by bacteria from high to low were as follows; D11, D8, D12, D10, D9, E2 and E1 degraded 43%, 27%, 21%, 18%, 12%, 12% and 9%, respectively. After three days of incubation, the rate of degradation of 1% alkane hydrocarbons by bacteria was determined by GC-MS analysis. It has been determined that 1% hexadecane decomposes approximately 65% by D9, 70% by D11, 38% by D12 and 76% by E1. Pentadecane was found to be decomposed 3% by D7, 29% by D8, and 48% by E2. In addition, as a result of three days of incubation, the rate of bacterial degradation of hydrocarbon mixtures (total 1%) consisting of hexadecane and pentadecane, singly or as a mixture, was determined. While individually D8, D11 and E2 degraded 31%, 43% and 53% respectively, in mixtures biodegradation of hydrocarbon mixtures was as high as 60% by D8+D11, 49% by D11+E2, 61% by D8+E2 and 72% by D8+D11+E2. The results of the sequence analysis of the alkM gene of PCR products obtained using the first primer (primer 1) designed for D11 and E2 strains showed 93,06% and 92,72% alkM gene similarity to that of Acinetobacter sp. BUU8 (A6N7F9), respectively. The sequence analysis of the alkb gene PCR products using the other primer (primer 3) designed for D11and E2 strains showed 94,48% and %95,52% alkB gene similarity to that of Psudomonas citronellolis (A0A024A3Z2) and Pseudomonas mendocina (A4XPE8), respectively.
Açıklama
Anahtar Kelimeler
Petrol kirliliği, Bakteriyel karakterizasyon, 16S rRNA gen dizisi analizi, Hidrokarbon biyodegradsyonu, AlkB ve AlkM gen analizi, Oil pollution, Bacterial characterization, 16S rRNA gene sequence analysis, Hydrocarbon biodegradation, AlkB and AlkM gene analysis
Kaynak
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Scopus Q Değeri
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Sayı
Künye
Eren, A. (2022). Petrol ve hidrokarbon parçalayan bakterilerin izolasyonu ve biyoteknolojik potansiyellerinin belirlenmesi. Yayımlanmamış doktora tezi, Dicle Üniversitesi, Diyarbakır.
