Ham petrolle kirlenmiş topraklarda istiridye mantarı (Pleurotus ostreatus)'nın mikoremediasyon kapasitesinin araştırılması
The research of the mycoremediation capacity of the oyster mushroom in the crude oil contaminated soils
Yazar: CEYDA ZAZOĞLU
Danışman: PROF. DR. MUSTAFA SAİT YAZĞAN
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Çevre Bilimleri Ana Bilim Dalı / Çevre Bilimleri ve Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Biyoteknoloji = Biotechnology ; Çevre Mühendisliği = Environmental Engineering
Dizin: Beyaz çürükçül mantarlar = White rot fungus ; Biyoremediasyon = Bioremediation ; Ham petrol = Crude oil ; Kayın mantarı = Pleurotus ostreatus

Özet:
Çevrenin sentetik organik bileşiklerle kirlenmesi dünya genelinde büyük bir sorun haline gelmiştir. Biyosferde doğal olarak bulunmayan bu sentetik bileşiklere ksenobiyotik denir. Bu sentetik bileşiklerin çoğu doğal mikroflora ve fauna tarafından kolayca parçalanamazlar. Endüstriyel ve çevresel biyoteknolojiye dayalı biyolojik yaklaşımlar, atık üretimi azaltımı, atıkların temizlenmesi ve bu atıkların kullanışlı ya da zararı olmayan, kolayca ayrışabilen kimyasal formlara dönüşümü üzerinde çalışan "temiz teknolojiler" konusunun geliştirilmesine odaklanır. Temiz teknolojiler atıkların iyileştirilmesi için canlı organizmaların metabolik yollarını kullanmayı hedefler. Bu tarz biyolojik yöntemlerden birisi de mikoremediasyon (fungal remediasyon) yöntemidir. Mantarlar ve diğer funguslar, geniş sayıda atık/kirleticinin parçalanması için enzimatik birer mekanizma olarak görev alır. Biyoremediasyon aracı olarak mantarların, daha çok bazitli mantarların, günümüzde iyileştirme amacıyla kullanımı daha yaygın hale gelmektedir.
Bu tez çalışmasında fungusları, özellikle beyaz-çürükçül mantarları, kullanmanın getireceği beklentilerden birisi olan yapay olarak kirletilmiş toprakların temizlenmesi araştırılmıştır. Bu beyaz-çürükçül mantarlar hücre-dışı lignin modifiye edici enzimler salgılamalarından ötürü, çok çeşitli sayıda kirletici moleküllerin ayrıştırılmasında oldukça etkilidirler. Düşük substrat spesifiteleri vardır (çok sayıda substrata etki edebilir), bu yüzden lignine geniş çapta benzeyen birçok molekül üzerinde etkili olabilirler. Sistemlerinde lignini parçalamak için kulanılan enzimler; lignin peroksidaz (LiP), manganez peroksidaz (MnP), çeşitli H2O2 üreten enzimler ve lakkaz enzimini içerir. Ayrıştırma süreci kirlenmiş bölgelere kıymık, saman ve mısır koçanı gibi karbon kaynakları eklenerek artırılabilir. 
Bu çalışmada 25 ve 50 günlük süreler boyunca, 5, 10 ve 15 g (% 2.5, % 5 ve % 7.5) Irak (light) ham petrolü ile yapay olarak kirletilen ve steril edilen toprakların, beyaz-çürükçül bir fungus olan istiridye mantarı (Pleurotus ostreatus) tarafından giderimi incelenip, ham petrol içerisindeki Toplam Petrol Hidrokarbon (TPH) seviyelerindeki değişimler, önceden oluşturulan standart eğriye göre belirlenmiştir. 
Ticari komposttan saf kültür hazırlanması, tohum ve saman kompostu ile mantar kültürü yapımı, sterilizasyon ve pastörizasyon aşamaları, deney kurulumu, ham petrol konsantrasyonlarının eklenmesi, toprak ve misel örneklerinin ekstraksiyon, distilasyonları, ekstrakte olmuş organik madde miktarı hesaplamaları ve standart eğrinin oluşturulması 5. Bölümde anlatılmıştır. Bölüm 6'da deneysel sonuçlar tartışılıp, tartışma ve öneriler Bölüm 7'de verilmiştir.
Deneyde hem toprak, hem misel örneklerindeki TPH miktarları Irak (light) ham petrolü kullanılarak UVF spektrofotometrede bakılıp bunlar arasındaki korelasyonlar STATISTICA 6.0 istatistiksel analiz programıyla hesaplanmıştır. 
Deney Seti I 15 g ve Deney Seti II 10 g toprak örneklerinde gözlenen TPH bulgularının, kendi kontrol gruplarından daha yüksek olduğu gözlenmiştir. Set II misel TPH miktarlarının Set I misel TPH miktarlarına göre gerçekleşen artışı, 5, 10 ve 15 g konsantrasyonları için sırasıyla % 14.5, % 62.5 ve % 57.1 olarak elde edilmiştir. Bu verilere göre misele geçen miktar en yüksek olarak 10 g örneklerinde görülmüştür. 
Kümelenme (Cluster) Analizi sonuçlarına göre 25. ve 50. günlerdeki setler arasında tek bir kümelenme altında toplanan birbiriyle oldukça ilişkili bir uyumun olduğu anlaşılmıştır.
Bu tez çalışmasına paralel yürütülen, üç farklı konsantrasyonda üç farklı ağır metal üzerinde P. ostreatus'un mikoremediasyon etkisinin araştırıldığı benzer bir deneyde 60 günün sonunda mantarın vejetatif gövde büyümesi (pin oluşumu), bu deneyde ise 50. gün sonunda sadece primordia oluşumu gözlenmiştir.

Abstract:
The pollution of the environment with synthetic organic compounds has become a major problem all around the world. These synthetic compounds are called as xenobiotics which do not occur naturally in the biosphere. Many of these synthetic compounds are not easily degraded by the natural microflora and fauna. Biological approaches based on the industrial and environmental biotechnology are focusing on the development of "clean technologies" which emphasizes on reducing waste generation, treatment and conversion of waste in some useful or non-virulent, easily degradable chemical forms. Further, these clean technologies focus on the use of metabolic pathways of living organisms for the remediation of waste. 
Different technologies for soil and groundwater cleanup of petroleum hydrocarbons have been developed in the last three decades. Each technology has certain restrictions and advantages in the applied field. One such biological method is mycoremediation (fungal remediation). The mushrooms and other fungi act as enzymatic machinery for degradation of a wide variety of waste/pollutant. However mushrooms, basidiomycetous fungi, are becoming more popular nowadays for remediation purposes as bioremediation tools. Therefore their cultivation all around the globe has become more popular. 
Mushroom cultivation is carried out successfully on many industrial wastes. The use of mushrooms as mycoremediation tools in the bio-transformation of industrial wastes to protein-rich fruiting bodies provides mushroom itself while it helps to solve the problems that are caused by the disposal of pollutants, at the same time.
Fungi that are used by humans for thousands of years for a variety of applications, plant polymers such as cellulose, hemicellulose and lignin are one of the main parsers in any ecosystem. Mushrooms have the ability to mineralize, release many elements and ions and store toxic substances in their bodies. They can facilitate the energy exchange between underground and aboveground systems. It has been proven that the mushrooms modify the soil permeability and soil ion exchange and eliminate the harmful effects of contaminated soil. Edible and/or medicinal mushrooms, also aquatic fungi play a role as natural healers.
Fungal remediation is one of the most complex areas in applied remediation engineering. Over the last 20 years, many mycologists and engineers have tried using mushrooms for the purpose of the reduction of organic compounds and have achieved positive results from the studies.
Much of the information known about the interaction between mushrooms and the waste materials are mostly based on studies conducted in the laboratory. However, mushrooms have been used in the treatment of a wide variety of waste and wastewater in the last two decades and their role in remediating different numbers of hazardous and toxic compounds in soil and in the sediments have been determined. Removal of metals, degradation and mineralization of phenols and chlorinated phenolic compounds, petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorinated insecticides and pesticides, dyes by mushrooms have been proven.
Oyster mushroom (P. ostreatus) is a fungus which can be produced easily and grows on various plant residues as substrate. Also, it is one of the most successfully colonizing species in soil and is often used in bioremediation studies. Pleurotus spp. can reduce lignocellulosic compounds and convert into protein-rich biomass. It also helps the management of agricultural waste which constitutes disposal problems. Oyster mushrooms grown on different agricultural waste shows higher colonization rate, early development and sporophore efficiency compared to other fungal species. Due to the ability of keeping up with the different agro-climatic conditions, cultivation of this mushroom has increased greatly in the last few years.
Soil pollution by petroleum hydrocarbons, usually occurs during the supply or discharge of oil that is caused by the flowing leakage from storage tanks. Most of the oil component is dangerous to human health and to soil biota, therefore the measurement of total petroleum hydrocarbons (TPH) is the first and most common hazard indicator necessary for peripheral diagnose.
Petroleum hydrocarbons are known to have entered the environment frequently in various ways and in large volumes. One of the largest and naturally occuring ways is the leaks from natural deposits that cause the appearance of petroleum oils in aquatic environments. Other methods comprise production, storage and transmission steps which constitute a significant potential for the accidental spread of petroleum hydrocarbons. Numerous cases have been documented on the oil leakages from underground storage tanks and the pipelines for the last two decades.
Crude oil is a colloidal mixture of myriad of hydrocarbon and non-hydrocarbon materials. The source material for nearly all oil products is the crude oil.
In this study, the remediation process by a white-rot fungi, Pleurotus ostreatus of the sterilized soils that are previously contaminated with 5, 10 and 15 g of Iraq (light) crude oil artificially has been observed and the changes in the Total Petroleum Hydrocarbon (TPH) levels in the crude oil according to the pre-established standard curve of oil has been determined during the period of 25 and 50 days. 
Preparing the pure culture from the commercial compost, mushroom cultivation process by making grain and straw spawn, sterilization and pasteurization steps, experimental design, adding crude oil concentrations, extractions and distillations of soil and mycelium samples, calculation of extracted organic matters and creating a standart curve are explained in Section 5. Experimental results are discussed in Section 6. Discussions and recommendations are given in Section 7.
Both soil and mycelium samples are extracted in order to evaluate the levels of TPH that are present in the related samples. The correlations between them are calculated via STATISTICA 6.0, a statistical analysis program.
The TPH findings in the experimental design Set I 15 g and Set II 10 g soil samples are observed to be higher than their control groups. The increase in the amount of TPH levels in the Set II mycelium samples according to the amount of TPH levels in the Set I mycelium samples are respectively 14.5 %, 62.5 % and 57.1 % for 5, 10 and 15 g concentrations. With respect to these data, the highest amount that has passed to mycelium was seen in the samples of 10 g.
Considering the results of Cluster Analysis, each set results (25th and the 50th days) collected under one single cluster which is understood to be a fairly compliance for the overall set results is occuring.
In a similar experiment, which was carried out correspondingly with this thesis study, investigating the mycoremediation effect of P. ostreatus on three different heavy metals with three different concentrations, mushroom fruiting body vegetation was observed at the end of 60 days. In this study, only mushroom primordia formation was observed at the end of 50 days.