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T.C. Bilecik Şeyh Edebali Üniversitesi Fen Bilimleri Enstitüsü Biyoteknoloji Anabilim Dali Termofilik Aktin

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T.C. Bilecik Şeyh Edebali Üniversitesi Fen Bilimleri Enstitüsü Biyoteknoloji Anabilim Dali Termofilik Aktin T.C. BİLECİK ŞEYH EDEBALİ ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOTEKNOLOJİ ANABİLİM DALI TERMOFİLİK AKTİNOMİSETLERİN İZOLASYONU, MOLEKÜLER KARAKTERİZASYONU VE KOMPOST KARIŞIMLARINDA ETKİLERİNİN BELİRLENMESİ YÜKSEK LİSANS TEZİ SAADET GİZEM ERTEKİN TEZ DANIŞMANI DOÇ.DR. LEVENT DEĞİRMENCİ İKİNCİ TEZ DANIŞMANI DR.ÖĞR.ÜYESİ FADİME ÖZDEMİR KOÇAK BİLECİK, 2020 10369927 T.C. BİLECİK ŞEYH EDEBALİ ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOTEKNOLOJİ ANABİLİM DALI TERMOFİLİK AKTİNOMİSETLERİN İZOLASYONU, MOLEKÜLER KARAKTERİZASYONU VE KOMPOST KARIŞIMLARINDA ETKİLERİNİN BELİRLENMESİ YÜKSEK LİSANS TEZİ SAADET GİZEM ERTEKİN TEZ DANIŞMANI DOÇ.DR. LEVENT DEĞİRMENCİ İKİNCİ TEZ DANIŞMANI DR.ÖĞR.ÜYESİ FADİME ÖZDEMİR KOÇAK BİLECİK, 2020 10369927 BEYAN “Termofilik Aktinomisetlerin İzolasyonu, Moleküler Karakterizasyonu ve Kompost Karışımlarında Etkilerinin Belirlenmesi” adlı yüksek lisans tez projesinin hazırlık ve yazımı sırasında bilimsel ahlak kurallarına uyduğumu, başkalarının eserlerinden yararlandığım bölümlerde bilimsel kurallara uygun olarak atıfta bulunduğumu, kullandığım verilerde herhangi bir tahrifat yapmadığımı, tezin herhangi bir kısmının Bilecik Şeyh Edebali Üniversitesi veya başka bir üniversitede başka bir tez çalışması olarak sunulmadığını beyan ederim. Bu tez çalışmasında Bilecik Şeyh Edebali Üniversitesi Bilimsel Araştırma Projesi (BAP) “Kompost Karışımlarında Hızlandırıcı Olarak Kullanılacak Termofilik Aktinomisetlerin Seçimine Farklı Parametrelerin Etkisi” adlı projeden destek alınmıştır. DESTEK ALINMIŞTIR X DESTEK ALINMAMIŞTIR Destek alındı ise; Destekleyen Kurum: Bilimsel Araştırma Projesi (BAP) Desteğin Türü Proje Numarası 1- BAP (Bilimsel Araştırma Projesi) X 2- TÜBİTAK 2019-02. BŞEÜ. 01-01 Diğer…………….. Saadet Gizem ERTEKİN ÖN SÖZ Tez çalışmam boyunca danışmanlığımı yürüten ve tez konumun belirlenmesinden çalışmanın sonuçlandırılmasına kadar değerli görüş ve önerilerini sunan, bilgi ve tecrübelerinden yararlandığım, yardımlarını ve desteklerini hiçbir zaman esirgemeyen, yoğun çalışmaları sırasında sabır gösterdikleri için Doç. Dr. Levent DEĞİRMENCİ ve Dr. Öğr. Üyesi Fadime ÖZDEMİR KOÇAK’a sonsuz teşekkürlerimi ve saygılarımı sunarım. Çalışmalarım boyunca bilgilerinden yararlandığım, desteklerini hiçbir şekilde esirgemeyen ve her koşulda yanımda olan Özge KAYGUSUZ ve Gülçin ÇETİN KILIÇASLAN’a sonsuz teşekkür ederim. Tez çalışmamda ve laboratuvar çalışmalarımda yanımda olup maddi manevi destek veren ve yalnız bırakmayan Ayten KUMAŞ’a sonsuz teşekkür ederim. Yüksek lisans eğitimim de dahil olmak üzere hayatımın her anında maddi ve manevi destekleriyle yanımda olan çok kıymetli babam Edip ERTEKİN, çok kıymetli annem Ülay ERTEKİN, canım abilerim Turgut ve Aykut ERTEKİN’e sonsuz teşekkür ederim. Yoğun tempolu bu süreçte desteklerini esirgemeyen, beni hiç yalnız bırakmayan, zor anlarımı paylaştığım, maddi ve manevi yanımda olan Hazel KARADAĞ, Şule Aybüke YAVUZ, Sevinç KARAÇAM ve Serhat TANIR’a sonsuz teşekkür ederim. Saadet Gizem ERTEKİN i ÖZET TERMOFİLİK AKTİNOMİSETLERİN İZOLASYONU, MOLEKÜLER KARAKTERİZASYONU VE KOMPOST KARIŞIMLARINDA ETKİLERİNİN BELİRLENMESİ “Termofilik Aktinomisetlerin İzolasyonu, Moleküler Karakterizasyonu ve Kompost Karışımlarında Etkilerinin Belirlenmesi” konulu tez çalışması kapsamında kompost karışımlarında kullanılmak üzere Streptomyces sp. cinsi mikroorganizmaların seçimi enzim temelli ve parametre temelli prosedürlerin uygulanmasıyla gerçekleştirilmiştir. Tez çalışması kapsamında ilk aşama Aktinobakterilerin kompost karışımlarından izolasyonudur. Mikroorganizma kaynağı olarak toprak ve aktif çamurun kullanıldığı kompostlardan farklı sıcaklıkta alınan numuneler ile izolasyon dilüsyon plaka yöntemiyle gerçekleştirilmiştir. İzolasyon, yoğun ekim, tek koloni ve saflaştırma aşamalarından sonra elde edilen 84 izolata ilk olarak enzim testleri uygulanmış ve enzim sonuçları ile morfolojik özellikleri benzer olan izolatlar ayırt edilerek, toplam incelenen mikroorganizma sayısı 21’e düşürülmüştür. 21 izolat ile ilk olarak detaylı morfolojik analizler izolatların farklı besiyerlerinde üreme davranışlarının gözlenmesiyle gerçekleştirilmiştir. 21 adet izolattan 15 adeti 16S rRNA analizi ile tanımlanmıştır. Enzim temelli seçim kriteri uygulanan izolatlardan üç tanesi kompost karışımlarında kullanılmak üzere seçilmiştir. Parametre temelli seçim kriterinde ilk olarak izolatların farklı sıcaklık ve pH değerlerinde üremeleri incelenmiş, farklı karbon kaynaklarında üremeleri ile üre hidroliz yetenekleri kıyaslanmıştır. Tez kapsamında kompost karışımlarında kullanılacak mikroorganizmaların seçimi için tercih edilen enzim testleri selüloz, nişasta ve ksilan degradasyonudur. Enzim temelli seçim kriteri sonunda mikroorganizma çalışmalarında sırasıyla N3C07, T4A05 ve N4A13, kompost denemelerinde A, B ve C kodlarıyla verilen mikroorganizmalar kompost karışımlarında kullanılmışlardır. Kompost denemeleri sonucunda özellikle tekli mikroorganizma kullanımında yüksek organik madde kayıpları belirlenmiştir. Mikroorganizmların ikili kullanıldıkları sistemlerde organik madde kaybı kontrolle hemen hemen aynıdır. Üçlü sistemde ise organik madde kaybı kontrolün oldukça gerisinde kalmıştır. Kompost olgunluğunun belirlenmesinde kullanılan Çimlenme İndeks (GI %) verileri kompostların tekli sistemlerde salatalık bitkisi ile uyumsuz olduğunu göstermiştir. Buna karşılık denemelerde kullanılan ikinci bitki olan buğdayın perfomansı yüksektir. Sonuçlar seçilen mikroorganizmaların kontrole kıyasla daha etkin olduklarını ve kompost verimini arttırdıklarını göstermiştir Anahtar kelimeler: Kompost, Streptomyces sp., Organik Madde, Enzim Testi, Çimlenme İndeksi ii ABSTRACT ISOLATİON AND MOLECULAR CHARACTERİZATİON OF THERMOPHİLİC ACTİNOMYCTES AND INVESTİGATİON OF THEİR EFFECTS İN COMPOST MİXTURES Postgraduate thesis about “Isolation and Molecular Characterization of Thermophilic Actinomyctes and Investigation of their Effects in Compost Mixtures” investigated enzyme and parameter based Streptomyces sp. selection criteria for use in compost mixtures. Isolation of Actinobacteria from compost mixtures was the first step in the present study. Soil and activated sludge, were utilized as microorganism source and micoorganisms sampled at different temperatures were isolated via dilution plate technique. 84 isolates were obtained after consecutive isolation, incubation, single colony reducing, and purficiation steps and these were initailly tested for their enymatic activity. Enzyme tests and morphological analyses were interpreted in accordance to reduce total number of microorganisms to 21. Detailed morphological analyses were utilized in comparing micoorganism production in the presence of various medium. 15 isolates were further analyzed via 16S rRNA sequencing and three isolates were selected following the results of enzyme tests. Parametre based selection tests were composed of microorganism development in the presence of various temperature and pH values, growth on different carbon sources and urea hydrolyses of microorganisms were also determined in the course of studies. Cellulose, starch and ksilane degradation were preferred for enzyme tests and micoroganisms coded as N3C07 (A), T4A05 (B) and N4A13 (C) were utilized in compost experiments. Highest organic matter degradation was obtained in the presence of single microorganism utilization. Binary systems indicated organic matter degradation close to control system. Organic matter degradation in the presence of triple system was lower than control. Germination Index utilized for determination of compost maturity revealed low GI % values for cucumber while high performance was obtained with wheat. Results indicated that micororganisms had been more effective than control. The increase in composting effciency via microorganism utilization as the highlight of thesis study Key Words: Compost, Streptomyces sp., Organic Matter, Enzyme Test, Germination İndex iii İÇİNDEKİLER ÖN SÖZ ...................................................................................................................................... i ÖZET ......................................................................................................................................... ii ABSTRACT ............................................................................................................................. iii TABLOLAR LİSTESİ ........................................................................................................... vii ŞEKİLLER LİSTESİ ............................................................................................................ viii KISALTMALAR VE SİMGELER LİSTESİ ....................................................................... xi 1.GİRİŞ ...................................................................................................................................... 1 2. LİTERATÜR ÖZETİ .......................................................................................................... 4 2.1. Aktinobakterilerin Genel Özellikleri ........................................................................... 4 2.2. Termofilik Aktinobakterilerin Genel Özellikleri ........................................................ 6 2.3. Streptomyces Cinsi Sistematiği ve Genel Özellikleri ................................................... 7 2.4. Mikroorganizmalara Uygulanan Enzim Testleri ...................................................... 10 2.5. Kompostlaşma ve Etki Eden Parametreler ............................................................... 18 2.6. Prokaryotik Taksonomi
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