Asetat ortamında büyütülen Cereibacter sphaeroides O.U. 001, Rhodopseudomonas palustris 7850 ve Cupriavidus necator H16 ile polihidroksibütirat (PHB) üretimi ve üretilen polimerlerin karakterizasyonu
Yükleniyor...
Dosyalar
Tarih
2023
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Dünyada nüfus artışı, yenilenemeyen kaynakların azalışı ve günlük hayatımızın bir parçası olan plastik ürenlerin çoğalması, petrol türevli plastiklerin doğada kalbolmaması sonucu oluşan çevre kirliliği güncel problemler arasındadır. Biyo uyumlu, parçalanabilir ve yenilenebilir özellikleriyle termoplastik bir polimer olan polihidroksibütirat (PHB) geleneksel plastiklerin birçok özelliğine sahiptir. Bu biyopolimer sentetik karbon bazlı polimerlere alternatif olarak uygundur. Alcaligenes, Rhodobacter, Bacillus, Rhizobium, Pseudomonas gibi farklı mikroorganizmalar tarafından sentezlenmektedir. Monomerlerin birleştirilmesi ve sentezi olarak enzimatik aşamaların gerçekleşmesi ile polimer biyosentezi gerçekleşir. Polihidroksibütirat (PHB) biyosentezini β-ketotiyolaz, asetoasetil-CoA, PHB polimeraz/sentaz enzimleri ve phbA, phbB, phbC genleri sağlar. PHB bakteriler, topraktaki mikroorganizmalar ve funguslar tarafından uygun koşullar altında parçalanarak karbondioksit, enerji ve su açığa çıkarmaktadır. Biyoparçalanabilir özelliğinden kaynaklı olarak tıp, ziraat, tekstil ve paketleme gibi birçok alanda kullanılmaktadır. Üretimde kullanılması açısından uygun substrat ve besi ortamı kullanıldığıda maliyet azaltılabilmektedir. Cereibacter sphaeroides, Rhodopseudomomas palustris ve Cupriavidus necator 70 mM asetat 2 mM ile sınırlandırılmış amonyum ortamında büyümeleri ve PHB üretimi incelendi. Asetat oranı 10, 25, 40, 55, 70, 85 ve 100 mM ortamlarda optik dansitelerine (OD) bakılarak karar verildi. 70 mM asetat besiyerinde C. sphaeroides, R. palustris ve C. necator’da sırasıyla 4.3, 5.7 ve 3.0 OD değerleri aynı sıra ile 9.6, 9.8 ve 9.1 pH değerleri ölçüldü. PHB granülleri Sudan Black B ve Nile red gibi lipofilik boyalarla tespit edildi. Sudan Black B boyaması ışık mikroskobunda bakıldığında bakteriler içerisinde PHB yoğunluğuna göre mor, mavi, siyah renkler arasında renkler gözlemlendi. Nile red ile floresans mikroskobunda bakıldığında ise küçük noktalar şeklinde kırmızı bir ışıma gözlemlendi. PHB’nin en iyi çözücüsü olan kloroform ile ekstraksiyonu yapıldığında C. sphaeroides’in 14,6 mg, R. palustris’in 31,1 mg, C. necator’un 5,4 mg PHB kuru ağırlıkları tartıldı. PHB verimi incelendiğinde sırasıyla bakterilerde; C. sphaeroides %0,054, R. palustris % 0,11, C. necator % 0,034 değerlerine ulaşıldı. Asetat ortamında PHB üretimine en yüksek seviyede R. palustris’de ulaşılmıştır. Nükleer manyetik rezonans (NMR) spektroskopisi yöntemi ile PHB karakterizasyonu yapılmış, C. sphaeroides ve R. Palustris bakterilerinden saf PHB elde edildiği, C. necator’da elde edilen PHB’nin ise diğer bakterilere kıyasla saf olmadığı ve bazı kirliliklerin de olduğu tespit edilmiştir. Bu çalışma kapsamında C. sphaeroides, R. palustris ve C. necator’da asetat besiyerinde PHB üretimi ve üretim sonucunca PHB verimi incelenmiştir.
The increase in population in the world, the decrease in non-renewable resources, the increase of plastic products that are a part of our daily life, and the environmental pollution caused by the fact that petroleum-derived plastics do not die in nature are among the current problems. Polyhydroxybutyrate (PHB), a thermoplastic polymer with biocompatible, degradable and renewable properties, has many of the properties of conventional plastics. This biopolymer is suitable as an alternative to synthetic carbon-based polymers. It is synthesized by different microorganisms such as Alcaligenes, Rhodobacter, Bacillus, Rhizobium, Pseudomonas. Polymer biosynthesis takes place with the realization of enzymatic steps as the assembly and synthesis of monomers. The biosynthesis of polyhydroxybutyrate (PHB) is provided by β-ketothiolase, acetoacetyl-CoA, PHB polymerase/synthase enzymes and phbA, phbB, phbC genes. PHB is broken down by bacteria, microorganisms and fungi in the soil under appropriate conditions, releasing carbon dioxide, energy and water. Due to its biodegradable feature, it is used in many fields such as medicine, agriculture, textile and packaging. Costs can be reduced when appropriate substrate and nutrient medium are used in production. The growth and PHB production of Cereibacter sphaeroides, Rhodopseudomomas palustris and Cupriavidus necator in ammonium medium limited with 70 mM acetate 2 mM were examined. The acetate ratio was determined by looking at their optical densities (OD) in 10, 25, 40, 55, 70, 85 and 100 mM medium. OD values of 4.3, 5.7 and 3.0, pH values of 9.6, 9.8 and 9.1, respectively, were measured in C. sphaeroides, R. palustris and C. necator in 70 mM acetate medium. PHB granules were detected with lipophilic dyes such as Sudan Black B and Nile red. When the Sudan Black B staining was examined under the light microscope, colors between purple, blue and black were observed according to the PHB concentration in the bacteria. When viewed under a fluorescence microscope with Nile red, a red glow in the form of small dots was observed. When PHB was extracted with chloroform, which is the best solvent, 14.6 mg of C. sphaeroides, 31.1 mg of R. palustris, and 5.4 mg of PHB of C. necator were weighed. When the PHB yield was examined, respectively, in bacteria; C. sphaeroides 0.054%, R. palustris 0.11%, C. necator 0.034%. The highest level of PHB production in acetate medium was reached in R. palustris. PHB characterization was made by nuclear magnetic resonance (NMR) spectroscopy method, it was determined that pure PHB was obtained from C. sphaeroides and R. palustris bacteria, PHB obtained from C. necator was not pure compared to other bacteria and there were some impurities. Within the scope of this study, PHB production in acetate medium in C. sphaeroides, R. palustris and C. necator and PHB yield as a result of production were investigated.
The increase in population in the world, the decrease in non-renewable resources, the increase of plastic products that are a part of our daily life, and the environmental pollution caused by the fact that petroleum-derived plastics do not die in nature are among the current problems. Polyhydroxybutyrate (PHB), a thermoplastic polymer with biocompatible, degradable and renewable properties, has many of the properties of conventional plastics. This biopolymer is suitable as an alternative to synthetic carbon-based polymers. It is synthesized by different microorganisms such as Alcaligenes, Rhodobacter, Bacillus, Rhizobium, Pseudomonas. Polymer biosynthesis takes place with the realization of enzymatic steps as the assembly and synthesis of monomers. The biosynthesis of polyhydroxybutyrate (PHB) is provided by β-ketothiolase, acetoacetyl-CoA, PHB polymerase/synthase enzymes and phbA, phbB, phbC genes. PHB is broken down by bacteria, microorganisms and fungi in the soil under appropriate conditions, releasing carbon dioxide, energy and water. Due to its biodegradable feature, it is used in many fields such as medicine, agriculture, textile and packaging. Costs can be reduced when appropriate substrate and nutrient medium are used in production. The growth and PHB production of Cereibacter sphaeroides, Rhodopseudomomas palustris and Cupriavidus necator in ammonium medium limited with 70 mM acetate 2 mM were examined. The acetate ratio was determined by looking at their optical densities (OD) in 10, 25, 40, 55, 70, 85 and 100 mM medium. OD values of 4.3, 5.7 and 3.0, pH values of 9.6, 9.8 and 9.1, respectively, were measured in C. sphaeroides, R. palustris and C. necator in 70 mM acetate medium. PHB granules were detected with lipophilic dyes such as Sudan Black B and Nile red. When the Sudan Black B staining was examined under the light microscope, colors between purple, blue and black were observed according to the PHB concentration in the bacteria. When viewed under a fluorescence microscope with Nile red, a red glow in the form of small dots was observed. When PHB was extracted with chloroform, which is the best solvent, 14.6 mg of C. sphaeroides, 31.1 mg of R. palustris, and 5.4 mg of PHB of C. necator were weighed. When the PHB yield was examined, respectively, in bacteria; C. sphaeroides 0.054%, R. palustris 0.11%, C. necator 0.034%. The highest level of PHB production in acetate medium was reached in R. palustris. PHB characterization was made by nuclear magnetic resonance (NMR) spectroscopy method, it was determined that pure PHB was obtained from C. sphaeroides and R. palustris bacteria, PHB obtained from C. necator was not pure compared to other bacteria and there were some impurities. Within the scope of this study, PHB production in acetate medium in C. sphaeroides, R. palustris and C. necator and PHB yield as a result of production were investigated.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Cereibacter Sphaeroides, Cupriavidus Necator, Polihidroksibütirat (PHB), Rhodopseudomonas Palustris, Cupriavidus Necator, Polyhydroxybutyrate (PHB), Rhodopseudomonas Palustris, Cereibacter Sphaeroides
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Akpınar, B. N. (2023). Asetat ortamında büyütülen Cereibacter sphaeroides O.U. 001, Rhodopseudomonas palustris 7850 ve Cupriavidus necator H16 ile polihidroksibütirat (PHB) üretimi ve üretilen polimerlerin karakterizasyonu. (Yayımlanmamış yüksek lisans tezi). Necmettin Erbakan Üniversitesi, Fen Bilimleri Enstitüsü Moleküler Biyoloji ve Genetik Anabilim Dalı, Konya.
