Diazotrofik Koşullarda Çoğaltılan Rhodobacter Sphaeroides O.U.001 ile Hidrojen Üretimi ie nifH Gen İfade Analizi
Yükleniyor...
Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Fotosentetik bakteri grubundan olan mor kükürtsüz (PNS) bakteriler hidrojen (H2) üretimi, azot
(N2) ve karbondioksit (CO2) fiksasyonu gibi zengin metabolik aktivitelere sahip olduklarından farklı
fizyolojik koşullarda yaşamlarını sürdürebilmektedirler. Bu özelliklerinden dolayı PNS bakterileri
temel metabolik olayların araştırılmasında model organizma olarak kullanılmıştır. Fotosentetik ve mor
kükürtsüz bakterilerden biri olan Rhodobacter sphaeroides O.U.001 de N2 fiksasyonu, H2 ve
polihidroksibutirat (PHB) üretimi gibi çok yönlü metabolizmaya sahip olup tüm genom dizisinin
bilinmesi nedeniyle araştırmalarda sıkça kullanılan model bir bakteridir. Bu çalışmanın amacı,
diazotrofik (N2’nin sabitlendiği) koşullarda kültüre edilen R. sphaeroides ile hidrojen üretimi
gerçekleştirmektir. Bu kapsamda bakteriler karbon kaynağı olarak malat’ın (15 mM) ve azot kaynağı
olarak N2’nin kullanıldığı besiyerinde çoğaltılmıştır. Kontrol grubu olarak ise karbon kaynağı olarak
malat’ın (15 mM) ve azot kaynağı olarak glutamat’ın (2 mM) kullanıldığı besiyeri (non diazotrofik
ortam) kullanılmıştır. Hidrojen üretimlerine ek olarak azot fiksasyonu sonucu oluşan amonyum
miktarları da ölçülmüştür. Ayrıca, bu iki koşul altında çoğaltılan bakterilerde nifH geninin ekspresyon
seviyesi kantitatif Polimeraz Zincir Reaksiyonu (qPCR) ile tespit edilmiştir. Ek olarak, bu iki farklı
fizyolojik koşulun bakteri morfolojisine etkisi alan emisyonlu taramalı elektron mikroskobu (FE-SEM)
ile incelenmiştir. Sonuçlara göre, diazotrofik koşullarda 13,95 mL hidrojen üretilirken, non diazotrofik
koşullarda 11,05 mL hidrojen üretimi tespit edilmiştir. Bu sonuca paralel olarak, diazotrofik
koşullardaki nifH gen ekspresyonu non diazotrofik koşullardaki gen ekspresyonunun 2,3 katı olarak
hesaplanmıştır. Diazotrofik ve non diazotrofik kültürlerde ölçülen amonyum miktarları da sırasıyla 5,05
mg/L ve 3,38 mg/L olarak bulunmuştur. Bu iki koşulda çoğaltılan bakterilerin FE-SEM görüntüleri
incelendiğinde ise bakterilerin morfolojilerinde belirgin bir fark gözlemlenmemiştir. Sonuç olarak,
diazotrofik büyüme koşullarında kültüre edilen R. sphaeroides ile daha yüksek miktarlarda hidrojen,
nifH gen ifadesi ve sabitlenmiş amonyum tespit edilmiştir. Bu sonuç, azotun hidrojen üretiminden
sorumlu olan nitrojenaz enziminin aktivite veya miktarını stimüle ettiği şeklinde yorumlanmıştır.
Gelecekteki çalışmalarda enzim miktar ve aktivite tayinleri ile bu bulguların desteklenmesi
öngörülmektedir.
Purple nonsulfur (PNS) bacteria from the photosynthetic bacteria group can survive in different physiological conditions as they have rich metabolic activities such as hydrogen (H2) production, nitrogen (N2) and carbon dioxide (CO2) fixation. Because of these properties, PNS bacteria have been used as a model organism in the investigation of basic metabolic events. Rhodobacter sphaeroides O.U.001, one of the photosynthetic and purple nonsulphur bacteria, is frequently used in research as a model bacterium because it has a versatile metabolism such as N2 fixation, H2 and polyhydroxybutyrate (PHB) production and its whole genome sequence is known. The aim of this study is to perform hydrogen production with R. sphaeroides cultured under diazotrophic (N2 fixation) conditions. In this context, bacteria were propagated in a medium using malate (15 mM) as a carbon source and N2 as a nitrogen source. As the control group, medium (non-diazotrophic medium) containing malate (15 mM) as carbon source and glutamate (2 mM) as nitrogen source was used. In addition to hydrogen production, ammonium amounts formed as a result of nitrogen fixation were also measured. In addition, the expression level of the nifH gene in bacteria grown under these two conditions was determined by quantitative Polymerase Chain Reaction (qPCR). Finally, the effects of these two different physiological conditions on bacterial morphology were investigated by field emission scanning electron microscopy (FE-SEM). According to the results, 13.95 mL of hydrogen was produced under diazotrophic conditions, while 11.05 mL of hydrogen was produced under non-diazotrophic conditions. In parallel with this result, nifH gene expression in diazotrophic conditions was calculated as 2.3 times the gene expression in non-diazotrophic conditions. Ammonium amounts measured in diazotrophic and non-diazotrophic cultures were found to be 5.05 mg/L and 3.38 mg/L, respectively. When the FE-SEM images of bacteria grown in these two conditions were examined, no significant difference was observed in the morphology of the bacteria. As a conclusion, higher amounts of hydrogen, nifH gene expression and fixed ammonium were detected in R. sphaeroides cultured under diazotrophic growth conditions. This result was interpreted as nitrogen stimulating the activity or amount of nitrogenase enzyme, which is responsible for hydrogen production. In future studies, it is projected that these findings will be supported by enzyme amount and activity tests.
Purple nonsulfur (PNS) bacteria from the photosynthetic bacteria group can survive in different physiological conditions as they have rich metabolic activities such as hydrogen (H2) production, nitrogen (N2) and carbon dioxide (CO2) fixation. Because of these properties, PNS bacteria have been used as a model organism in the investigation of basic metabolic events. Rhodobacter sphaeroides O.U.001, one of the photosynthetic and purple nonsulphur bacteria, is frequently used in research as a model bacterium because it has a versatile metabolism such as N2 fixation, H2 and polyhydroxybutyrate (PHB) production and its whole genome sequence is known. The aim of this study is to perform hydrogen production with R. sphaeroides cultured under diazotrophic (N2 fixation) conditions. In this context, bacteria were propagated in a medium using malate (15 mM) as a carbon source and N2 as a nitrogen source. As the control group, medium (non-diazotrophic medium) containing malate (15 mM) as carbon source and glutamate (2 mM) as nitrogen source was used. In addition to hydrogen production, ammonium amounts formed as a result of nitrogen fixation were also measured. In addition, the expression level of the nifH gene in bacteria grown under these two conditions was determined by quantitative Polymerase Chain Reaction (qPCR). Finally, the effects of these two different physiological conditions on bacterial morphology were investigated by field emission scanning electron microscopy (FE-SEM). According to the results, 13.95 mL of hydrogen was produced under diazotrophic conditions, while 11.05 mL of hydrogen was produced under non-diazotrophic conditions. In parallel with this result, nifH gene expression in diazotrophic conditions was calculated as 2.3 times the gene expression in non-diazotrophic conditions. Ammonium amounts measured in diazotrophic and non-diazotrophic cultures were found to be 5.05 mg/L and 3.38 mg/L, respectively. When the FE-SEM images of bacteria grown in these two conditions were examined, no significant difference was observed in the morphology of the bacteria. As a conclusion, higher amounts of hydrogen, nifH gene expression and fixed ammonium were detected in R. sphaeroides cultured under diazotrophic growth conditions. This result was interpreted as nitrogen stimulating the activity or amount of nitrogenase enzyme, which is responsible for hydrogen production. In future studies, it is projected that these findings will be supported by enzyme amount and activity tests.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Azot, Diazotrofik, Hidrojen, Rhodobacter sphaeroides, Nitrogen, Diazotrophic, Hydrogen
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Sert, M. (2022). Diazotrofik koşullarda çoğaltılan Rhodobacter sphaeroides O.U.001 ile hidrojen üretimi ve nifH gen ifade analizi. (Yayımlanmamış yüksek lisans tezi). Necmettin Erbakan Üniversitesi, Fen Bilimleri Enstitüsü Moleküler Biyoloji ve Genetik Anabilim Dalı, Konya.
