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Understanding Carbon Metabolism in Hydrogen Production by Pns Bacteria

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Understanding Carbon Metabolism in Hydrogen Production by Pns Bacteria UNDERSTANDING CARBON METABOLISM IN HYDROGEN PRODUCTION BY PNS BACTERIA A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY EZGİ MELİS DOĞAN IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMICAL ENGINEERING AUGUST 2016 Approval of the Thesis; UNDERSTANDING CARBON METABOLISM IN HYDROGEN PRODUCTION BY PNS BACTERIA submitted by EZGİ MELİS DOĞAN in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department, Middle East Technical University by, Prof. Dr. Gülbin Dural Ünver Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Halil Kalıpçılar Head of the Department, Chemical Engineering Asst. Prof. Dr. Harun Koku Supervisor, Chemical Engineering Dept., METU Examining Committee Members: Prof. Dr. Ufuk Bölükbaşı Chemical Engineering Dept., METU Asst. Prof. Dr. Harun Koku Chemical Engineering Dept., METU Assoc. Prof. Dr. Serkan Kıncal Chemical Engineering Dept., METU Assoc. Prof. Dr. Demet Çetin Department of Primary Education, Gazi University Asst. Prof. Dr. Eda Çelik Akdur Chemical Engineering Dept., Hacettepe University Date: 17.08.2016 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name: Ezgi Melis Doğan Signature : iv ABSTRACT UNDERSTANDING CARBON METABOLISM IN HYDROGEN PRODUCTION BY PNS BACTERIA Doğan, Ezgi Melis M.Sc., Chemical Engineering Department Supervisor: Asst. Prof. Dr. Harun Koku August 2016, 164 pages In biological hydrogen production systems using purple non-sulfur bacteria (PNS bacteria), a thorough understanding of the metabolism of these microorganisms plays a vital role in assessing and improving efficiency and productivity. This metabolism is very complex, and the result of the interplay of several systems and components such as the photosystems, carbon flow and enzymatic reactions. Mathematical models are sought to represent the complex metabolism of these bacteria, which in turn can be used to interpret and enhance the phenomenological equations obtained from experiment, and ultimately aid the design of large-scale bioreactors. The aim of this study is to analyze the metabolism of PNS bacteria using contemporary tools and techniques (Flux Balance Analysis), with emphasis on carbon flow. The thesis mainly concerns the modeling of the metabolism of PNS bacteria, focusing on Rhodopseudomonas palustris which utilizes sucrose as a carbon source and glutamate v as a nitrogen source in a growth medium with a low N/C ratio. For this purpose, the metabolic model in the present work was verified with the experimental results which were previously performed based on the same conditions considered by the model. Two objective functions, namely, the maximal growth rate of biomass and maximum hydrogen production rate were investigated in particular. The distribution of fluxes in R. palustris showed s linear increase in the specific growth rate of biomass with increasing glutamate uptake rate. The biomass growth was found constant when initial sucrose concentration was changed and a strong function of glutamate uptake rate. A decrease in H2 production was observed at higher photon fluxes and PHB was antagonistically produced to H2 production. Acetic acid and formic acid were found the most and least effective organic acid for H2 production, respectively. The distribution of modeled fluxes will help explain the capability of the hydrogen production and growth on sucrose of R. palustris. Keywords: Metabolic Engineering, Purple Non-Sulphur Bacteria, Biological Hydrogen Production, Sucrose, Mathematical Model vi ÖZ MOR KÜKÜRTSÜZ BAKTERİLERİN HİDROJEN ÜRETİMİNDEKİ KARBON METABOLİZMASININ ANLAŞILMASI Doğan, Ezgi Melis Yüksek Lisans, Kimya Mühendisliği Bölümü Tez Yöneticisi : Y. Doç. Dr. Harun Koku Ağustos 2016, 164 sayfa Mor kükürtsüz bakterilerle biyolojik hidrojen üretiminin verimi ve üretilebilirliğini artırmak için bu bakterilerin metabolik faaliyetlerini anlamak çok büyük bir öneme sahiptir. Bu metabolizma oldukça karmaşıktır ve birden fazla metabolizma öğesinin ortak faaliyeti sonucu oluşur. Bu öğeler genel olarak fotosistem, karbon akışı ve enzimatik reaksiyonlardan oluşmaktadır. Bu metabolizmayi ve elemanlarını yorumlamak için matematiksel modeller kullanılmaktadır. Matematiksel modeller, deneyler aracılığıyla elde edilen olgusal denklemleri yansıtır ve karmaşık bir metabolizmanın daha basit bir şekilde ifade edilmesini sağlar. Böylelikle büyük ölçekli fotobiyoreaktor tasarımı süreçleri kolaylaştırılabilir. Bu tezin asıl amacı çağdaş yöntemler ve teknikler kullanarak ve karbon akışına odaklanarak, mor kükürtsüz bakterilerin metabolizmalarını analiz etmektir. Bu tez genel olarak mor kükürtsüz bakterilerden Rhodopseudomonas palustris bakteri türü üzerine vii odaklanarak bir metabolizma modeli ortaya çıkarmayı hedeflemektedir. Karbon kaynağı olarak sukroz ve azot kaynağı olarak glutamat esas alınmıştır. Bakteri koşulları icin ortamda bulunan karbon miktarının nitrojen miktarına oranı düşük kabul edilmiştir. Bu nedenle, bu araştırma konusu daha önce model için ifade edilen koşullarda sukroz ile yapılan deney sonuçları ile test edilerek, sonuçları yorumlamak amacıyla metabolik akı analizinin uygulanmasını içerir. Metabolik akı analizi, bakterinin maksimum büyüme hızı ve maksimum hidrojen üretme hızı olmak üzere, iki farklı amaç fonksiyonunu incelemektedir. R. palustris in elde edilen akı dağılımından, bakteri büyüme hızının glutamat alım hızına paralel bir şekilde değiştiği gözlemlenmiştir. Ortamdaki sukroz konsantrasyonu değiştirildiğinde, bakteri büyüme hızı değişiklik göstermemiştir. Yüksek foton akılarında, hidrojen üretim hızında azalma, PHB üretim hızında artma görülmüştür. Asetik asit ve formik asit, sırasıyla, hidrojen üretimini artırmak için en çok ve en az etkili organik asitler olarak belirlenmiştir. Yapılan metabolik akı analizin sonuçları olarak elde edilen akı dağılımı, R. palustris’in sükroz ile hidrojen üretim kapasitesini çalışmak için kullanılabilecektir. Anahtar Kelimeler: Metabolizma Mühendisliği, Mor Kükürtsüz Bakteriler, Biyolojik Hidrojen Üretimi, Sukroz, Matematik Model viii To my family, ix ACKNOWLEDGEMENTS I would like to thank sincerely to my supervisor Asst. Prof. Dr. Harun Koku and I would like to express my gratitude to Prof. Dr. İnci Eroğlu for their guidance, support, suggestions and more importantly, motivation throughout this study. I would like to give my special thanks to Prof. Dr. Meral Yücel and Prof. Dr. Ufuk Gündüz for their valuable ideas, aids and support. I am thankful to Emrah Sağır who provided experimental data used in the evaluation of this present work. I also thank to Muazzez Gürgan from Biology department for her advices in this study. I am grateful deeply to my fiancé, Feyyaz Güner who gave motivation and encouragement to me during my thesis with his endless support and love. Lastly, I sincerely thank all members of my family, especially my parents for their encouragement, support and love throughout my thesis. This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) as project numbered ‘114M436’. x TABLE OF CONTENTS ABSTRACT................................................................................................................v ÖZ..............................................................................................................................vii ACKNOWLEDGEMENTS.........................................................................................x TABLE OF CONTENTS............................................................................................xi LIST OF TABLES ...................................................................................................xiv LIST OF FIGURES ..................................................................................................xvi LIST OF ABBREVIATIONS ..................................................................................xix CHAPTERS 1. INTRODUCTION.......................................................................................1 1.1 Hydrogen Production Techniques..................................................2 1.2 Techniques of Bio-hydrogen Production.......................................5 1.2.1 Bio-photolysis.................................................................6 1.2.2 Dark Fermentation..........................................................6 1.2.3 Photo Fermentation.........................................................7 1.2.4 Integrated systems...........................................................8 1.3 The Role of Metabolic Engineering for Hydrogen Production…...9 1.4 Objective and Scope.....................................................................10 2. LITERATURE SURVEY..........................................................................13 2.1 Characteristics of Purple Non-Sulfur Bacteria.............................13 2.2 Overview of the Metabolism in Hydrogen Production by PNS Bacteria........................................................................................16 2.2.1 Carbon Flow.................................................................18 2.2.2 Enzyme Systems...........................................................20 2.2.3 Photosynthetic Unit.......................................................24
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