SCALE UP OF PANEL PHOTOBIOREACTORS FOR HYDROGEN … · A THESIS SUBMITTED TO . THE GRADUATE SCHOOL...

SCALE UP OF PANEL PHOTOBIOREACTORS FOR HYDROGEN

PRODUCTION BY PNS BACTERIA

A THESIS SUBMITTED TO

THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

OF

MIDDLE EAST TECHNICAL UNIVERSITY

BY

SEVLER GÖKÇE AVCIOĞLU

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR

THE DEGREE OF MASTER OF SCIENCE

IN

CHEMICAL ENGINEERING

SEPTEMBER 2010

Approval of the thesis:



submitted by S. GÖKÇE AVCIOĞLU in partial fulfillment of the requirements for

the degree of Master of Science in Chemical Engineering Department, Middle

East Technical University by,

Prof. Dr. Canan Özgen __________________

Dean, Graduate School of Natural and Applied Sciences

Prof. Dr. Gürkan Karakaş __________________

Head of Department, Chemical Engineering

Prof. Dr. İnci Eroğlu __________________

Supervisor, Chemical Engineering Department, METU

Dr. Ebru Özgür __________________

Co-supervisor, Hydrogen Research Laboratory, METU

Examining Committee Members:

Prof. Dr. Göknur Bayram __________________

Chemical Engineering Department, METU

Prof. Dr. İnci Eroğlu __________________

Chemical Engineering Department, METU

Doç. Dr. Bülent Akay __________________

Chemical Engineering Department, AU

Dr. Ebru Özgür __________________

Hydrogen Research Laboratory, METU

Dr. Başar Uyar __________________

Chemical Engineering Department, KU

Date: 07.09.2010

iii

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 wok.

Name, Last name: S. Gökçe Avcıoğlu

Signature: _________________

iv

ABSTRACT



Avcıoğlu, S. Gökçe

M. Sc., Department of Chemical Engineering

Supervisor: Prof. Dr. İnci Eroğlu

Co-Supervisor: Dr. Ebru Özgür

September 2010, 209 pages

Production of hydrogen from biomass through the use of dark and

photofermentative bacteria will be applicable in the future and a promising route.

The aim of this study is to develop and to scale-up solar panel photobioreactors for

the biological hydrogen production by photosynthetic purple non sulfur (PNS)

bacteria on artificial substrates and on real dark fermentation effluent of molasses.

The parameters studied are light intensity, temperature, feed stock, feed rate, pH, cell

density, light and dark cycle and carbon to nitrogen ratio on hydrogen production.

Continuous hydrogen production has been achieved on artificial medium and dark

fermentor effluent of molasses containing acetate and lactate by Rhodobacter

capsulatus wild type and (hup-) mutant strains in panel photobioreactors in indoor

and outdoor conditions by fed batch operation. Laboratory (from 4 to 8 liters) and

large scale (20 L) panel photobioreactors by using various designs and construction

materials were developed. In this photobioreactors continuous hydrogen production

was achieved by feeding. Na2CO3 can be used as buffer to keep the pH stable during

v

long term operation on molasses dark fermentor effluent. The adjustment of the

feedstock by dilution and buffer addition were found to be essential for the long term

stability of pH, biomass and H2 production for both in indoor and outdoor

applications.

Keywords: Biohydrogen, photofermentation, panel photobioreactors,

Rhodobacter capsulatus, molasses

vi

ÖZ

PNS BAKTERİ İLE HİDROJEN ÜRETİMİ İÇİN PANEL REAKTÖRÜNÜN

GELİŞTİRİLMESİ

Avcıoğlu, S. Gökçe

Yüksek Lisans., Kimya Mühendisliği Bölümü

Tez Yöneticisi: Prof. Dr. İnci Eroğlu

Ortak Tez Yöneticisi: Dr. Ebru Özgür

Eylül 2010, 209 sayfa

Fermentatif ve fotosentetik mikroorganizmaların kullanılması sayesinde

biyokütleden hidrojen üretimi, gelecekte uygulanabilecek ve umut vadeden bir

yöntemdir. Bu çalışmanın amacı, fotosentetik, mor, sülfürsüz bakteri ile tanımlı

besiyerinden ve fermantasyon atığı olan melastan biyolojik hidrojen üretimi için

panel tipi güneş biyoreaktörlerini geliştirmek ve boyut büyütmektir. Işık şiddeti,

sıcaklık, besiyeri kompozisyonu, besleme hızı, pH, hücre yoğunluğu, aydınlık ve

karanlık devre, karbonun azota oranının hidrojen üretimine etkisi çalışılmıştır. Asetat

ve laktat içeren tanımlı besiyeri ve melasın termofilik fermantasyon atığında

Rhodobacter capsulatus bakterisinin doğal ve mutant türü ile panel tipi

biyoreaktörlerde aydınlatılarak ve güneş ışığında sürekli hidrojen üretimi

gerçekleştirilmiştir. Laboratuar (4 L’den 8 L’ye kadar) ve pilot (20 L) ölçekte panel

biyoreaktörleri, çeşitli tasarım ve yapı malzemeleri kullanılarak geliştirilmiştir. Bu

reaktörlerde sürekli hidrojen üretimi besleme yapılarak gerçekleştirilmiştir. Na2CO3,

termofilik fermantasyon atığı olan melasta yapılan uzun süreli uygulamalarda pH’ın

sabit tutulması için tampon çözelti olarak kullanılabilir. Besiyeri kompozisyonunun

vii

seyreltme ve tampon çözelti eklenerek ayarlanmasının, pH, biyokütle ve hidrojen

üretiminde uzun süreli kararlılığın hem aydınlatılarak hem de güneş ışığında

gerçekleştirilen uygulamalar için önemli olduğu saptanmıştır.

Anahtar Kelimeler: Biyohidrojen, fotofermantasyon, panel biyoreaktörleri,

Rhodobacter capsulatus, melas

viii

In memories of

Soner Balta, Öner Balta and Güneş Korkmaz

ix

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude’s to my supervisor Prof. Dr. İnci

Eroğlu for her faith in me from the first time we have met, endless supports, advices,

kindness, friendship, positive intent, giving countless chance for continuing my

studies, motivation during my thesis. I would like to thank to my co-supervisor Dr.

Ebru Özgür for guiding me in studying with biological systems and giving me

invaluable advices about improvement of laboratory techniques and my instructors

Prof. Dr. Ufuk Gündüz and Prof. Dr. Meral Yücel for their valuable contributions

and suggestions on the biological systems.

I also thank Dr. Başar Uyar, Elif Genç for their suggestions on reactor design,

and considerable contributions to my study, friendship and recommendations.

I also would like to thank to my co-working laboratory mates in hydrogen

Research Laboratory; Nilüfer Afşar, Dominic Deo Androga, Muazzez Gürgan,

Emrah Sağır, Gülşah Pekgöz, Burcu Özsoy, Kamal Elkahlout, Begüm Peksel and Efe

Boran for endless support and friendship. I also gratefully thank especially Endam

Özkan and Pelin Sevinç for invaluable collaboration and friendship. I also would like

to thank to Şerife Topçu for collaboration in providing hygiene in the laboratory.

I gratefully thank the technical assistance of Gülten Orakçı and Dr. Mustafa

Esen Martı for guidance in HPLC analysis, and Kerime Güney for elemental

analysis.

I am thankful for Dr. Truus de Vrije from Wageningen UR, Food and

Biobased Research group, The Netherlands for supplying the molasses dark

fermentor effluent.

This study has been supported by HYVOLUTION the EU 6th

Framework

Integrated Project 019825: Non-thermal production of pure hydrogen from biomass.

I also thank to my mother, my father, my brother and Turgay letting me be as

I am, and being with me for better for worse, supporting me by giving invaluable

advices in all my Master of Science study.

x

I would like to thank to Aydan Bulut, Osman Karslıoğlu, Başar Çağlar, Arzu

Kanca, İsmet Kızılrmak, Nilgün Ercan, Bülent Bozali, İhsan Karababa, and Ramazan

Gök for endless camaraderie, friendship and invaluable brain storming on profession

in my Chemical Engineering studies and in engineering politics. I am also very

grateful to Yelda, Mina, Aycan, Oya, Nisan, Gülen, Selen, Ceren and Setenay for

sharing invaluable life experiences, friendship with me and guiding and encouraging

me in all my thesis steps.

At last I would like to thank to Soner Balta, Öner Balta and Güneş Korkmaz,

Nergis Balta, Durmuş Balta, Gülfer Akkaya and Tuncay Yılmaz. I am indebted to

you for your concrete and intangible contributions to my life and personality.

xi

TABLE OF CONTENTS

ABSTRACT ................................................................................................................ iv

ÖZ ............................................................................................................................... vi

ACKNOWLEDGEMENTS ........................................................................................ ix

TABLE OF CONTENTS ............................................................................................ xi

LIST OF TABLES .................................................................................................. xviii

LIST OF FIGURES ................................................................................................ xxiii

LIST OF SYMBOLS AND ABBREVIATIONS ................................................. xxxiii

CHAPTERS

1. INTRODUCTION ................................................................................................. 1

2. LITERATURE SURVEY ...................................................................................... 6

2.1. Hydrogen as the Future Energy Carrier .......................................................... 6

2.2. Hydrogen Production Technologies ............................................................... 8

2.3. Biological Hydrogen Production .................................................................... 9

2.3.1. Dark Fermentation ............................................................................... 9

2.3.2. Photofermentation .............................................................................. 10

2.3.3. Integrated Systems ............................................................................. 15

2.4. Flat Panel Photobioreactors .......................................................................... 16

2.4.1. Temperature Distribution ................................................................... 17

2.4.2. Light intensity Distribution ................................................................ 18

2.4.3. Feeding, Cooling and Mixing ............................................................ 18

2.4.4. Applications of Flat Panel Photobioreactors ...................................... 19

2.4.5. Scale up of the Panel Photobioreactors .............................................. 25

2.5. Objective of the Study .................................................................................. 25

3. MATERIALS AND METHODS ......................................................................... 28

3.1. The Microorganisms ..................................................................................... 28

3.2. Culture Media ............................................................................................... 28

3.2.1. Growth Medium ................................................................................. 29

xii

3.2.1.1. Pre-cultivation ........................................................................ 29

3.2.1.2. Storage.................................................................................... 30

3.2.2. Hydrogen Production Media .............................................................. 30

3.2.2.1. Artificial Media ...................................................................... 30

3.2.2.2. Real Dark Fermentor Effluent of Molasses ........................... 31

3.3. Experimental Set-up...................................................................................... 31

3.3.1. Small Scale Experiments.................................................................... 32

3.3.1.1. Photobioreactors ..................................................................... 32

3.3.1.2. Sterilization of Photobioreactors and Media .......................... 32

3.3.1.3. Start-up ................................................................................... 33

3.3.1.4. Temperature Indication and Control ...................................... 33

3.3.1.5. Light Intensity Measurement ................................................. 33

3.3.1.6. Sampling and Feeding ............................................................ 33

3.3.1.7. Shutdown ............................................................................... 33

3.3.2. Experiments with Panel Photobioreactors ......................................... 34

3.3.2.1. Experimental Set-up ............................................................... 34

3.3.2.2. Sterilization of Photobioreactors and Media .......................... 39

3.3.2.3. Start-up ................................................................................... 39

3.3.2.4. Temperature Indication and Control ...................................... 40

3.3.2.5. Light Intensity Measurements ................................................ 40

3.3.2.6. Sampling and Feeding ............................................................ 41

3.3.2.7. Shutdown ............................................................................... 41

3.4. Analyses ........................................................................................................ 41

3.4.1. pH Measurement ................................................................................ 41

3.4.2. Spectrophotometric Analysis ............................................................. 41

3.4.2.1. Cell Concentration ................................................................. 41

3.4.2.2. Bacteriochlorophyll a Measurement ...................................... 42

3.4.2.3. TOC Analysis ......................................................................... 42

3.4.2.4. TN Analysis ........................................................................... 43

3.4.2.5. Ammonia Analysis ................................................................. 43

3.4.2.6. COD Analysis ........................................................................ 44

3.4.3. Gas Chromatography Analysis .......................................................... 44

xiii

3.4.3.1. Gas Analysis .......................................................................... 44

3.4.4. HPLC Analyses .................................................................................. 45

3.4.4.1. Organic Acid Analysis ........................................................... 45

3.4.5. Elemental Analysis............................................................................. 45

4. RESULTS AND DISCUSSIONS ........................................................................ 46

4.1. Selection of Construction Materials for Panel Photobioreactors .................. 46

4.1.1. Hydrogen Permeability ...................................................................... 46

4.1.2. Selection of Cooling Coil Material .................................................... 53

4.1.3. Panel Photobioreactor Design ............................................................ 55

4.2. Continuous Hydrogen Production on Defined Medium by R. capsulatus

Wild Type and Mutant Strains ...................................................................... 56

4.2.1. Indoor Lab-Scale Panel Photobioreactor ........................................... 56

4.2.1.1. Temperature and Light Intensity Distribution........................ 57

4.2.1.2. Effect of Feed Composition on Long Term Operation ..............

....................................................................................................... 59

4.2.1.3. Effect of Increased Glutamate Amount in the Feed on

Hydrogen Production .................................................................... 63

4.2.1.4. Effect of Removing Lactate from the Feeding Media on


4.2.1.5. Effect of Increasing Acetate Amount in the Feeding Media on


4.2.1.6. Effect of C/N Ratio on Hydrogen Production ........................ 66

4.2.2. Outdoor Lab-Scale Panel Photobioreactors ....................................... 67

4.2.2.1. Variation in Photobioreactor Temperature............................. 68

4.2.2.2. Effect of Feed Composition on Long Term Operation ..............

....................................................................................................... 71

4.2.3. Outdoor Pilot-Scale Panel Photobioreactor ....................................... 79

4.2.4. Parameters Affecting Prolonged Hydrogen Production ..................... 83

4.2.4.1. Effect of Percentage of Activated Bacteria at Start-up ..............

....................................................................................................... 83

4.2.4.2. Determining the Feeding Strategy for Continuous Operation ...

....................................................................................................... 84

xiv

4.3. Hydrogen Production on Dark Fermentor Effluent of Molasses .................. 85

4.3.1. Effect of Buffers ................................................................................. 86

4.3.2. Continuous Biohydrogen Production with R. capsulatus wild type ......

............................................................................................................... 90

4.3.2.1. Indoor Experiments ................................................................ 91

4.3.2.2. Continuous Hydrogen Production in Outdoor Experiment ........

....................................................................................................... 99

4.3.2.3. Long Term Stability of Continuous Hydrogen Production in

Outdoor Experiment .................................................................... 104

4.3.3. Long Term Stability of Continuous Biohydrogen Production with R.

capsulatus hup- in Outdoor Experiments ............................................ 115

4.4. Logistic Growth Model ............................................................................... 123

4.5. Comparison of Photofermentation Efficiency of Define Medium with the

Molasses DFE ............................................................................................. 134

5. CONCLUSIONS ................................................................................................ 136

REFERENCES ......................................................................................................... 138

APPENDICES ......................................................................................................... 153

A. COMPOSITION OF THE MEDIA AND SOLUTIONS ................ 153

A.1. Composition of the Minimal Medium ................................. 153

A.2. Composition of the Growth Medium ................................... 154

A.3. Composition of the Hydrogen Production Medium ............. 154

A.4. Composition of the Trace Elements Solution ...................... 155

A.5. Composition of the Vitamin Solutions ................................. 155

A.6. Composition of the Fe-Citrate Solution ............................... 155

B. PROPERTIES of CONSTRUCTION MATERIAL ........................ 156

B.1. Properties of Plexiglas.......................................................... 156

B.2. Properties of PVC Rigid Sheet ............................................. 156

B.3. Properties of Aluminum 6061-T6 Tubing............................ 157

B.4. Properties of Ball Valves ..................................................... 157

B.5. Properties of Adaptors.......................................................... 158

C. LIGHT ABSORBTION SPECTRA of Rhodobacter capsulatus ..........

...................................................................................................................... 159

xv

D. CALIBRATION CURVE OF DRY CELL WEIGHT VERSUS

OPTİCAL DENSITY AT 660nm ................................................................ 160

D.1. Calibration Curve of Dry Cell Weight versus Optical Density

of Rhodobacter capsulatus wild type ............................................... 160

D.2. Calibration Curve of Dry cell Weight versus Optical Density

of Rhodobacter capsulatus hup-mutant ........................................... 161

E. SAMPLE CHROMATOGRAM FOR GAS ANALYSIS ............... 162

E.1. Sample Chromatogram for Gas Analysis ............................. 162

F. SAMPLE HPLC CHROMATOGRAM OF ORGANIC ACID

ANALYSIS AND CALIBRATION CURVE OF ACETIC ACID ............. 163

F.1. Sample Chromatogram of Organic Acid Analysis............... 163

F.2. Sample HPLC calibration Curve for Lactic Acid ................ 164

F.3. Sample HPLC calibration Curve for Formic Acid ............... 164

F.4. Sample HPLC calibration Curve for Acetic Acid ................ 165

F.5. Sample HPLC calibration Curve Propionic Acid ................ 165

F.6. Sample HPLC calibration Curve for Butyric Acid .............. 166

G. PERMEABILITY of HYDROGEN THROUGH SOLIDS ............. 167

H. SOLUBILITY of HYDROGEN in WATER ................................... 169

I. MEASURING HYDROGEN PERMEABILITY THROUGH

REACTOR MATERIAL ............................................................................. 170

J. DEFINITION of GLOBAL SOLAR RADIATION ........................ 171

K. SAMPLE CALCULATIONS FOR EVALUATION OF THE

ANALYSIS .................................................................................................. 172

K.1. Sample Calculation for Dry Cell Weight ............................. 172

K.2. Sample Calculation for Bacteriochlorophyll a Content ....... 172

K.3. Sample Calculation for Acetic Acid Concentration ............. 173

L. SAMPLE CALCULATIONS FOR EVALUATION OF THE

EXPERIMENTAL DATA ........................................................................... 174

L.1. Sample Calculation for Permeability Correction ................. 174

L.2. Sample Calculation for Solubility Correction ...................... 175

L.2.1 Sample Calculation for Solubility Correction in Batch

Operation .............................................................................. 175

xvi

L.2.1 Sample Calculation for Solubility Correction in

Continuous Operation .......................................................... 175

L.3. Sample Calculation for Molar Productivity ......................... 176

L.4. .Sample Calculation for Molar Percentage Yield ................ 177

L.5. Sample Calculation for Acetate Conversion Efficiency ...... 177

L.6. Sample Calculation for Light Conversion Efficiency .......... 178

L.6.1 Light Conversion Efficiency for Indoor Experiments ...

.............................................................................................. 178

L.6.2 Light Conversion Efficiency for Outdoor Experiments .

.............................................................................................. 178

L.7. Sample Calculation for COD Removal Efficiency .............. 179

M. EXPERIMENTAL DATA ............................................................... 180

M.1. Experimental Data of Selection of Material of Construction for

Cooling Coil ..................................................................................... 180

M.2. Experimental Data of Indoor Continuous Photobioreactor (8

L) on Defined Medium (24.08.07) .................................................. 181

M.3. Experimental Data of Outdoor Continuous Photobioreactors (8


M.4. Experimental Data of Outdoor Continuous Photobioreactors (8


M.5. Experimental Data of Outdoor Continuous Pilot-Scale

Photobioreactor (20 L) on Defined Medium .................................... 193

M.6. Experimental Data on the Effect of Buffer on the Molasses

DFE .................................................................................................. 194


ml) on the Molasses DFE ................................................................. 196


L) on the Molasses DFE ................................................................... 197

M.9. .Experimental Data of Outdoor Continuous Photobioreactor (4

L) with Cooling by Rb. capsulatus wild type on the Molasses DFE

(27.07.09) ........................................................................................ 198

xvii

M.10. .Experimental Data of Outdoor Continuous Photobioreactor (4

L) with Cooling by Rb. capsulatus wild type on the Molasses DFE

(17.08.09) ........................................................................................ 199

M.11. Experimental Data of Outdoor Continuous Photobioreactor (4

L) with Cooling by Rb. capsulatus hup- on the Molasses DFE

(30.07.09) ........................................................................................ 204

xviii

LIST OF TABLES

TABLES

Table 2.1 Biological hydrogen production processes ............................................. 9

Table 2.2 Hydrogen productivities, operational conditions and operating modes

of the panel photobioreactors studied in indoor and outdoor conditions

on malate, acetate, lactate and olive mill wastewater by PNS bacteria in

the literature .......................................................................................... 24

Table 3.1 C/N ratios according to the concentrations of the substrates in the

hydrogen production media used in the experiments ........................... 32

Table 4.1 Hydrogen permeability of PMMA, Glass, Polyurethane (PU), Polyvinyl

Chloride (PVC), Low Density Polyethylene (LDPE) ......................... 50

Table 4.2 Hydrogen, oxygen and nitrogen permeability of PMMA, Glass,

Polyurethane (PU), Polyvinyl Chloride (PVC), Low Density

Polyethylene (LDPE) .......................................................................... 52

Table 4.3 Hydrogen permeability of Aluminum (Al), Copper (Cu), Stainless Steel

316 (SS 316), Stainless Steel 304 (SS 304) ......................................... 53

Table 4.4 Productivity, and molar yield values obtained during the fed batch

operation of the indoor 8 L photobioreactor runs with Rb. capsulatus

wild type on defined medium containing acetate (40-80 mM), lactate

(7.5 mM) and glutamate (2-10 mM) ................................................... 64

Table 4.5 Feed content, average biomass concentration, productivity, and molar

yield values obtained in the indoor 8 L photobioreactor runs with Rb.

capsulatus wild type on defined medium containing acetate (40-80

mM), lactate (7.5 mM) and glutamate (2-10 mM) in the Phase II and III

.............................................................................................................. 65



xix


mM), lactate (7.5 mM) and glutamate (2-10 mM) in the Phase III and

IV .......................................................................................................... 66

Table 4.7 Feed content, average biomass concentration, productivity and molar



mM), lactate (7.5 mM) and glutamate (2-10 mM) in the Phase IV and V

.............................................................................................................. 67

Table 4.8 C/N ratio, productivity and molar yield values obtained in the indoor 8

L photobioreactor runs with Rb. capsulatus wild type on defined

medium containing acetate (40-80 mM), lactate (7.5 mM) and

glutamate (2-10 mM) in the Phase II, III, IV and V ............................. 68

Table 4.9 The productivity and molar yield of all phases obtained in the outdoor 8

L photobioreactor runs with Rb. capsulatus wild type on defined

medium containing acetate (40-80 mM), lactate (7.5 mM) and

glutamate (2-10 mM) ........................................................................... 76


L photobioreactor (Run310807) runs with Rb. capsulatus wild type on

defined medium containing acetate (40-80 mM), lactate (7.5 mM) and

glutamate (2-10 mM) ........................................................................... 80

Table 4.11 Feed content, percentage of activated bacteria, duration of batch period,

biomass concentration at the end of batch period and hydrogen

productivity at start-up in three photobioreactors run with Rb.


mM), lactate (7.5 mM) and glutamate (2-10 mM) at start-up, batch

period .................................................................................................... 84

Table 4.12 Operational mode, feed content, average biomass concentration,

hydrogen productivity, and yield of three photobioreactors run with Rb.


mM), lactate (7.5 mM) and glutamate (2-10 mM) at Phase II .................

.............................................................................................................. 85

Table 4.13 Composition of the molasses dark fermentor effluent ......................... 87

xx

Table 4.14 The average biomass concentration, and average pH, total hydrogen

production in the photobioreactors ....................................................... 90

Table 4.15 Productivity, yield, light conversion efficiency and the composition of

evolved gas in indoor photobioreactors run on the molasses DFE

supplemented with different concentrations of phosphate and carbonate

buffer, using Rb. capsulatus wild type ................................................. 91

Table 4.16 Comparison of the hydrogen productivity values obtained in this study

with similar studies in the literature .................................................... 100

Table 4.17 Growth modeling of the Rb. capsulatus DSM 1710 and Rb. capsulatus

hup- on defined medium in panel photobioreactors ........................... 130


hup- on the molasses dark fermentor effluent .................................... 134

Table 4.19 Comparison of yields and productivities of continuous hydrogen

production on defined medium and the molasses DFE experiments in

panel photobioreactors ....................................................................... 135

Table A.1 Composition of the minimal medium ................................................. 153

Table A.2 Composition of the growth medium ................................................... 154

Table A.3 Composition of the hydrogen production medium (40 mM Acetate/2

mM Glutamate) ................................................................................. 154

Table A.4 The composition of trace element solution ........................................ 155

Table A.5 The composition of vitamin solution .................................................. 155

Table B.1 Physical properties of Plexiglas ......................................................... 156

Table B.2 Properties of PVC Rigid Sheet ........................................................... 157

Table B.3 Properties of Aluminum 6061-T6 Tubing .......................................... 157

xxi

Table M.1 pH, OD and cumulative H2 production values of selection of cooling

coil research ........................................................................................ 180

Table M.2 Temperature variation on the surfaces of indoor continuous

photobioreactor (8 L) on defined medium ......................................... 181

Table M.3 Light Intensity variation on the surfaces of indoor continuous

photobioreactor (8 L) on defined medium ......................................... 182

Table M.4 Cumulative H2 production, pH, OD, and organic acid values of indoor

continuous photobioreactor (8 L) on defined medium ....................... 182

Table M.5 Cumulative H2 production, pH, OD, and organic acid values of outdoor





continuous pilot scale photobioreactor (20 L) on defined medium .... 193

Table M.8 pH, OD, and cumulative gas production values obtained at different

concentrations of Na2CO3 and KH2PO4 ............................................. 194

Table M.9 Cumulative H2 production pH, OD values of indoor photobioreactor

(500 ml) on the molasses DFE ........................................................... 196

Table M.10 Cumulative H2 production pH, OD an organic acid values of indoor

continuous photobioreactor (4 L) on the molasses DFE .................... 197

Table M.11 Cumulative H2 production pH, OD an organic acid values of outdoor

continuous photobioreactor (4 L) with cooling by Rb. capsulatus wild

type on the molasses DFE (27.07.09) ................................................. 198


continuous photobioreactor (4 L) with cooling by Rb. capsulatus wild

type on the molasses DFE (18.07.09) ................................................. 199

xxii


continuous photobioreactor (4 L) with cooling by Rb. capsulatus hup-

on the molasses DFE (30.07.09) ....................................................... 204

xxiii

LIST OF FIGURES

FIGURES

Figure 2.1 Energy demand according to the energy carriers over the last 200 years.

Orange, renewables; red, nuclear fission; blue, hydro power; grey,

natural gas; dark grey, crude oil; black, coal; green, biomass ................ 7

Figure 2.2 Development of the oil price without correction for inflation .................

................................................................................................................ 7

Figure 2.3 Hydrogen production by purple non-sulfur bacteria. UQ: Ubiquinone,

PS: photosystem, OR: oxidoreductase, Fd: ferrodoxin ........................ 13

Figure 2.4 Schematic of metabolic pathways of H2 evolution/uptake and

competing reactions. A: Nitrogenase complex, catalyzing H2 evolution,

B: Membrane-bound uptake hydrogenase, responsible for H2uptake

(recycling), C: PHB synthase, involved in biosynthesis of poly-3-

hydroxybutyrate granules ..................................................................... 14

Figure 2.5 Three-component integrated biological system for H2 production ..........

.............................................................................................................. 16

Figure 3.1 Anaerobic medium preparation bottle .................................................. 29

Figure 3.2 Pre-cultivation bottle and anaerobic media preparation ....................... 30

Figure 3.3 Experimental set up of small-scale experiments .................................. 32

Figure 3.4 Experimental set up of 8 L laboratory scale experiments .................... 35

Figure 3.5 Picture of indoor 8 L laboratory scale experiment ............................... 36

Figure 3.6 Picture of outdoor 8 L photobioreactor started at 31.08.07 (PBR 1) ......

.............................................................................................................. 36

xxiv

Figure 3.7 Picture of outdoor 8 L photobioreactors started at 31.08.07 (PBR 1) and

26.09.07 (PBR 2) as they were in the greenhouse ................................ 37

Figure 3.8 Experimental set up of the laboratory (4 L) and pilot scale (20 L)

photobioreactors with internal cooling coil .......................................... 37

Figure 3.9 Picture of outdoor continuous photobioreactors (4 L) with internal

cooling coil run by Rb. capsulatus hup-(PBR 1) and Rb. capsulatus

wild type (PBR 2) on the molasses DFE started at 30.07.08 and

17.08.07, respectively ........................................................................... 38

Figure 3.10 Pictures of PBR1 and PBR2 in the greenhouse .................................... 38

Figure 3.11 Picture of outdoor pilot scale photobioreactor (20 L) with internal

cooling coil runs by Rb. capsulatus hup- on defined medium containing

30 mM acetate and 2 mM glutamate at the start-up period .................. 39

Figure 4.1 Comparison of permeability coefficients of hydrogen and oxygen of

PC, fluorocarbon polymers, and PET materials, Acrylics (Blake, 2005)

.............................................................................................................. 47

Figure 4.2 Comparison of permeability coefficients of hydrogen that was found

out experimentally and already in the literature. Chosen materials are

Plexiglas (PMMA), Glass, Polyurethane (PU), Polyvinyl Chloride

(PVC) and Low Density Polyethylene (LDPE) which have 3.5, 2.5, 1.5,

1.5 and 0.3 mm wall thicknesses, respectively ..................................... 49

Figure 4.3 Hydrogen, oxygen, and nitrogen permeability coefficients of Plexiglas

(PMMA), Glass, Polyurethane (PU), Polyvinyl Chloride (PVC) and

Low Density Polyethylene (LDPE) in the literature, respectively ....... 50

Figure 4.4 Hydrogen permeability coefficients of Aluminum (Al), Copper (Cu),

Stainless Steel 316 (SS 316), Stainless Steel 304 (SS 304) that was in

the literature .......................................................................................... 52

Figure 4.5 The pH, growth, and cumulative H2 production in the photobioreactor

which didn’t have any cooling coil material and the photobioreactors

(50 ml) containing Polyurethane (PU), Aluminum (Al), Polyvinyl

Chloride (PVC), Copper (Cu), Stainless Steel 316 (SS 316), Stainless

Steel 304 (SS 304) ............................................................................... 54

xxv

Figure 4.6 Temperature distributions of the surfaces of the indoor photobioreactor

runs with Rb. capsulatus wild type on defined medium containing

acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM).

Starting date of the experiment was 24.08.07 ...................................... 57

Figure 4.7 Light intensity distribution on the surfaces of the indoor

photobioreactor runs with Rb. capsulatus wild type on defined medium

containing acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10

mM). Starting date of the experiment was 24.08.07 ............................ 58

Figure 4.8 The pH variation in the indoor photobioreactor runs with Rb.


mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the

experiment was 24.08.07. Feeding was started on 8th

day of the

experiment ............................................................................................ 59

Figure 4.9 Cumulative hydrogen production and biomass in the indoor



mM). Starting date of the experiment was 24.08.07. Feeding was

started on 8th

day of the experiment ..................................................... 60

Figure 4.10 Organic acids concentration in the indoor photobioreactor runs with Rb.




day of the

experiment ............................................................................................ 61

Figure 4.11 Variations in temperature in the outdoor photobioreactor runs with Rb.




day of the

experiment ............................................................................................ 69



mM), lactate (7.5 mM) and glutamate (2-10 mM) Starting date of the


day of the

experiment ............................................................................................ 70

Figure 4.13 Daily global solar radiation energy during outdoor period of the

photobioreactors (Run260907 and Run310807) run with Rb. capsulatus

xxvi


(7.5 mM) and glutamate (2-10 mM). Starting date of the data is

31.08.07 ................................................................................................ 71

Figure 4.14 pH variation in the outdoor photobioreactor runs with Rb. capsulatus


(7.5 mM) and glutamate (2-10 mM) Starting date of the experiment

was 26.09.07. Feeding was started on 15th

day of the experiment ...........

.............................................................................................................. 72

Figure 4.15 Growth and cumulative hydrogen production in the outdoor



mM). Starting date of the experiment was 260907. Feeding was started

on 15th

day of the experiment ............................................................... 73

Figure 4.16 Organic acid consumption in the outdoor photobioreactor runs with Rb.




day of the

experiment ............................................................................................ 74



(7.5 mM) and glutamate (2-10 mM). Starting date of the experiment


day of the experiment ......... 76

Figure 4.18 Growth and hydrogen production in the outdoor photobioreactor runs

with Rb. capsulatus wild type on defined medium containing acetate

(40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date

of the experiment was 31.08.07. Feeding was started on 6th

day of the

experiment ............................................................................................ 77





day of the

experiment ............................................................................................ 78

Figure 4.20 The pH variation in outdoor pilot-scale photobioreactor runs with Rb.

capsulatus hup- on defined medium containing 30 mM acetate and 2

xxvii

mM glutamate at the start-up period. Starting date of the experiment


day of the experiment ......... 80

Figure 4.21 Growth and hydrogen production in outdoor pilot-scale photobioreactor

runs with Rb. capsulatus hup- on defined medium containing 30 mM

acetate and 2 mM glutamate at the start-up period. Starting date of the


day of the

experiment ............................................................................................ 81

Figure 4.22 Organic acid consumption in outdoor pilot scale photobioreactor runs

with Rb. capsulatus hup- on defined medium containing 30 mM acetate

and 2 mM glutamate at the startup period. Starting date of the


day of the

experiment ............................................................................................ 82

Figure 4.23 pH, biomass, and cumulative hydrogen production in the

photobioreactors run by Rb. capsulatus wild type on media containing

the molasses DFE supplemented with 5, 10 and 15 mM of Na2CO3 and

KH2PO4 buffer ...................................................................................... 88

Figure 4.24 pH variation in the 500 ml indoor continuous photobioreactor runs on

the molasses DFE using Rb. capsulatus wild type. Feeding was started

at 4th

day ............................................................................................... 92

Figure 4.25 Growth and daily hydrogen production in the 500 ml indoor continuous

photobioreactor runs on the molasses DFE using Rb. capsulatus wild

type. Feeding was started at 4th

day ...................................................... 93

Figure 4.26 Organic acid consumption in the 500 ml indoor continuous

photobioreactor runs on the molasses DFE using Rb. capsulatus wild

type. Feeding was started at 4th

day ...................................................... 94

Figure 4.27 pH variations in indoor continuous photobioreactor runs by Rb.

capsulatus wild type on the molasses DFE. Feeding was started at the

3rd

day ................................................................................................... 96

Figure 4.28 Biomass and daily hydrogen production in indoor continuous

photobioreactor runs by Rb. capsulatus wild type on the molasses DFE.

Feeding was started at the 3rd

day ........................................................ 97

xxviii

Figure 4.29 Organic acid consumption in indoor continuous photobioreactor runs

by Rb. capsulatus wild type on the molasses DFE. Feeding started at

the 3rd

day ............................................................................................. 98

Figure 4.30 Variation in pH in outdoor continuous photobioreactor during hydrogen

production runs by Rb. capsulatus wild type on the molasses DFE.

Starting date of the experiment was 27.07.09. Feeding was started at 3rd

day ...................................................................................................... 100

Figure 4.31 Variation in growth and hydrogen production in outdoor continuous



day ...................................................................................................... 101

Figure 4.32 The maximum and minimum temperatures attained in outdoor

continuous photobioreactor runs by Rb. capsulatus wild type on the

molasses DFE. Starting date of the experiment was 27.07.09. Feeding

was started at 3rd

day .......................................................................... 102

Figure 4.33 Daily global solar radiation energy versus daily hydrogen production in

outdoor continuous photobioreactor runs by Rb. capsulatus wild type

on the molasses DFE. Starting date of the experiment was 27.07.09.

Feeding was started at 3rd

day ............................................................ 103

Figure 4.34 Organic acid consumption in outdoor continuous photobioreactor

during hydrogen production runs by Rb. capsulatus wild type on the


was started at 3rd

day .......................................................................... 104

Figure 4.35 Long term stability of pH in outdoor continuous photobioreactor during

hydrogen production runs by Rb. capsulatus wild type on the molasses

DFE. Starting date of the experiment was 17.08.09. Feeding was started

at 6th

day ............................................................................................. 106

Figure 4.36 Growth and daily hydrogen production in outdoor continuous


Starting date of the experiment was 17.08.09. Feeding was started at 6th

day ...................................................................................................... 107

xxix

Figure 4.37 Variation in temperature in outdoor continuous photobioreactor runs by

Rb. capsulatus wild type on the molasses DFE. Starting date of the

experiment was 17.08.09. Feeding was started at 6th

day .................. 109




Feeding was started at 6th

day............................................................. 110

Figure 4.39 Organic acid consumption in outdoor continuous photobioreactor runs

by Rb. capsulatus wild type on the molasses DFE. Starting date of the


day .................. 111

Figure 4.40 Variation in COD, TOC, TN and NH4+ in outdoor continuous


Starting date of the experiment was 17.08.09. Feeding was started at 6th

day ...................................................................................................... 113

Figure 4.41 Variation in concentrations of Mg, Zn, Co, Mn, Fe, Ni, Cu, Ca, and Na

in outdoor continuous photobioreactor runs by Rb. capsulatus wild type



day............................................................. 114

Figure 4.42 The hourly hydrogen production, temperature and the solar radiation in


on the molasses DFE .......................................................................... 115

Figure 4.43 Long term stability of pH in outdoor continuous photobioreactor runs

by Rb. capsulatus hup- on the molasses DFE. Starting date of the


day ................ 116

Figure 4.44 Growth and hydrogen production in outdoor continuous

photobioreactor runs by Rb. capsulatus hup- on the molasses DFE.

Starting date of the experiment was 30.07.09. Feeding was started at

11th

day...……………………… ....................................................... 117


Rb. capsulatus hup- on the molasses DFE. Starting date of the


day ................ 118

xxx


outdoor continuous photobioreactor runs by Rb. capsulatus hup- on the


was started at 11th

day ........................................................................ 119


by Rb. capsulatus hup- on the molasses DFE. Starting date of the


day ................ 120

Figure 4.48 The COD, TOC, TN and NH4+ concentrations in outdoor continuous



11th

day ............................................................................................... 121


in outdoor continuous photobioreactor runs by Rb. capsulatus wild type



day........................................................... 122

Figure 4.50 The hourly hydrogen production, temperature in outdoor continuous



11th

day ............................................................................................... 123

Figure 4.51 The logistic model for the growth of Rhodobacter capsulatus (DSM

1710) studied in indoor continuous photobioreactor (8 L) on defined

medium (24.08.07-28.01.08) ............................................................. 125


1710) studied in outdoor continuous photobioreactor (8 L) on defined

medium (26.09.07-23.01.08) ............................................................. 126


1710) studied in outdoor continuous photobioreactors (8 L) on defined

medium (31.08.07) ............................................................................ 127

Figure 4.54 The logistic model for the growth of Rhodobacter capsulatus hup-

studied in outdoor continuous pilot-scale photobioreactor (20 L) on

defined medium .................................................................................. 128

xxxi

Figure 4.55 The logistic model for the growth of Rhodobacter capsulatus DSM

1710 studied in indoor continuous photobioreactor (500 ml) on the

molasses DFE ..................................................................................... 130

Figure 4.56 The logistic model for the growth of Rhodobacter capsulatus DSM

1710 studied in indoor continuous photobioreactor (4 L) on the

molasses DFE (24.04.09-03.05.09) ................................................... 131

Figure 4.57 The logistic model for the growth of Rhodobacter capsulatus

(DSM1710) studied in outdoor continuous photobioreactor (4 L) with

cooling on the molasses DFE (27.07.09) .......................................... 132

Figure 4.58 The logistic model for the growth of Rhodobacter capsulatus

(DSM1710) studied in outdoor continuous photobioreactor (4 L) on the

molasses DFE (17.08.09-11.10.09) ................................................... 132

Figure 4.59 The logistic model for the growth of Rhodobacter capsulatus hup-

studied in outdoor continuous photobioreactor (4 L) on the molasses

DFE (30.07.09-11.10.09) .................................................................. 133

Figure C The light absorption spectrum of Rhodobacter capsulatus ................ 159

Figure D.1 Calibration curve of dry weight versus OD660 and the regression trend

line for Rhodobacter capsulatus (DSM 1710) (Uyar, 2008). An optical

density of 1.0 at 660nm corresponds to a cell density of 0.54 gram dry

cell weight/liter of culture of Rhodobacter capsulatus (DSM 1710) ......

............................................................................................................ 160


line for Rhodobacter capsulatus mutant (Ozturk, 2005). An optical

density of 1.0 at 660nm corresponds to a cell density of 0.47 gram dry

cell weight/liter of culture of Rhodobacter capsulatus hup- mutant .. 161

Figure E.1 Sample chromatogram for gas analysis .............................................. 162

Figure F.1 Sample chromatogram of organic acid analysis. Retention times of

lactic, formic, acetic, propionic and butyric acid are 20.4, 21.7, 23.6,

27.6, 33.4, respectively ....................................................................... 163

Figure F.2 Sample HPLC calibration curve for lactic acid .................................. 164

xxxii

Figure F.3 Sample HPLC calibration curve for formic acid ................................ 164

Figure F.4 Standard HPLC calibration curve of acetic acid ................................ 165

Figure F.5 Standard HPLC calibration curve of propionic acid .......................... 165

Figure F.6 Standard HPLC calibration curve of butyric acid .............................. 166

Figure J.1 Diffuse, direct and reflected radiation ................................................ 171

xxxiii

LIST OF SYMBOLS

A: Irradiated area (m2)

Acetyl-CoA: Acetyl Coenzyme A

ADP: Adenosine di-Phosphate

ATP: Adenosine tri-Phosphate

bchla: Bacterirochlorophyll a

C: Concentration (mM)

Cbchl a:Bacteriochlorophyll a concentration of the culture (mg bchl a/Lc)

D: Sum of days elapsed from January 1

DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen

E: Energy of sun (J)

I: Light intensity (W/m2)

kc: Specific growth rate constant obtained by logistic model, (h-1

)

L: Length (m)

m: Slope of the calibration curve (mg/Lc)

MW: Molecular weight (g mole-1

)

N: Number of moles (mole)

OD: Optical density

P: Pressure (Pa)

PBR: Photobioreactor

PHB: Polyhydroxybutyrate

PMMA: Poly(methyl methacrylate)

PNS: Purple Non-Sulfur

QH2: Molar productivity of hydrogen (mmolesH2/Lc.h)

r: Correlation coefficient (dimensionless)

R: Ideal gas constant (dm3.Pa.mmole

-1.K

-1)

S: Standard error (dimensionless)

t: time (h)

xxxiv

T: Temperature (0C)

V: Volume (L)

x: Mole fraction (dimensionless)

X: Bacterial concentration (g dcw/Lc)

Y: Yield (%)

Greek Letters:

ε: Extinction coefficient

μmax: Specific growth rate constant obtained by exponential model, (h-1

)

η: Efficiency (%)

∆Hc: Combustion enthalpy of hydrogen (j/mole)

σD: Solar constant (W/m2)

θ: Solar declination

Subscripts:

c: Culture

i: initial

f: final

in: Feed

LC:Light Conversion

o: Overall

out: Effluent

max: Maximum

e: Experimental

m: Model

ACT: Actual

THEO: Theoretical

xxxv

LIST OF ABBREVIATIONS

AA: Acetic acid

BA: Butyric acid

C/N: Carbon to Nitrogen Ratio

COD: Concentration of chemical oxygen demand (mg/L)

DFE: Dark Fermentor Effluent

FA: Formic acid

FID: Flame Ionization Detector

FM: Fresh media

GC: Gas Chromatography

hup-: Uptake hydrogenase deficient (mutant)

HPLC: High Performance Liquid Chromatography

LA: Lactic acid

LC: Light Conversion

LDPE: Low density polyethylene

NA: Not available

PA: Propionic acid

SRWW: Sugar refinery waste water

SS: Stainless Steel

TN: Total nitrogen (mg/L),

TOC: Total organic carbon (mg/L),

1

CHAPTER 1

INTRODUCTION

Provision of the energy demand, depletion of the common energy resources and

the global climate change emerged with increasing fossil-based CO2 emission

constitutes the main challenges of the mankind in this century. In this point of view

hydrogen will become more and more important throughout the next decades.

Because hydrogen is the most abundant element in the universe and water was the

main product in combustion (Das and Veziroglu, 2001). This makes it invaluable as a

clean, renewable, harmless energy carrier of the future. Furthermore it has high

energy content considering its molecular weight. Higher heating value of hydrogen is

142 MJ/kg and greater than the higher heating value of natural gas which is 28.1

MJ/kg (Hawkins and Joffe, 2005).

There are several methods to produce hydrogen from both renewable and non-

renewable resources. From non-renewable resources steam methane reforming,

partial oxidation of hydrocarbons, coal gasification, and biomass gasification are

regarded as the most common methods (Pilavachi et al., 2009). Steam methane

reforming supply for more than a half of world hydrogen production that’s why it is

the principal industrial process for the production of hydrogen. Gasification of coal is

also a commercial technology for production of hydrogen in the processes of

ammonia and methanol synthesis. However, coal gasification process has been

successfully implemented in regions that do not have access to natural gas (Mueller-

Langer et al., 2007). Hydrogen production technologies basing on fossil fuels also

increase the CO2 emission and faces with the exhaustion of the resources. Obviously,

hydrogen can be an ideal energy carrier if it is produced from renewables. The

electrolysis combined with photovoltaics, wind power, hydropower, geothermal

power and biological hydrogen production from biomass are also alternative

2

developing methods to the non-renewable ones as clean hydrogen production

techniques.

Using hydrogen as an energy carrier brings with it both technological and

infrastructural development. So long as it is possible to burn hydrogen directly as a

fuel, it is also possible to use fuel cells as a direct electrical energy conversion

device. There are four main areas of fuel cell applications: auxiliary power units

which provide additional on board power for vehicles, portable fuel cells which may

be used to replace batteries in portable electronic devices such as computers and

mobile phones, generators, stationary power for essential services, and fuel cell

vehicles (Ekins and Hughes, 2009).

Biological hydrogen is produced via direct photolysis, photofermentation, and

dark fermentation processes (Nath and Das, 2004) employing microalgae and

cyanobacteria, photosynthetic purple bacteria, or dark fermentative bacteria.

However the integration of dark and photofermentation driven by thermophile and

photosynthetic bacteria is the most promising biological process.

Two-step biohydrogen production with dark fermentation in the first step and

photofermentation in the second step offers a highly efficient biohydrogen

production process, as the maximum conversion of substrates to H2 becomes

possible. In dark fermentation, biomass is converted to hydrogen, CO2 and organic

acids by thermophilic or mesophilic dark fermentative bacteria and in

photofermentation organic acids derived from dark fermentation are converted to

more hydrogen by purple non-sulfur (PNS) bacteria. Such an approach has been

employed by the EU 6th

Framework Integrated Project HYVOLUTION, which aims

to develop a 2-stage bioprocess for the cost effective production of pure hydrogen

from biomass, ranging from energy crops to bioresidues from agro-industries

(Claassen and de Vrije, 2006).

Biomass sources used in this integrated process can be basically defined as

organic matter includes wood and wood wastes and residues, crops, trees, plants,

grasses, aquatic plants and algae, agricultural and forestry wastes, sludge, animal

wastes and residues, organic waste materials and municipal solid waste, etc.

(Muradov and Veziroglu, 2008).

3

Sugar beet molasses is a by-product of the sugar industry that is obtained as thick

syrup during the crystallization of sucrose. It contains a high amount of sucrose (ca.

50%), and is rich inorganic nitrogen, vitamins and salt that may support bacterial

growth. It has been described as a suitable feedstock for dark fermentation under

mesophilic conditions (Li et al., 2009, Guo et al., 2008, Ren et al., 2006, Ren et al.,

2007). It has been reported that sequential operation of thermophilic dark

fermentation effluent (DFE) of the molasses can be used as a substrate for the

photofermentative hydrogen production using different PNS bacterial strains, in

batch mode. The highest yield and productivity was obtained with the uptake

hydrogenase deficient (hup-) mutant strain of Rhodobacter capsulatus (Ozgur et al,

2010).

The microorganism, Rhodobacter capsulatus is a non-sulfur purple bacterium

which is rod-shaped, odorless, has very faint peach-like taste in addition to that it can

develop in a media over a pH range at least from 6 to 8.5, in the optimum

temperature value of 30 0C and grow on propionate, glucose, fructose, alanine,

glutamic acid and most fatty acids and grow without mannose, leucine, thiosulfate,

ethanol, glycerol, mannitol, sorbitol, tartarate, citrate and gluconate (Van Niel et al.,

1944).

There are two key enzymatic systems on the focus of photoproduction of

hydrogen in photosynthetic bacteria; the nitrogen fixation system and the hydrogen

metabolism. In nitrogen fixation system molecular nitrogen is reduced to ammonia

and hydrogen is produced with the consumption of ferrodoxin and ATP by the

leadership of nitrogenase enzyme (Meyer et al., 1978). In case where there is no

molecular nitrogen or in nitrogen limiting conditions four times more hydrogen is

produced consuming the same amount of ATP by nitrogenase enzyme. In hydrogen

metabolism hydrogenase enzymes catalyze the activation or reversible oxidation of

molecular hydrogen (Vignais et al., 1985). To prevent recycling of produced

hydrogen uptake hydrogenase operon was inactivated and thus the hydrogen

productivity yield is increased with the mutant Rhodobacter capsulatus hup– strain

(Ozturk et al., 2006).

Photobioreactors is termed as the technical systems for the production of the

phototrophic microorganisms (Pulz, 2001). They are classified according to the

4

differences in their geometry such as: flat panel and tubular according to their

position; horizontal, inclined, vertical or spiral and manifold connection and

serpentine, and with respect to operational modes such as: air or pump mixed, two-

phase reactors or multiphase reactors (Tredici, 2004). Air-lift reactors, bubble

columns and flat panels due to its simple construction and operation show clear

advantages as: the mass transfer is high in these systems and short liquid circulation

times can be obtained however, limited shear action and power input are necessary in

these photobioreactors for mixing, heat elimination and mass and light transfer

(Barbosa, 2003).

The photobioreactors are run in batch, continuous or fed-batch mode. Several

important control parameters are taken into account while running a photobioreactor

as: light intensity, temperature, pH, and biomass concentration, feed and feed rate.

Most of these parameters are related to each other and they affect the stability of the

continuous system. They need to be measured on-line, if possible.

In this research, the aim is to develop and to scale-up of solar panel

photobioreactors for the biological hydrogen production by photosynthetic purple

non sulfur (PNS) bacteria on artificial substrates and on real dark fermentation

effluent of molasses. The parameters studied are light intensity, temperature, feed

stock, feed rate, pH, cell concentration, light and dark cycle and carbon to nitrogen

ratio on hydrogen production. Continuous hydrogen production has been achieved on

artificial medium containing acetate and lactate by Rhodobacter capsulatus wild type

and an uptake hydrogenase deleted (hup-) mutant strain up to five months in panel

photobioreactors in indoor and outdoor conditions. In this photobioreactors

continuous hydrogen production was achieved by feeding. For scale up purposes, the

performance of 4 - 8 L panel photobioreactors has been compared with 20 L panel

photobioreactor. Various construction materials and designs for panel

photobioreactors were studied. Real dark fermentation effluent of molasses has been

used as feedstock as well as the defined medium. It can be utilized for growth and

hydrogen production by Rhodobacter capsulatus wild type and hup- mutant strains.

Besides it contains vitamins and minerals which is vital for biological activity of

microorganisms consequently there is no need to add additional nutrients. The

adjustment of the feedstock by dilution and buffer addition were found to be essential

5

for the long term stability of pH, biomass and H2 production for both in indoor and

outdoor applications. Na2CO3 can be used as buffer to keep the pH stable during long

term operation on the molasses dark fermentor effluent. Photofermentative hydrogen

production on dark fermentation effluent of the molasses has been achieved up to

five months in continuous outdoor panel photobioreactors by fed batch operation.

The seasonal changes in the climatic conditions such as light intensity and

temperature had a great effect on hydrogen productivity and yield. It has been

concluded that the carbon to nitrogen (C/N) ratio of the feedstock should be kept

within an optimal range for long term applications. Photobioreactor temperature was

controlled around 35 0C with chilled water passing through an internal cooling coil in

outdoor experiments. In outdoor conditions temperature control get the most

important parameter and it brings along with a cooling or a heating requirement.

6

CHAPTER 2

LITERATURE SURVEY

2.1. Hydrogen as the Future Energy Carrier

Since the beginning of the twenty-first century the limitations of the fossil

age with regard to the continuing growth of energy demand, the peaking mining rate

of oil, the growing impact of CO2 emissions on the environment and the dependency

of the economy in the industrialized world on the availability of fossil fuels became

very obvious. So, a major change in the energy economy from fossil energy carriers

to renewable energy fluxes is necessary (Züttel et al., 2010). The energy demand

according to the energy carriers over the last 200 years is shown in Figure 2.1. The

energy demand increased from 5 to 120 x 1012

kWhyr-1

in the last 200 years. Since

the steam engine was found in 1800ies the population of human beings increased by

a factor of 6, but the energy consumption increased by a factor of 80 and the

worldwide average continuous power consumption today is 2 kW per capita (Züttel

et al., 2010). Nevertheless the energy demand is supplied mostly (over 80%) by

fossil fuels (coal, oil, gas) and fuels make up approximately 67% of the global

energy market but in contrast global electricity demand occurs for only 33% (Hoffert

et al., 1998). The demand for fossil fuels has a strong impact on social, political and

economic interactions between the various countries. For example, two-thirds of the

crude oil reserves are located in the Middle East region, but most of it is consumed in

USA, Europe and Japan (Züttel et al., 2010). The development of oil price without

correction for inflation is shown in Figure 2.2. Since the World War 2, oil price

increase started with US oil peak then it continued with the Iran revolution, Iraq war

and the last economic crises.

7

Figure 2.1 Energy demand according to the energy carriers over the last 200 years.

Orange, renewables; red, nuclear fission; blue, hydro power; grey, natural gas; dark

grey, crude oil; black, coal; green, biomass (Züttel et al., 2010).

Figure 2.2 Development of the oil price without correction for inflation (Züttel et al.,

2010).

8

Because of the reasons mentioned above there is a great deal of effort made to

develop future hydrogen economy. Hydrogen has been identified as one of the most

promising fuels for the future (Abraham, 2002) because it is an excellent energy

carrier being the lightest, simplest and most abundant element in the universe. The

amount of energy produced during hydrogen combustion process (with a low heating

value on mass basis) is 2.4, 2.8 and 4 times higher than that of methane, gasoline and

coal, respectively (Marban et al., 2007). On combustion, it produces water as the

main product, thus considered a clean non-polluting fuel. The developing H2

economy is almost entirely dependent upon the use of carbon-based non-renewable

resources steam reformation of natural gas (approximately 48%), petroleum refining

(approximately 30%), coal gasification (18%) and nuclear powered water electrolysis

(4%) (Gregoire-Padro, 2005). This means that 90% of the H2 we use is currently

derived from fossil fuels (Hankamer et al., 2007). Hydrogen is a renewable fuel only

if it is produced directly from solar light or indirectly via electricity from a renewable

source, wind power, hydro power or biomass.

2.2. Hydrogen Production Technologies

There are several methods for producing hydrogen from both renewable and

non-renewable sources. Hydrogen production processes from non-renewable

resources such as natural gas, oil and coal is the most common way used up to now.

Among all the processes, steam reforming of natural gas and gasification of coal are

the most common methods used worldwide. However, considering the continuous

increase in oil consumption and having trouble on finding new oil reserves, it is

predicted that new energy sources other than fossil fuels have to be found as soon as

possible.

Hydrogen production processes from renewable sources is developing

abruptly in the last few decades. Biomass and water are used as substrates; wind and

hydro power, solar energy are used as energy sources in these new technologies.

There are several methods for converting biomass to hydrogen but biological

hydrogen production via dark and/or photo fermentation is the renewable developing

technology among the others. In fermentative hydrogen production processes

9

biomass or organic wastes are converted to H2 and CO2 under dark or illuminated or

radiated conditions. Water is used as substrate in electrolysis to be converted to H2

and O2 in addition to electrical current. Wind power, hydro power or solar energy

can be served as an energy source for electrolysis of water then the hydrogen

production via electrolysis became renewable.

2.3. Biological Hydrogen Production

Biological methods of hydrogen production (direct photolysis,

photofermentation, and dark fermentation processes) by microorganisms as

microalgae and cyanobacteria, photosynthetic purple bacteria or dark fermentative

bacteria are preferable to chemical methods because of the possibility to use sunlight,

CO2 and organic wastes as substrates for environmentally benign conversions, under

moderate conditions (Redwood et al., 2009). Table 2.1 shows the different biological

hydrogen production processes (Nath and Das, 2004).

Table 2.1 Biological Hydrogen Production Processes

Process Reactions Microorganism

Direct

Biophotolysis

2H2O H2 + O2 Microalgae

Cyanobacteria

Dark

fermentation C6H12O6+6H2O 12H2+2CH3COOH+2CO2

Fermentative

Bacteria

Photo

fermentation 2CH3COOH + 2H2O 4H2 + 2CO2

Photosynthetic

bacteria

2.3.1. Dark Fermentation

In dark fermentation process, biomass is converted to hydrogen, CO2 and

organic acids by thermophile dark fermentative bacteria. Fermentation reactions can

be operated at mesophilic (25 –40 0C), thermophilic (40–65

0C), extremely

thermophilic (65–80 0C), or hyperthermophilic (>80

0C) temperatures (Levin et al.,

2004). Production of hydrogen can be carried out by a wide variety of

hv

hv

10

microorganisms such as strict anaerobes (clostridia, ruminococci, and archaea),

facultative anaerobes (Escherichia coli and Enterobacteraerogenes) and aerobes

(e.g. Alcaligeneseutrophus and Bacillus licheniformis) when kept under anoxic

conditions (Nandi and Sengupta, 1998). Many types of organic compounds, ranging

from polymers to monomeric carbohydrates, fats and amino acids are known to be

sources for hydrogen (Claassen et al., 1999). With respect to the whole process of

thermophilic heterotrophic fermentation, the partial hydrogen pressure in the gas

phase should be kept low (<2 kPa), otherwise the growth rate may decline (Kelly

1988) and, depending on the strain, the product formation may shift from acetate to

lactate (Janssen and Morgan 1992) or to alanine (Kergen and Stams 1994), thus

decreasing the final yield of hydrogen per mole of glucose. At concentrations higher

than 2 kPa, hydrogen may even inhibit growth (Schröder et al., 1994; Schafer and

Schönheit, 1991). In addition to volatile fatty acids (VFAs), anaerobic fermentation

also leads to the formation of alcohols such as ethanol, butanol and lactate (Levin et

al., 2004).

The greatest challenge for fermentative biohydrogen is that the hydrogen

yield (%17) is low (Lee et al., 2010). To increase the H2 yield, combination of dark

fermentation and microbial electrolysis cells (MECs) can be applied by which the H2

yield can be improved up to 81% (9.6 moleH2/mole glucose) if the MEC can capture

80% of its donor–substrate electrons as H2 (Lee et al., 2010).

Dark fermentation systems are described with 2-4 orders of magnitude higher

hydrogen production rates than photofermentation and biophotolysis systems but at

very low substrate conversion efficiency and low H2 enrichment of biogas.

Photofermentation systems have the next highest production rates and outperform

dark fermentation in substrate conversion efficiency and purity of biogas. A dual

system of dark and photofermentation is promising and the production rates for dual

systems could theoretically be at least as productive as the most productive dark

fermentation system.

11

2.3.2. Photofermentation

In photofermentation process photosynthetic bacteria use organic acids as the

primary carbon source for photosynthetic growth and hydrogen production by

harvesting sunlight under anaerobic conditions. Photosynthetic bacteria are found to

be the most promising as compared to other microbial system mainly due to the

following reasons (Das and Basak, 2007):

(i) High substrate to product conversion yield

(ii) Lack of oxygen-evolving activity, which is desirable for

biohydrogen production

(iii) Ability to use a wide wavelength of light (400-950 nm)

(iv) Capability to use organic substrates (derived from wastes) for

hydrogen generation that also helps in the bioremediation process.

Photosynthetic bacteria are divided into two groups as purple sulfur and

purple non-sulfur bacteria. The purple sulfur bacteria are obligate anaerobic

autotrophs, which utilize H2, H2S, and elemental sulfur, whereas non sulfur

Rhodospirillum and Rhodopseudomonas could not use sulfur and are capable of

growing aerobically on organic substrate in absence of light (Nandi and Sengupta,

1998).

Rhodobacter capsulatus can grow in media over a pH range of 6.0-8.5 and

temperature range from 25 to 39.5 0C. Lactate was found to be readily utilizable and

acetate (+ CO2) is used by only after an “adaptation” period of 2 days (van Niel,

1944; Weaver et al., 1975). Furthermore photoheterotrophic growth of Rhodobacter

capsulatus supported by a number of organic substrates which include sugars,

pyruvate, fatty acids, and dicarboxylic acids of the citric acid cycle, however, it is not

supported with ethanol (+ CO2), glycerol (+ CO2), citrate, gluconate, mannose, or

tartrate (Weaver et al., 1975).

The formation of molecular hydrogen results from the reduction of protons

from an electron donor by hydrogenase and nitrogenase enzyme complex (Vignais et

al., 1985). The hydrogenase enzymes catalyze the following reversible reaction

which shows the reversible oxidation of molecular hydrogen.

12

2H+ + 2e

- H2 (2.1)

In the presence of H2 and an electron acceptor, hydrogenase will act as a H2

uptake enzyme; in the presence of an electron donor of low potential, it may use the

protons from water as electron acceptors and release H2 (Vignais et al., 2007).

Three different classes of hydrogenases have been identified so far: [Fe]-

hydrogenase, [NiFe]-hydrogenase, and [NiFeSe]-hydrogenase. In major cases, it is

evident that [NiFe]-hydrogenase is responsible for hydrogen uptake while [Fe]-

hydrogenase catalyzes the hydrogen production processes (Cammack, 1999). [Fe]-

hydrogenase is highly sensitive towards oxygen and possesses 100-fold more activity

than [NiFe]-hydrogenase (Adams 1990). However, nitrogenase enzyme is

responsible for producing hydrogen from protons and simultaneously fixing nitrogen

in the PNS bacteria. Nitrogenase produces H2 as a byproduct. Three different types

of nitrogenase have been identified so far, and their mode of hydrogen-producing

activity using ATP is given below (McKinlay and Harwood, 2010):

Mo-nitrogenase:

N2 + 8H+ + 8e

- + 16ATP 2NH3 + H2 + 16ADP + 16Pi (2.2)

Absence of N2:

8H+ + 8e

- + 16ATP 4H2 + 16ADP + 16Pi (2.3)

V-nitrogenase:

N2 + 12H+ + 12e

- + 24ATP 2NH3 + 3H2 + 24ADP + 24Pi (2.4)

Fe-nitrogenase:

N2 + 24H+ + 24e

- + 48ATP 2NH3 + 9H2 + 48ADP + 48Pi (2.5)

The resulting stoichiometry between N2 fixation and biohydrogen production

depends on the type of nitrogenase, varying from 1 mole H2 generated per mole N2

fixed by the common Mo-containing nitrogenase to 9 mole H2 per mole N2 in the

case of the highly O2-sensitive Fe-type nitrogenase (Lee et al., 2010).

13

In Rhodobacter capsulatus, Fe-nitrogenase is repressed by Mo, and its

expression relies on many of the same regulatory proteins that are used for Mo-

nitrogenase (Masepohl et al., 2002). Nitrogenase is rapidly repressed by nitrogen

compounds, such as NH3 because N2 fixing needs ATP to happen (Dixon and Kahn,

2004). For many purple non-sulfur bacteria, this repression can be bypassed by using

glutamate as the nitrogen source (Harwood, 2008).

Hydrogen production by purple non-sulfur bacteria is shown in Figure 2.3

(McKinlay and Harwood, 2010).

Figure 2.3 Hydrogen production by purple non-sulfur bacteria. UQ: Ubiquinone, PS:

photosystem, OR: oxidoreductase, Fd: ferrodoxin.

Electrons are transferred via ubiquinone (UQ) to the photosystem (PS) where

they are energized by light. Electrons are repeatedly energized and cycled through

the photosynthetic electron transport chain to produce a proton gradient. Energy from

the proton gradient is used to transfer electrons from the photosynthetic electron

transport chain to Ferrodoxin via oxidoreductases (OR). The proton gradient is also

used to generate ATP. Ferrodoxin and ATP are then used to generate H2 via

nitrogenase (N2ase).

Hydrogen evolution in this process is competitive with synthesis of

polyhydroxyalkanates and hydrogen recycling by uptake hydrogenase (hup)

(Tsygankov 2006). The PNS bacteria, Rhodobacter capsulatus, were recently

improved for hydrogen production by eliminating polyhydroxyalkanoate (PHA)

14

synthesis and knocking out the uptake hydrogenase (Mathews and Wang, 2009). The

other improvement strategy used in PNS bacteria involved the genetic modification

of the electron transfer chains in Rhodobacter capsulatus (Ozturk et al., 2006).

Figure 2.4 shows schematic of metabolic pathways of H2 with competing reactions.

Figure 2.4 Schematic of metabolic pathways of H2 evolution/uptake and competing

reactions. A: Nitrogenase complex, catalyzing H2 evolution, B: Membrane-bound

uptake hydrogenase, responsible for H2 uptake (recycling), C: PHB synthase,

involved in biosynthesis of poly-3-hydroxybutyrate granules (Franchi et al., 2005).

Another improvement can be, as done for microalgae, minimizing, or

truncating, the chlorophyll antenna size of the photosystems which can improve

photosynthetic solar energy conversion efficiency and productivity up to 3-fold

(Melis, 2009). Inhomogeneity of the light distribution in the reactor also contributes

to lowering the overall light conversion efficiency. This may be overcome by using

co-cultures having different light utilization characteristics (Das et al., 2008).

Factors determining optimal hydrogen production, such as pH, temperature,

light intensity and wavelength, concentration of electron donor, age of culture, cell

density and nutritional history of the cells, may differ from optimal factors for

15

growth. Therefore, separation of microbial growth and product formation has often

improved hydrogen production yields (Hillmer and Gest, 1977; Sasikala et al., 1993).

The biofilm technique has been proved to be an effective cell immobilization

method in the field of photobiological H2 production. For this purpose a groove-type

photobioreactor was developed and it was shown that a groove structure with large

specific surface area was beneficial to cell immobilization and biofilm formation of

the photosynthetic bacteria on photobioreactor surface as well as light penetration

(Zhang et al., 2010).

2.3.3. Integrated Systems

Integrated systems are better than any of the biological hydrogen production

systems as they combine advantages of each other and help overcome disadvantages.

This may lead to increased hydrogen yields. Producing hydrogen from complex

organic substrates present in municipal, agricultural, and industrial wastewaters can

be accomplished by a high yield through combination of dark and photofermentation.

Organic acids, principally acetate, produced during a first stage dark

fermentation of sugars (glucose, sucrose) to hydrogen, are converted to more

hydrogen at the second stage increasing the overall hydrogen yield.

In the first stage of the integration of dark and photofermentation that

molasses used as biomass the yield per mole of hexose has been found to be

maximally 4 mole of hydrogen as given in the following fermentation (Claassen et

al., 1999):

C6H12O6(l)+12H2O(l) 4H2(g)+2CO2(g)+2CH3COOH(l) (2.6)

The ∆G'o of this reaction is -206 kJ/mole, which is sufficient to allow

microbial growth (Claassen et al., 1999).

In the photofermentation stage the yield per mole of hexose has been found to

be maximally 8 mole of hydrogen as given in the following fermentation:

2CH3COOH(l) + 4H2O(l) 8H2(g) + 4CO2(g) (2.7)

hv

16

The ∆G'o of this reaction is +104.6 kJ/mole. This thermodynamic barrier is

overcome by light energy.

Another three component proposal is shown in Figure 2.5 that includes dark

fermentation, oxygenic and anoxygenic fermentation (Melis and Melnicki, 2006).

Figure 2.5 Three-component integrated biological system for H2 production.

As shown in Figure 2.6, the role of the photobioreactors is to produce H2,

using both oxygenic and non-oxygenic photosynthesis, and to accumulate cell

biomass. The biomass is then used as substrate for dark fermentation by microbes

that are able to convert it into H2 and CO2, while waste organic acids from the latter

are fed back into the photobioreactors to stimulate biomass accumulation (Ghirardi et

al., 2008).

Immobilized-cell systems have also an alternative to suspended-cell systems

in continuous operations for enhanced biohydrogen production because they are

capable of maintaining higher biomass concentrations and could operate at higher

dilution rates without biomass washout (Show et al., 2008). Biomass immobilization

can be achieved through forming granules, biofilm, or gel-entrapped bioparticles,

which have been employed in different reactor systems (Zhang et al., 2008).

2.4. Flat Panel Photobioreactors

A flat panel photobioreactor (PBR) consists of a frame covered by a

transparent plate on both sides (Gebicki et al., 2009). Flat plate photobioreactors

17

were first described in the 1980s (Samson and Leduy, 1985) and thereafter

researched and developed by Tredici et al. (1991) and Tredici and Materassi (1992).

Vertical flat type PBRs has important advantages for mass production of

photoautotrophic microorganisms because lamination of the culture in correct

orientation to the light source is the best mode by which to expose a culture to

illumination (Richmond and Cheng-Wu, 2001). Flat plates can be easily cleaned

from both outside and inside (Cheng-Wu et al., 2001) so the operational costs

decreases and the life of the photobioreactor extends.

The efficiency of a PBR is determined by the integration of: light capturing,

light transportation, light distribution, and light usage (Zijffers et al., 2008). In

addition to efficient use of light, controlled temperature, well defined feeding rate,

cooling procedure and mixing potential of the photobioreactors are also the critical

parameters for a high performance and long term study.

2.4.1 Temperature Distribution

Temperature distribution on the surface of the PBR is highly related with the

light intensity and orientation of the PBR according to the light sources. If the PBR is

illuminated by an artificial light source (a lamp) the light intensity distribution on the

surface of the PBR may not be uniform. This results in insufficiently illuminated

areas on the surface, especially at the edges of the photobioreactor. When PBRs are

illuminated from both sides, more uniform light intensity distribution can be

achieved however, temperature distribution may not be uniform. The temperature

especially at the center of the PBR may be higher than that at the edges.

In outdoor experiments light source is the sun and PBRs are illuminated more

effectively because the exposed light to the surface of the PBR is more uniform so

the temperature distribution on the surfaces are closely related to the angle of the

impinging light on the reactor surface. Parallel plates placed in an east-west-facing

orientation that the distance between the plates is not narrow are an effective method

for a homogeneous temperature distribution on the surfaces (Zhang et al., 2001).

18

2.4.2 Light Intensity Distribution

The light energy that is absorbed by the microorganism has several

destinations. In case of purple non-sulfur bacteria producing hydrogen

photoheterotrophically, these are:

(1) Biomass growth,

(2) Biomass maintenance,

(3) Hydrogen generation, and

(4) Photosynthetic heat dissipation (Hoekama et al., 2006).

At high densities, self-shading causes the light to be progressively dissipated

as it passes through the culture until, at bottom, there is complete darkness; this

partially compensates for the limitation imposed by the light saturation effect

(Tredici et al., 1998). That spatial light dilution causing more light to be available to

the single cell is demonstrated by the higher productivity and lower pigment content

that characterize cultures illuminated from both sides (Tredici and Zitelli, 1998). The

effect of doubling illumination from either one to both sides, decreases, as expected,

light conversion efficiency, but is made up by increasing productivity (Zitelli et al.,

2000). Flat panel photobioreactors run in three or four parallel plates side by side, the

distance between the plates should not be narrow because shading always influences

irradiance levels on the surface of flat panel reactors that are close together (Pulz et

al., 1995). To prevent exponential decrease in the energy of light passing through an

absorbing medium the light path of the photobioreactor should be short (Tsygankov,

2000). Generating turbulence in photobioreactors provides moving the culture cells

in and out of the light (Weissman et al., 1987) so it enforced the homogeneous light

distribution in the reactor and with it the light utilization (Meiser et al., 2004).

2.4.3 Feeding, Cooling and Mixing

Stabilizing the cell concentration, obtaining constant substrate, nutrient and

buffer concentrations in the photobioreactor are the purposes of feeding. Biomass

concentration in the photobioreactor increases at low dilution rates, and vice versa

(Samson and Leduy, 1985). Moreover, feeding provides new substrate, nutrients for

19

the microorganism so continuous product formation can be obtained. By continuous

feeding the pH in the photobioreactor can be stabilized with buffer addition. High

dilution rates also prevent biofilm formation on the surface of the photobioreactor

but it can also lead to washout. Recycling of culture suspension increases

productivity of the photobioreactor (Samson and Leduy, 1985).

In mesophilic systems culture temperature is an important parameter to be

controlled in outdoor conditions. Water-spray systems are often used to avoid

overheating. However, the cooling capacity of spray systems is limited and its

application is only possible under certain environmental conditions. So another

cooling apparatus is necessary to control the temperature in the photobioreactor.

Sierra et al. (2008) stated that the heat transfer coefficient of the internal heat

exchanger is much higher than the coefficient of the external surface of the reactor.

So the internal heat exchangers are useful to control the temperature of the culture in

flat panel photobioreactors. A cooling coil inserted into the photobioreactor is the

most efficient method in outdoor conditions to control the temperature.

Vigorous mixing of the culture suspension increases the flashing-light effect,

facilitates movement of produced gas through the photobioreactor, and prevents the

possibility of concentration gradient in the photobioreactor that’s why the

productivity increases (Zhang et al., 2002). A modicum of turbulence can prevent

stratification which could expose for long periods (presumably tens to hundreds of

seconds) to high irradiance (Weismann et al., 1987). However, mixing leads to

higher running costs. Besides, higher mixing rates may cause cell damage. The flat

panel photobioreactors are not suitable for mechanical mixing from inside because

their light path is too narrow for an agitator. On the other hand mixing with

compressed gasses is very effective in panel photobioreactors.

2.4.4 Application of Flat Panel Photobioreactors

First flat panel photobioreactor were described by Samson and Leduy(1985)

and by Ramos de Ortega (1986) and much researched by Tredici et al. (1991), who

introduced the idea to make reactors from commercially available panels of

20

transparent sheets partitioned to form alveoli-narrow internal channels (Hu et al.,

1996).

Kim et al. (1982a) studied on batch hydrogen production by Rhodobacter

sphaeroides in two panel photobioreactors of 33 L for 25 days in outdoor conditions.

One of these photobioreactors (Culture A) was placed vertically to the ground and

the other (Culture B) was inclined at 30 0C to receive more sunlight. At the end of 25

days of hydrogen production rate was 0.021 LH2/Lc.h in culture A and 0.023

LH2/Lc.h in culture B.

Kim et al. (1987a) studied semi-continuous hydrogen production in a flat

panel photobioreactor of 6 L for 47 days in outdoor conditions. Hydrogen production

rate of 47 days of the experiment was 0.049 LH2/Lc.h.

Kitajima et al. (1998) examined the effect of reactor depth with agitation on

the course of hydrogen production by Rhodobacter sphaeroides RV on lactate for 15

days in batch indoor experiments under simulated condition of solar energy by 12

hours light 12 hours dark cycle using five cylindrical plane type PBR with different

depths (1, 3, 5, 10, 20cm). Light receiving area in this photobioreactors was 490 cm2.

Hydrogen productivity in the photobioreactors according to the depths were 0.019,

0.042, 0.022, 0.0054, 0.00046 LH2/Lc.h, respectively.

Modigell and Holle (1998) studied on the reactor development to determine

the influence of light, irradiation, substrate source and concentration, and operational

mode on hydrogen production by Rhodospirillum rubrum with a modular outdoor

bioreactor in the form of hollow channel plates made of acrylic glass that are

connected at the top and at the bottom to form a loop and erected vertically and

placed at east-west position. Constant hydrogen production was observed with

exchange of the half of the medium every fifth day for almost two months. In this

study maximum hydrogen production was reached to 2 LH2/m2.h.

Arai et al. (1998) investigated hydrogen production by Rhodobacter

sphaeroides RV using lactate and propionate as carbon sources. They used outdoor

flat bioreactors. The experiments were held in winter season in Japan, from October

1994 to March 1995. The bioreactors were made of acrylic resin and had the

irradiated area of 20 x 44 cm2, with inner thickness of 5 cm and capacity of 4.4 L.

Under an average outer temperature of 13.4 0C during 30 days from November to

21

December 1994, hydrogen production rate was 0.0125 LH2/Lc.h. In the middle of

winter of 1995, the average temperature was 5.2 0C and hydrogen production rate

achieved was 0.15 LH2/Lc.h.

Miyake et al (1999a) studied simulation of a daily sunlight illumination

pattern for photobiological hydrogen production by Rhodobacter sphaeroides RV on

lactate in indoor and outdoor panel photobioreactors with an irradiation area of 159

cm2, a working volume of 0.7 L and a 4.5 cm light path. The experiments, 3 batch

cultures, were carried out over a 3 day period in August 14 to 17 in 1996, Tsukuba,

Japan. From sunrise to sunset, the orientation of the photobioreactor was adjusted

every 30 min in a southward-facing direction at an angle of 300 from the horizontal

plane. The light intensity was increased then decreased in 12 hours in 1, 3 and 6 steps

in order to simulate daylight. Maximum hydrogen production rates for outdoor and

indoor (in 1 step) experiments were 0.042, 0.049 LH2/Lc.h.

Hoekama et al (2002) studied anaerobic photoheterotrophic cultivation of

Rhodopseudomonas sphaeroides HCC 2037 on acetate in a 2.4 L pneumatically

agitated flat panel photobioreactor with gas re-circulation in indoor conditions. In

this study it was cleared that pneumatic agitation with nitrogen or argon inhibits

bacterial growth at any flow rate between 0.33 and 6.66 LL-1

.min-1

.

Hoekama et al (2006) studied photosynthetic efficiency of photoheterotrophic

formation of hydrogen from acetate by Rhodobacter capsulatus NCIMB 11773 in a

panel type PBR with a working volume of 2.4 L in indoor conditions. They

developed a model for unsteady state behavior of the system from D-stat

experiments. They predicted from their model that hydrogen production rate could

possibly be increased up to 2.2 mmolesH2/Lc.h if biomass concentration is 4.4

gdcw/Lc.

Eroglu et al (2008) studied performance of an 8 L flat panel solar bioreactor

on hydrogen production by Rhodobacter sphaeroides O.U.001 using malate, acetate,

lactate and olive mill wastewater as carbon sources in a cooled solar bioreactor

which has a working volume of 6.5 L. Maximum hydrogen production obtained in

outdoor conditions according to the substrates were 0.010, 0.008, 0.002, 0.003

LH2/Lc.h.

22

Uyar et al (2008) also studied on performance of scaling up of panel

photobioreactor made of acrylic sheets and PVC frame on hydrogen production by

Rhodobacter capsulatus DSM 155 in indoor conditions. The photobioreactors were

illuminated by 500 W halogen lamps at a distance of 90 cm. Acetate and lactate were

used as carbon sources in these batch experiments. Average hydrogen productivity

obtained in the 5 L and 25 L photobioreactor was 0.0061, 0.0065 LH2/Lc.h,

respectively.

Androga et al (2009) studied on continuous hydrogen production by wild

type, mutant and heat adapted strains of Rhodobacter capsulatus DSM 1710 in 8 L

panel photobioreactor which were carried out in semi-continuous mode in indoor and

outdoor conditions in winter 2008, Ankara, Turkey. In all experiments

photobioreactors were placed perpendicular to the horizontal plane. In indoor

experiments photobioreactors were illuminated with tungsten lamps. In outdoor

conditions maximum hydrogen productivity (0.009 LH2/Lc.h) was obtained by

Rhodobacter capsulatus hup- on acetate for 25 days. In indoor conditions the

maximum hydrogen productivity (0.018 LH2/Lc.h) was obtained by Rhodobacter

capsulatus hup- on 40 mM acetate and 4 mM glutamate for 12 days.

Gebicki et al (2010) investigated the flat panel and tubular reactors in outdoor

conditions in Germany in the summertime for their applicability for H2 production by

means of purple non-sulfur bacteria Rhodobacter capsulatus. Four parallel

photobioreactors (25 L) were installed on a construction vertically so the total

volume of the photobioreactors reached to 100 L. All the experiments were

performed as fed-batch. The mean hydrogen productivity for panel photobioreactor

was 0.025 LH2/Lc.h while 8 m2 of the illuminated reactor surface of the panel reactor

could be installed on 1 m2 of ground space.

The hydrogen productivity, operational conditions and operating modes of the

panel photobioreactors studied in indoor and outdoor conditions on different

substrates by PNS bacteria in the literature is summarized in Table 2.2.

23

Tab

le 2

.2 H

ydro

gen

pro

duct

ivit

ies,

oper

atio

nal

condit

ions

and o

per

atin

g m

odes

of

the

pan

el p

hoto

bio

reac

tors

stu

die

d i

n i

ndoor

and o

utd

oor

condit

ions

on m

alat

e, a

ceta

te, la

ctat

e an

d o

live

mil

l w

aste

wat

er b

y P

NS

bac

teri

a in

the

lite

ratu

re

Ref

. B

act

eria

S

ub

stra

te

PB

R

Volu

me

(L)

Op

erati

on

al

Con

dit

ion

s

Op

erati

ng

Mod

e

Du

rati

on

(days)

Pro

du

ctiv

ity

(LH

2/(

Lc.h

)

Pro

du

ctiv

ity

(mm

ole

sH2/(

Lc.h

)

Kim

et

al.

1982

Rb. sp

haer

oid

es

B5/A

L

acta

te

Pan

el

33

Outd

oor

Bat

ch

25

0.0

21

0.9

3

Kim

et

al.

1982

Rb. sp

haer

oid

es

B5/B

L

acta

te

Pan

el

33

Outd

oor

Bat

ch

25

0.0

24

1.0

6

Kim

et

al.

1987

Rb. sp

haer

oid

es

B6

L

acta

te

Pan

el

6

Outd

oor

Sem

i-

Conti

nuous

47

0.0

49

2.1

9

Kit

ajim

a et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

0.5

In

doo

r B

atch

9

0.0

19

0.8

9

Kit

ajim

a et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

1.5

In

doo

r B

atch

9

0.0

42

1.8

8

Kit

ajim

a et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

2.5

In

doo

r B

atch

15

0.0

22

0.9

7

Kit

ajim

a et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

5

Indoo

r B

atch

15

0.0

054

0.2

4

Kit

ajim

a et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

10

Indoo

r B

atch

15

0.0

0046

0.0

21

24

Tab

le 2

.2 (

Con

tin

ued

)

Ara

i et

al.

1998

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

4.4

O

utd

oor

NA

30

0.0

13

0.5

6

Miy

ake

et a

l.

1999

Rb .sp

haer

oid

es

RV

L

acta

te

Pan

el

0.7

O

utd

oor

Bat

ch

3

0.0

42

1.8

8

Miy

ake

et a

l.

1999

Rb. sp

haer

oid

es

RV

L

acta

te

Pan

el

0.7

In

doo

r B

atch

3

0.0

49

2.1

9

Hoek

ama

et a

l.

2006

Rb. ca

psu

latu

s

NC

IMB

11773

A

ceta

te

Pan

el

2.4

In

doo

r C

onti

nuous

D-s

tatc

40

0.0

5a

2.2

0a

Ero

glu

et

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26

2.4.5 Scale-up of the Panel Photobioreactors

For the successful scale-up of the photobioreactors, the efficiency at which

(solar) light energy is directed to biomass growth and hydrogen production is the

most important optimization parameter (Hoekama et al., 2002). It is also necessary to

consider the plant design, material cost, strength of the reactor to achieve a high-

performance pilot scale device (Zhang et al., 2002).

In an application, the flat panel photobioreactors were scaled up to 1 m in

both height and width (Janssen et al., 2002) in order to reduce deflection of the plates

and to guarantee the gas-tightness of the enclosed volume (Gebicki et al., 2009).

Large-scale photobioreactors that can be operated under fixed and optimal

conditions with strong possibilities for sterilization, and with contamination risks that

are as low as possible remain to be developed in the future (Janssen et al., 2002).

Uyar et al (2008) studied with the largest (25 L) flat panel photobioreactor

made of PMMA and PVC frame for photobiological hydrogen production by

Rhodobacter capsulatus in batch experiments. The mean hydrogen productivity was

0.0065 LH2/Lc.h in the scale-up of photofermentation process up to 25 L on

acetate/lactate/glutamate (40/7.5/2 mM) medium.

Gebicki et al (2010) was also studied with 25 L panel photobioreactors in fed

batch mode in outdoor conditions in Germany for H2 production by Rhodobacter

capsulatus DSM 155. In this study four parallel photobioreactors (25 L) were

installed on a construction vertically so the total volume of the photobioreactors

reached up to 100L. The mean hydrogen productivity obtained in this experiment

was 0.025 LH2/Lc.h.

2.5 Objective of the Study

In this research, the aim is to develop and to scale-up solar panel

photobioreactors for the biological hydrogen production by photosynthetic purple

non sulfur (PNS) bacteria on artificial substrates and on real dark fermentation

effluents of molasses. The parameters studied are light intensity, temperature, feed

stock, feed rate, pH, cell density, light and dark cycle and C/N ratio on hydrogen

production. Continuous hydrogen production has been achieved on artificial medium

27

containing acetate and lactate by Rhodobacter capsulatus wild type and an uptake

hydrogenase deleted (hup-) mutant strain in panel photobioreactors in indoor and

outdoor conditions by fed batch operation. For scale up purposes, the performance of

4 - 8 L panel photobioreactors has been compared with 20 L panel photobioreactor.

Real dark fermentation effluent of molasses has been used as feedstock as well as the

defined medium. The adjustment of the feedstock by dilution and buffer addition

were found to be essential for the long term stability of pH, biomass and H2

production for both in indoor and outdoor applications. Photofermentative hydrogen

production on dark fermentation effluent of molasses has been achieved up to five

months in continuous outdoor panel photobioreactors. The seasonal changes in the

climatic conditions such as light intensity and temperature had a great effect on

hydrogen productivity and yield. It has been concluded that the carbon to nitrogen

(C/N) ratio of the feedstock should be kept within an optimal range for long term

applications. Na2CO3 can be used as buffer to keep the pH stable during long term

operation on the molasses dark fermentor effluent. Sugar and the other nutrients in

the dark fermentor effluent of molasses were also utilized for biomass growth by Rb.

capsulatus hup- strain.

28

CHAPTER 3

MATERIALS and METHODS

3.1 The Microorganism

Rhodobacter capsulatus wild type (DSM 1710) strain which is obtained from

DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH –

German Collection of Microorganisms and Cell Cultures, Germany). Rhodobacter

capsulatus hup- which is a mutant strain lack of uptake hydrogenase enzyme that

was genetically modified by Dr. Yavuz Ozturk (GMBE, TUB_TAK-MAM, Gebze)

from Rhodobacter capsulatus MT1131 (Ozturk et al., 2006).

3.2 Culture Media

The culture media are classified as growth and hydrogen production media.

3.2.1 Growth Media

For growth of Rhodobacter capsulatus the modified form of the minimal

medium of the Biebl and Pfennig (1981) which contained vitamins, trace elements

and iron citrate in addition to acetic acid (20 mM) and sodium glutamate (10 mM)

without ammonium chloride and yeast extract was used. The complete components

of the minimal medium of the Biebl and Pfennig (1981) and the growth medium are

given in Appendix A.1 and A.2, respectively. The recipe of the trace elements,

vitamin and Fe-citrate solutions are given in Appendix A.4, A.5, A.6, respectively.

To prepare the medium firstly the ingredients were dissolved in distilled

water in a 1 liter glass bottle with plastic crown cap resistant to high temperatures,

secondly the pH of the medium was set to 6.3 and 6.4 by adding 5 mM NaOH. The

bottle is shown in Figure 3.1. Then the glass bottle containing the media was put in

the autoclave (Prior Clave) with leaving the cap a little bit open. Later the medium

29

was autoclaved for 15 minutes at 121 0C. The cap of the bottle was secured before

taking the bottle out of the autoclave. After cooling the media to the room

temperature, vitamin, trace elements and iron-citrate were added to the solution in a

Laminar Flow cabin. Finally the media was ready for pre-cultivation.

Figure 3.1 Anaerobic medium preparation bottle

3.2.1.1 Pre-cultivation

For pre-cultivation first of all a 55 ml serum bottle and an orange rubber

stopper were autoclaved 15 minutes at 121 0C and then taken into the Laminar Flow

cabin. Previously prepared growth media (45 ml) and 10% activated bacteria (5 ml)

were added into the bottle. After the cap of the bottle, and the septa were blazed

away, the bottle closed with septa and secured with aluminum ring seal. In order to

obtain an anaerobic atmosphere inside, the bottle was flushed with argon gas (99.995

% purity) with a flow rate of 100-150 ml/min for about 5 minutes. The sterile needles

were used for argon input and output and there was a 0.20 micron sterile filter in

front of the input needle on account of filtering the undesired materials in the argon

gas. The argon flushing step of the pre-cultivation procedure is shown in Figure 3.2.

30

Figure 3.2 Pre-cultivation bottle and anaerobic media preparation

At the end the bottles were put in a cooled incubator (Nuve) which was

adjusted to 30 0C. The light intensity was adjusted to 2000 lux (114 W/m

2 for

tungsten lamp) with 100W tungsten lamps (Uyar, 2008). The distance of the lamps

from the photobioreactor was arranged by measuring the light intensity in front of the

bottle with a lux meter (LX-105 Light Meter).

3.2.1.2 Storage

To store the bacteria for one month, pre-cultivated bacteria in a 55 ml serum

bottle was put +4 0C refrigerator. Pure bacterial strains mixed with sterile glycerol

were put in an eppendorf and stored at -80 0C in a deep freezer (Ugur).

3.2.2 Hydrogen Production Media

Hydrogen production media was either prepared as artificial media. Either

artificial media is prepared or adjusted real dark fermentor effluent of molasses is

used as hydrogen production medium.

3.2.1.1 Artificial Media

Substrates of the artificial hydrogen production media are similar to growth

media except the ratio of carbon to nitrogen. The preparation procedure is also the

same with growth media. In the initial experiments the artificial medium comprised

31

acetate (40 mM), lactate (7.5 mM) and glutamate (2 mM) was used. The carbon to

nitrogen ratio of this media was 56.25. Then glutamate amount in the media was

increased to 10 mM and the carbon to nitrogen ratio was decreased to 15.25. Later

the lactate was removed from the media thence it contained only acetate (40 mM)

and glutamate (10 mM). The composition of the artificial media containing 40 mM

acetate and 2 mM glutamate is given in Appendix A.3. The carbon to nitrogen ratio

was again decreased to 13 in this media. At the end the media contained 80 mM

acetate and 10 mM lactate was used. The carbon to nitrogen ratio was increased to 21

in this media and reached acceptable level. The substrates, concentrations and the

carbon to nitrogen ratio of hydrogen production media are tabulated in Table 3.1.

Table 3.1 C/N ratios according to the concentrations of the substrates in the

hydrogen production media used in the experiments

Substrates Concentration (mM)

Acetate 40 40 40 80

Lactate 7.5 7.5 - -

Glutamate 2 10 10 10

C/N ratio 56.25 15.25 13 21

3.2.1.2 Real Dark Fermentor Effluent of Molasses

The real dark fermentor effluent of molasses was sent by F&B (Food and

Biobased Research, Wageningen, the Netherlands). Molasses was the effluent of the

dark fermentation process which was carried by the thermophilic bacteria,

Caldicellusiruptor saccharolyticus. It was rich in variable nutrients, sugars and

fermentation by-products.

3.3 Experimental Set-up

There were small, laboratory and large-scale photobioreactors used in this study.

The experimental set-up and the photobioreactor design were changed according to

the aim of the study.

32

3.3.1 Small-Scale Experiments

3.3.1.1 Photobioreactors

Small-scale experiments were studied in indoor conditions and operated in

batch or continuous modes. Glass bottles were used as photobioreactors and the

volumes of the photobioreactors were varied from 55 ml to 500 ml. In these small-

scale experiments the evolved gas was collected by water displacement method in a

glass tube which was 40 ml. The connection between the photobioreactors and the

gas collection tubes were provided with capillary tubing. The experimental set up of

the small-scale experiments is shown in Figure 3.3.

Figure 3.3 Experimental set-up of small-scale experiments

3.3.1.2 Sterilization of the Photobioreactors and Media

The photobioreactors and the hydrogen production media were autoclaved for

15 minutes at 121 0C for sterilization.

33

3.3.1.3 Start-up

The photobioreactors were started up in a Laminar Flow cabin. First of all

40% of the bottle filled with hydrogen production media then 10% activated bacteria

were added. The bottle was closed and then shook vigorously for assure mixing.

Then the cap was opened in order to take start-up sample (5 ml). Then again the

bottle was closed very tightly. After flushing the bottle with argon gas it was put into

the incubator.

3.3.1.4 Temperature Indication and Control

The photobioreactors were put in a heating-cooling incubator in this way the

temperature of the photobioreactors could be kept constant at 30 0C. Even so a

portable temperature probe was also put in the incubator in order to check the

temperature.

3.3.1.5 Light Intensity Measurements

The light intensity of the photobioreactors was adjusted to 2000 lux (114

W/m2) with two 100 W tungsten lamps.

3.3.1.6 Sampling and Feeding

Every day 5 ml samples were taken from the photobioreactor for analysis.

The cap of the photobioreactor was not open during the sampling operation and

sterile syringes were used. Primarily 5 ml sample were taken from the

photobioreactor. Then the equivalent amount media -in batch operations distilled

water and in continuous operations the hydrogen production medium- were given

into the photobioreactor.

3.3.1.7 Shut down

At the end of the experiments after taking the last sample from the

photobioreactor 3% hydrogen peroxide was added to the photobioreactor. After

ensuring that the biological activity in the photobioreactor was exhausted the waste

34

in the photobioreactor was poured out to the drainage. Then the empty

photobioreactor was sterilized with 3% hydrogen peroxide again.

3.3.2 Experiments with Panel Photobioreactors

3.3.2.1 Experimental Set-up

The flat panel photobioreactors were manufactured by Yazgan Pleksi

Company, Ankara, Turkey. The photobioreactors were made of Plexiglas sheets. The

properties of the Plexiglas are given in Appendix B1.

The photobioreactors were constructed in two scales; laboratory (4 to 8 liters)

and large scale (20 liters) photobioreactors. The laboratory scale photobioreactors

were operated both in indoor and outdoor conditions and pilot scale photobioreactor

was operated only in outdoor conditions. The photobioreactors were constructed in

two different designs; photobioreactors (8 L) with non-cooling apparatus and

photobioreactors (4 L and 20 L) with internal cooling coil.

The dimensions of the laboratory (8 L) photobioreactors are 50 cm x 40 cm x

4 cm and they were constructed by combining the Plexiglas sheets (thickness of 6

mm) like a rectangular box then sealing the corners by liquefied acrylic.

The dimensions of the laboratory (4 L) and pilot scale photobioreactor (20 L)

with cooling coil are 45 cm x 45 cm x 2 cm and 100 cm x100 cm x 2 cm,

respectively. PVC rigid sheet (thickness of 2 cm) were used as frame (dimensions of

45 cmx45 cm and 100 cm x 100 cm) that connected the two Plexiglas sheets

(thickness of 10 mm) together with stainless steel socket cap bolts and nuts with

plastic insert. Teflon gaskets and stainless steel washers were used for sealing the

bolt holes. Sponge rubber cord was used as a sealing material between the Plexiglas

sheets and PVC frame. The properties of PVC unplasticized frame are given in

Appendix B.2. For internal cooling PVC tubing was used as cooling coil in

laboratory scale (4 L) and Aluminum T6 was used in pilot scale (20 L)

photobioreactors. The properties of Aluminum T6 are given in appendix B.3. Output

connections of the PVC cooling coil were made by push-in adaptors inserted in the

PVC frame and it was stacked to the Plexiglas sheets with suction pads.

35

Miniature ball valves were inserted on the top and one sidelong of the

photobioreactors in order to provide input and output functionality. The properties of

the valves are given in Appendix B.4. The evolved gas was collected in a cylindrical

gas column (volume of 8 L) made of Plexiglas. The level of the liquid in gas

collector in indoor experiments was monitored with a camera connected to a PC. The

Polyurethane tubing connections were made with the push-in fitting adaptors that

were screwed on the ball valves. The technical properties of the adaptors are given in

Appendix B.5. Photobioreactors were either placed on a bench in indoor or steel

frame in outdoor conditions.

Experimental set up of 8 L laboratory scale experiments is shown in Figure

3.4 and the picture of indoor and outdoor 8 L laboratory scale photobioreactor run

with Rb. capsulatus wild type on defined medium containing acetate (40-80 mM),

lactate (7.5 mM) and glutamate (2-10 mM) is given in Figures 3.5, 3.6 and 3.7.

Figure 3.4 Experimental set up of 8 L laboratory scale experiments

36

Figure 3.5 Picture of indoor 8 L laboratory scale experiment.

Figure 3.6 Picture of outdoor 8 L photobioreactor started at 31.08.07 (PBR 1).

PBR 1

37

Figure 3.7 Picture of outdoor 8 L photobioreactors started at 31.08.07 (PBR 1) and

26.09.07 (PBR 2) as they were in the green house.

Experimental set up of the laboratory (4 L) and pilot scale (20 L)

photobioreactors with internal cooling coil is given in Figure 3.8 and pictures of

outdoor continuous photobioreactors are shown in Figures 3.9, 3.10 and 3.11.

Figure 3.8 Experimental set up of the laboratory (4 L) and pilot scale (20 L)

photobioreactors with internal cooling coil.

PBR 1

PBR 2

38

Figure 3.9 Picture of outdoor continuous photobioreactors (4 L) with internal

cooling coil run by Rb. capsulatus hup- (PBR 1) and Rb. capsulatus wild type (PBR

2) on the molasses DFE started at 30.07.08 and 17.08.07, respectively.

Figure 3.10 Pictures of PBR 1 and PBR 2 in the green house.

PBR 1 PBR 2

PBR 1 PBR 2

39

Figure 3.11 Picture of outdoor pilot scale photobioreactor (20 L) with internal

cooling coil runs by Rb. capsulatus hup- on defined medium containing 30 mM

acetate and 2 mM glutamate at the start-up period.

3.3.2.2 Sterilization of the Photobioreactors and Media

The photobioreactors were sterilized with hydrogen peroxide. In the first step

the photobioreactor was filled with distilled water contained 3% hydrogen peroxide

(Degen et al., 2001). After waiting 24 h the photobioreactor was washed with

distilled water three times in order to ensure that there were no hydrogen peroxide in

it. The hydrogen production media was sterilized via autoclaving as described before.

3.3.2.3 Start-up

The laboratory and large-scale photobioreactors were started up in

unsterilized conditions. The sterilized hydrogen production medium and the 25%

activated bacteria were added to the photobioreactor with a funnel from the hole on

40

the top of the photobioreactor. After the filling operation the hole was closed with a

sterile septum. There was 1% empty space remained on the top of the

photobioreactor. Then the argon gas was flushed from the valve on the top of the

photobioreactor. After 10 minutes valves on the top of the photobioreactor were

closed. Then the photobioreactor was placed in the stainless steel frame.

3.3.2.4 Temperature Indication and Control

A portable temperature probe (Maxi-T) was inserted into the hole closed with

a septum which was on the top of the photobioreactor so the temperature variations

in the photobioreactor were monitored. Beside the surface temperature of the

photobioreactors was measured by using an infrared thermometer (Testo 830-T1).

The photobioreactor was placed in a room which the temperature was adjusted with

an air conditioner. The temperature of the air conditioner was set up to 20 0C-25

0C

so the photobioreactor temperature was controlled between 30 and 35 0C.

In outdoor experiments the temperature of the photobioreactor was measured

with a temperature probe (Elimko E680 Univesal Data Loggers/Scanners, Turkey)

plunged into the photobioreactor. A cooler (Pnöso, PSS Series Process Water Cooler,

Turkey) was used to cool the distilled water circulating through PVC tube inserted

into the photobioreactor connected to a computer.

3.3.2.5 Light Intensity Measurements

The illumination of the photobioreactor was provided with four 100 W

tungsten lamps placed two sides of the photobioreactor. The light intensity on the

photobioreactor was adjusted to 2000 lux to prevent uncontrolled temperature

increase. The light intensity measurements were done with a lux meter (Lutron).

In outdoor experiments the light intensity was measured instantly by the lux

meter. The total global solar radiation data were also taken from the National

Meteorology Institute of Turkey.

41

3.3.2.6 Sampling and Feeding

First the sample (5 ml) was taken from the valve which was inserted in the

middle of the sidelong of the photobioreactor. Then 10% effluent was taken from the

bottom valve of the photobioreactor. This step made an apparent vacuum in the

photobioreactor. Then 10% hydrogen production medium or dark fermentor effluent

of molasses was given to the photobioreactor from the middle valve on the side of

the photobioreactor. At the end the pressure in the photobioreactor came to its initial

value.

3.3.2.7 Shutdown

As the experiment was finished the last sample were taken. Then 3%

hydrogen peroxide was added to the photobioreactor to stop the biological activity in

the photobioreactor. Then the waste was poured out to the drainage and the

photobioreactor was sterilized again.

3.4 Analyses

3.4.1 pH Measurement

The pH of the culture sample was measured with a pH meter (Mettler Toledo

3311). 2 ml culture was adequate for the measurement. Before the measurements the

pH electrode was calibrated with the calibration solutions which were set to 7, 4 and

9, respectively. If the calibration gave 80% - 120% accuracy the measurement would

be reliable.

3.4.2 Spectrophotometric Analysis

3.4.2.1 Cell Concentration

The cell concentration was obtained by measuring the optical density of the

culture sample using a visible spectrophotometer (Shimadzu UV-1201). For this

purpose the absorbance of the culture at 660 nm was read. The light absorption

spectrum was given in Appendix C. Hydrogen production medium and dark

42

fermentor effluent of molasses was used as a blank solution according to the study.

Then the absorbance values were converted to dry cell weights by the help of the

calibration curve of dry cell weight (gdcw) versus OD660 both Rhodobacter

capsulatus wild type and mutant strains shown in Appendix D.1 and D.2,

respectively. The calibration factor that was found from the calibration curves for Rb.

capsulatus was 0.56 gdcw/Lc. A sample calculation for dry cell weight calculation is

given in Appendix J.1.

3.4.2.2 Bacteriochlorophyll a Measurement

For determination of bacteriochlorophyll a concentration, 1 ml culture sample

was centrifuged at 13400 rpm for 10 minutes in 1.5 ml vial. Then the supernatant

was discarded and 1 ml acetone-methanol mixture (7:2 v/v) was added for the

extraction of the bacteriochlorophyll a (Cohen-Bazire 1957). Later the mixture was

vortex 1 minute for homogenization and the homogenate was centrifuged again at

13400 rpm for 10 minutes to remove almost 92% of the proteins under ambient

conditions (Biel A.J, 1986). Bacteriochlorophyll a concentration was calculated from

the absorbance of the supernatant at 770nm.The extinction coefficient is 75 mM-1

cm-

1 (Clayton R.K., 1963) and molecular weight of bacteriochlorophyll a. The molecular

formula of bacteriochlorophyll a is C55H74N4O6Mg (Senge and Smith, 1995).

Acetone-methanol mixture was used as blank solution. A sample calculation was

given in Appendix J.2.

3.4.2.3 TOC Analysis

The total organic carbon of the sample is analyzed with a COD reactor

(WTW 3200) and a spectrophotometer (Hach-Lange). Direct method of low range

total organic carbon and reagent set were used. It is essential that the total organic

carbon level of the sample is within the range of the method. For this purpose 1 ml

sample of photofermentation effluent of molasses taken into a clean tube and it was

diluted 100 times in order to fit in the appropriate range. Then the proceedings of the

method 10129 are implemented. At sample is sparged under slightly acidic

conditions to remove the inorganic carbon. In the outside vial, organic carbon in the

43

sample was digested by persulfate and acid to form carbon dioxide. During digestion,

the carbon dioxide diffused into a pH indicator reagent in the inner ampule. The

adsorption of carbon dioxide into the indicator forms carbonic acid. Carbonic acid

changed the pH of the indicator solution which, in turn, changed the color. The

amount of color change was related to the original amount of carbon present in the

sample. Test results were measured at 430 nm as soon as possible. The reagent blank

was not preserved.

3.4.2.4 TN Analysis

The total nitrogen analyses were operated with a COD reactor (WTW 3200)

and a spectrophotometer (Hach-Lange). Persulfate digestion method of low range

total nitrogen and total nitrogen reagent set were used. Before starting the procedure

1ml photofermentation effluent of molasses were diluted 10 times in order to provide

appropriate range. Sodium metabisulfite was added after the digestion to eliminate

halogen oxide interferences. Nitrate then reacted with chromotropic acid under

strongly acidic conditions to form a yellow complex. Absorbance was measured

immediately at 410 nm. The reagent blank was used up to seven days for

measurements using the same lots of reagents. It was stored in the dark at room

temperature (18–25 0C). If a small amount of white flocculation appeared (usually at

the end of one week), the reagent blank was discarded and a new one was prepared.

3.4.2.5 Ammonia Analysis

The ammonia content of the sample is determined by using the

spectrophotometer (Hach-Lange) and the appropriate reagent set. The salicylate

method (10031) of high range ammonia nitrogen is performed without dilution of the

sample. For this purpose first ammonia compounds are combined with chlorine to

form monochloramine. Then monochloramine reacts with salicylate to form 5-

aminosalicylate. The 5-aminosalicylate is oxidized in the presence of a sodium

nitroprusside catalyst to form a blue colored compound. However, the blue color was

masked by the yellow color from the excess reagent present to give a green-colored

solution. Absorbance is measured immediately at 655 nm. To preserve the samples,

44

pH of the culture sample is reduced to 2 or less with at least 2 ml of hydrochloric

acid. Preserved samples can be stored up to 28 days at 4 0C or less. Before analysis

the samples are warmed to room temperature then neutralized to a pH of 7 with 5 N

sodium hydroxide.

3.4.2.6 COD Analysis

A spectrophotometer (Hach-Lange) and the appropriate reagent set between

20 and 1500 mg/L are used to determine the COD content of the sample. The

procedure of the reactor digestion method (8000) of chemical oxygen demand is

followed. The culture sample is diluted 10 times before analysis. By this procedure

the mg/L COD results are defined as the mg of O2 consumed per liter of the sample.

First, the sample was heated for two hours with a strong oxidizing agent, potassium

dichromate. Oxidizable organic compounds reacted, reducing the dichromate ion

(Cr2O72–

) to green chromic ion (Cr3+

) by this way the amount of Cr3+

produced is

determined. The COD reagent also contains silver as a catalyst and mercury ions to

complex chloride interferences. The absorbance is measured at 620 nm, as soon as

possible. The blank is used repeatedly for measurements using the same lot of vials.

It is stored in the dark. To monitor decomposition of the blank the absorbance is

measured at the appropriate wavelength (420 or 620 nm). At first the absorbance

mode is set to zero, using a vial containing 5 ml of deionized water. The absorbance

of the blank is then measured. If the value is lower than 0.01 absorbance units the

blank is used for measurements. The samples are treated with sulfuric acid to a pH of

less than 2 (about 2 ml per liter) and refrigerated at 4 0C then can be stored up to 28

days. Correction is needed in the test result for volume additions.

3.4.3 Gas Chromatography Analyses

3.4.3.1 Gas Analysis

For determination of the composition of the evolved gas 500 µl gas sample is

taken from the top of the gas column with a gas-tight syringe (Hamilton, 22 GA 500

μl gas tight No. 1750) then it is assayed by a gas chromatography (Agilent

Technologies 6890N) equipped with Supelco Carboxen 1010 column and a thermal

45

conductivity detector. Argon was used as a carrier gas at a flow rate of 26 ml/min.

The oven, injector and detector temperatures were 140 0C, 160

0C and 170

0C,

respectively. A typical gas analysis chromatogram is given in Appendix E.1.

3.4.4 HPLC Analyses

3.4.4.1 Organic Acid Analysis

Organic acid analysis is carried out using a HPLC (Shimadzu 10A series).

Liquid samples are filtered using a 45 μm nylon filters (Millipore, 13 mm) to get rid

of impurities. The filtered samples are analyzed by an Alltech IOA-1000 (300 mm x

7.8 mm) HPLC column. In the analysis, 0.085 M H2SO4 is used as the mobile phase

and the oven temperature is kept constant at 66 0C. A low gradient pump (Shimadzu

LC-10AT) with a degasser (Shimadzu DGU-14A) is used to maintain the mobile

phase flow rate at 0.4 ml/min. An auto sampler (Shimadzu SIL-10AD) injected 10 μl

sample into the system and a UV detector (Shimadzu FCV-10AT) with absorbance

set at 210 nm is used to determine the component separation. Peak values for the

samples are automatically recorded and concentrations are determined manually by

constructing calibration curves for different concentrations of pure organic acid

standards. The organic acids measured are lactic acid, formic acid, acetic acid,

propionic acid and butyric acid. Shown in Appendix F.1, F.2, F.3, F.4, F.5, and F.6

are sample HPLC chromatogram and calibration curves for lactic acid, formic acid,

acetic acid, propionic acid and butyric acid, respectively. Sample calculation of

acetic acid concentration is shown in Appendix J.3.

3.4.5 Elemental Analyses

The detailed elemental composition of the samples was done with atomic

absorption spectroscopy (Philips, PU9200X) in Department of Chemical Engineering

of Middle East Technical University, Ankara, Turkey.

46

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Selection of Construction Materials for Panel Photobioreactors

In photobiological processes cost, the ability to transmit light and, gas permeation

rates of the reactor materials are apparently important for materials of construction.

Because hydrogen, oxygen and nitrogen permeation rates, transmission of the

specific wavelengths of light required by the light conversion system, factors related

to strength and impact resistance are very important for process intensification. It is

essential to prevent leakage of produced hydrogen from inside to the outside of the

photobioreactor as well as prevent oxygen and nitrogen leakage into the system in

order to keep continuity of microbiological activity towards the hydrogen production.

Optical elements are the most important part of the reactor materials because they

are the most susceptible to weathering effects. The other reactor materials consist of

seals and gaskets, adhesives, piping, and valves are all have to meet rigorous cost and

performance standards. So, in this section, first hydrogen and oxygen permeability of

optical elements in the literature were reviewed then hydrogen permeability’s of

selected reactor materials in the literature and found out experimentally were

compared. At the end effect of type of cooling coil material on hydrogen production,

cell growth, and, pH, and effect of photobioreactor design on the process

intensification was researched.

4.1.1 Hydrogen Permeability

The rate of hydrogen loss and potentially nitrogen and oxygen entry into

reactors due to the permeability of the materials of construction is considered to be a

key performance and safety parameter (Blake, 2005). Figure 4.1 gives values for

hydrogen and oxygen permeability coefficients for polymers of interest for the

47

optical elements in photobiological systems. The figure also contains hydrogen and

oxygen values from the literature. Data for five materials shown on the right side of

the figure (indicated by PDL) are from the Polymer Data Library compilation

(Massey, 2003).

Figure 4.1 Comparison of permeability coefficients of hydrogen and oxygen of PC,

fluorocarbon polymers, and PET materials, Acrylics (Blake, 2005).

Blake (2005) concluded after researching hydrogen and oxygen permeability

of some optical elements that PCs are tough but “yellow” and crack outdoors over

time, PET formulations have not yet proven to be durable outdoors but have

favorable gas permeation properties, Kynar, a fluoropolymer (PVDF), has very good

outdoor durability and favorable gas permeation properties. But Kynar is not widely

used in the industry so it costs higher than plastics. Consequently the most proper

reactor materials for photobiological systems in outdoor conditions in terms of cost,

48

availability, and convenience to system requirements are the most widely used

acrylics, glass, and plastics.

The best candidates which are Plexiglas (PMMA), Low Density Polyethylene

(LDPE), Polyvinylchloride (PVC), and Polyurethane (PU) among acrylics and

plastics were chosen beside Glass as a possible reactor material in this study.

Because all chosen materials are cheap and most widely used in various

manufacturing fields all around the word.

PMMA is a transparent thermoplastic and a kind of synthetic polymer of

methyl methacrylate. It is a versatile material that has been used in a wide range of

fields and applications. It is alternative to polycarbonate and it has moderate

properties as easy handling and processing, and low cost. Low Density Polyethylene

(LDPE) is also a thermoplastic made of petroleum. Its most common use is in plastic

bags. It is also used for various molded laboratory equipments and tubing. PVC is the

third most widely produced plastic, after polyethylene and polypropylene (ACC

Resin Statistics Summary, 2009) and it is widely used in construction because it is

cheap, durable, and easy to assemble. Polyurethane is widely used high performance

adhesives and sealants, seals, gasket, and hard plastic parts. Glass is also essential in

science and industry. So hydrogen permeability of Plexiglas (PMMA), Glass,

Polyurethane (PU), Polyvinyl Chloride (PVC) and Low Density Polyethylene

(LDPE) was examined in order to choose the best photobioreactor material in

photobiological systems. Method for measuring hydrogen permeability through

chosen reactor materials is given in Appendix I. Figure 4.2 shows comparison of

permeability coefficients of hydrogen related to chosen materials that were found out

experimentally and the data already in the literature. The wall thicknesses of

Plexiglas (PMMA), Glass, Polyurethane (PU), Polyvinyl Chloride (PVC) and Low

Density Polyethylene (LDPE) used in the experiments were 3.5, 2.5, 1.5, 1.5 and

0.3mm, respectively. Definition of permeability and experimental method of

measuring hydrogen permeability through materials are given in Appendices G and I,

respectively.

49

Figure 4.2 Comparison of permeability coefficients of hydrogen that was found out

experimentally and already in the literature. Chosen materials are Plexiglas

(PMMA), Glass, Polyurethane (PU), Polyvinyl Chloride (PVC) and Low Density

Polyethylene (LDPE) which have 3.5, 2.5, 1.5, 1.5 and 0.3mm wall thicknesses,

respectively.

Results related to hydrogen permeability experiments and data already in the

literature are given in Table 4.1.

Table 4.1 Hydrogen permeability of PMMA, Glass, Polyurethane (PU), Polyvinyl

Chloride (PVC), Low Density Polyethylene (LDPE)

Hydrogen Permeability (mole/(m.s.Pa)

Material Experimental Literature Reference

PMMA 1.88E-14 8.09E-16 Orme, 2003

Glass 4.38E-16 2.00E-16 Souers et al., 1978

PU 2.78E-12 7.40E-15 Patricio et al., 2006

PVC (plasticized) 4.86E-11 6.29E-08 Mark et al., 1999

LDPE 2.30E-11 5.83E-15 Orme, 2003

1

10

100

1.000

10.000

100.000

1.000.000

PMMA Glass PU PVC LDPE

Per

mea

bil

ity

Co

effi

cien

t (1

016m

ole

/m.s

.Pa

)

Materials

Experimental Literature

50

Hydrogen permeability of Glass was experimentally found out the lowest and

PVC was found out the highest among the other chosen materials. In addition to that

experimental data obtained in this study for Glass is also similar with the data that

was already in the literature. As we compare the results of the hydrogen permeability

experiments in terms of Polyurethane (PU), Polyvinyl Chloride (PVC) and Low

Density Polyethylene (LDPE) we can see that hydrogen permeability of PVC is the

highest and PU is the lowest. In the literature hydrogen permeability of plasticized

PVC is also highest but hydrogen permeability values of PU and LDPE are very

similar and LDPE has lower hydrogen permeability than PU.

To choose suitable reactor material for photobiological systems nitrogen and

oxygen permeability values of the materials of construction are also important as

well as hydrogen permeability values. Figure 4.3 illustrates hydrogen, oxygen, and

nitrogen permeability coefficients of Plexiglas (PMMA), Glass, Polyurethane (PU),

Polyvinyl Chloride (PVC) and Low Density Polyethylene (LDPE) that are in the

literature.

Figure 4.3 Hydrogen, oxygen, and nitrogen permeability coefficients of Plexiglas

(PMMA), Glass, Polyurethane (PU), Polyvinyl Chloride (PVC) and Low Density

Polyethylene (LDPE) in the literature, respectively.

1

10

100

1.000

10.000

PMMA Glass PU PVC LDPE

Per

mea

bil

ity

Co

effi

cien

t (1

016 m

ole

/(m

.s.P

a))

Materials

H2 O2 N2

51

Data related to hydrogen, oxygen and nitrogen permeability coefficients of

chosen materials in the literature are given in Table 4.2.

Table 4.2 Hydrogen, oxygen and nitrogen permeability of PMMA, Glass,

Polyurethane (PU), Polyvinyl Chloride (PVC), Low Density Polyethylene (LDPE)

Permeability (mole/(m.s.Pa)

Material H2 O2 N2 Reference

PMMA 8.09E-16 1.11E-15 4.05E-16 Orme, 2003

Glass 2.00E-16 NA NA Souers et al., 1978

PU 7.40E-15 2.75E-15 8.43E-16 Patricio et al., 2006

PVC

(plasticized) 6.29E-08 9.44E-08 7.19E-08 Mark et al., 1999

LDPE 5.83E-15 2.12E-15 1.42E-15 Orme, 2003

The most suitable photobioreactor materials according to hydrogen, oxygen

and nitrogen permeability values for photobiological hydrogen production are

Plexiglas (PMMA) and Glass but Glass is brittle so it is not suitable for machining.

On the other hand Plexiglas (PMMA) is easy to work with and very strong so

Plexiglas (PMMA) was chosen as photobioreactor material.

Hydrogen permeability coefficient of cooling coil material is also important

in biological hydrogen production systems because produced hydrogen can permeate

through the coil into the cooling liquid and this is accounted for a hydrogen loss from

the photobioreactor. So flexible plastics and pliable metals are good candidates for a

cooling coil. Therefore Aluminum (Al), Copper (Cu), Stainless Steel 316 (SS 316),

Stainless Steel 304 (SS 304), and Polyvinyl Chloride (PVC) and Polyurethane (PU)

were chosen as candidates for cooling coil material. Hydrogen permeability values of

PVC and PU were given above so hydrogen permeability values only for Al, Cu, SS

316, and SS 304 in the literature are figure out in Figure 4.4 and also the

permeability values are tabulated in Table 4.3.

52

Figure 4.4 Hydrogen permeability coefficients of Aluminum (Al), Copper (Cu),

Stainless Steel 316 (SS 316), Stainless Steel 304 (SS 304) that was in the literature.

Table 4.3 Hydrogen permeability of Aluminum (Al), Copper (Cu), Stainless Steel

316 (SS316), Stainless Steel 304 (SS 304)

Permeability (mole/(m.s.Pa)

Material H2 Reference

Aluminum 18.0E-5 Steward, 1983

Copper 8.42E-7 Steward, 1983

Stainless Steel 316 2.36E-7 Steward, 1983

Stainless Steel 304 1.95E-6 Steward, 1983

According to the data Aluminum has the highest and Stainless Steel 316 has

the lowest hydrogen permeability coefficient. On the other hand PU and PVC has

lower hydrogen permeability values than Al, Cu, SS 316, SS 304 and PU has the

lowest hydrogen permeability value but PVC is more flexible than PU so it is

suitable for cooling coil material.

1

10

100

1.000

Al Cu SS 316 SS 304

Per

mea

bil

ity

*1

07 (

mo

l/m

.s.P

a1/2

)

Materials

53

4.1.2 Selection of Cooling Coil Material

When there is a big difference between day and night temperatures, cooling

or heating become inevitable in order to keep the temperature of the reactor in the

desired range. For this purpose a coil was designed to be inserted into the

photobioreactor. The effect of cooling coil material on pH, the biological growth and

its hydrogen production of R. capsulatus has been determined to select a suitable

construction material for the coil. Polyurethane (PU), Aluminum (Al), Polyvinyl

Chloride (PVC), Copper (Cu), Stainless Steel 316 (SS 316), and Stainless Steel

304(SS 304) were some of the materials that has been researched. The pH, growth

and cumulative hydrogen production by Rb. capsulatus was tested in small bottle

experiments containing a piece of coil material and compared with control

experiment which did not contain these materials. Figure 4.5 illustrates the pH, cell

concentration and cumulative hydrogen production in the photobioreactor which

didn’t have any cooling coil material and the photobioreactors (50 ml) containing

Polyurethane (PU), Aluminum (Al), Polyvinyl Chloride (PVC), Copper (Cu),

Stainless Steel 316 (SS 316), and Stainless Steel 304 (SS 304).The experimental data

are given in Appendix M.1. The materials tested had no significant effect on pH and

it was around 7.0 during the experiment.

The microorganism in the photobioreactor in which Polyurethane (PU) tubing

was inserted grew more than the others at fourth day but the trend of the cell

concentration in all photobioreactors was similar during the experiment. The growth

in all photobioreactors stabilized around 0.5 gdcw/Lc after a rapid increase in the first

4 days. Microbial growth was not affected adversely with any of the selected

materials. The maximum hydrogen production was observed in the photobioreactor

in which stainless steel 304 alloys (SS 304) was inserted. The hydrogen

productivities in the photobioreactors that PVC, Aluminum (Al) and the Stainless

Steel 316 alloy (SS 316) were inserted produced the same amount of hydrogen.

Hydrogen production in the Polyurethane (PU) inserted photobioreactor was lower

than the PVC inserted one. The lowest hydrogen production was observed in the

photobioreactor that was no cooling coil material in it which was named as control

and the photobioreactor containing Copper (Cu) in it.

54

Figure 4.5 The pH, growth, and cumulative H2 production in the photobioreactor

which didn’t have any cooling coil material and the photobioreactors (50 ml)

containing Polyurethane (PU), Aluminum (Al), Polyvinyl Chloride (PVC), Copper

(Cu), Stainless Steel 316 (SS 316), Stainless Steel 304 (SS 304).

5,0

5,5

6,0

6,5

7,0

7,5

8,0

8,5

9,0

9,5

10,0

0 1 2 3 4 5 6 7 8 9 10 11 12

pH

Control PU Al PVC

Cu SS 316 SS 304

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

0 1 2 3 4 5 6 7 8 9 10 11 12

Cel

l C

on

cen

tra

tio

n (

gd

cw/L

c)

0

20

40

60

80

100

120

140

160

180

0 1 2 3 4 5 6 7 8 9 10 11 12

Cu

mu

lati

ve

H2 P

rod

uct

ion

(m

l)

Time (days)

55

As a conclusion after investigating the effect of cooling coil material on pH,

cell concentration and hydrogen productivity, PVC was chosen as cooling coil

material for the outdoor photobioreactors.

4.1.3 Panel Photobioreactor Design

The economics of photobiological hydrogen production process is primarily

based on reactor material. Durability and cost have to be considered while choosing

the construction materials. Glass, Plexiglas and Low Density Polyethylene are most

common photobioreactor materials used in laboratory or prototype scales. Glass and

PMMA have a long life span (20 years), while LDPE has 3 years maximum life span

(Burgess et al., 2006). Although it has long life span glass is not workable and is

more fragile but Plexiglas is suitable for machining. So Plexiglas was chosen as a

photobioreactor material.

When designing the photobioreactor, a maximal ratio of illuminated surface

area to land space was aimed. Since the ratio of illuminated surface area per ground

space is higher in vertical reactors than in horizontal ones, vertical panel

photobioreactor has been selected. The maximum height of the panel is limited to 1

m and the thickness of the plates is limited to 10 mm in order to reduce the deflection

of the transparent plates and to guarantee the gas-tightness of the enclosed volume,

respectively.

The performance of a photobioreactor is strongly dependent on the light

availability for each single cell of the dense suspension of the microorganism within

the panels. Self-shading due to light absorption by the pigments causes an

exponential decay in light intensity with culture depth. To provide high light

intensity absorption, the depth of the photobioreactors was adjusted to 4 cm, but it is

then decreased to 2 cm (Nakada et al., 1998). Because, hydrogen production rate per

area decreases as the reactor depth becomes greater than 3 cm (Kitajima et al., 1999).

Double-sided illumination was applied in order to provide more homogeneous light

distribution in the photobioreactor. Besides light distribution investigations, mixing

effects have led to a panel depth of 2 cm. Evolving hydrogen bubbles produced by

the cells induce a slow convective flow in the narrow vertical volumes of the panels

56

and thereby the suspension is autogenously mixed by an air-lift effect. This effect has

already been reported by Kim et al. (1982), who observed an enhanced productivity

in an inclined flat panel photobioreactor.

4.2 Continuous Hydrogen Production on Defined Medium by Rb. capsulatus

Wild Type and Mutant Strains

Studies with defined medium primarily aimed at the determination of optimum

substrate concentrations providing maximum hydrogen production continuously both

in indoor and outdoor conditions. In the second step, establishing the optimum

feeding rate, minimizing the negative effect of temperature fluctuations on hydrogen

production and providing adequate light intensity were aimed. For this purpose 3

photobioreactors (8 liters each) without cooling; one in indoor and two in outdoor

were run in parallel periods.

4.2.1 Indoor Lab-Scale Panel Photobioreactor

An 8 L in indoor lab-scale panel photobioreactor (Run240807) was operated

on defined medium containing acetate and lactate as carbon source and glutamate as

nitrogen source. The experiment lasted for 167 days. The temperature and light

intensity distributions of both sides of the photobioreactor, pH, cell concentration,

and cumulative hydrogen production were recorded continuously. All gas

measurements were corrected by calculating permeability of hydrogen through

reactor surface and solubility of hydrogen in water and carryover amounts in

inoculum. Methods for calculating hydrogen permeability through solids and

solubility in water and are given in Appendix G and H, respectively. Organic acid

composition and gas composition were also analyzed throughout the experiment. In

order to find the optimum feed composition, it was changed from time to time.

Experimental data are given in Appendix M.2. The total global solar radiation data

was taken from the National Meteorology Institute in Ankara. The definition of total

global solar radiation is given in Appendix J.

57

4.2.1.1 Temperature and Light Intensity Distribution

The homogenous temperature and light intensity distribution in the

photobioreactor was important in order to obtain maximum hydrogen production

efficiency. For this reason the room temperature in which the photobioreactor run

was controlled by an air conditioner around 22 0C in hot days; 32

0C in cold days.

The light intensity was provided with 4 tungsten lamps placed two sides of the

photobioreactor within a distance of 15 cm from the photobioreactor. To obtain the

temperature and light intensity distribution data on the surfaces of the

photobioreactor the temperature and the light intensity were recorded in 20 points.

The temperature distribution of the surfaces of the photobioreactor run with Rb.

capsulatus wild type on defined medium is shown in Figure 4.6.

The temperature distribution on the surfaces was very similar on both sides.

The hottest zone of the surfaces was on the middle of the PBR where temperature

was around 32-34 0C. The sides of the both surfaces were cooler than the middle part

and the temperature was between 28 0C and 30

0C on these places. As a result, the

optimum temperature range for photo-production of hydrogen (30-40 0C) (Sasikala et

al., 1991b) was provided in the photobioreactor.

Figure 4.6 Temperature distributions of the surfaces of the indoor photobioreactor

runs with Rb. capsulatus wild type on defined medium containing acetate (40-80

mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the experiment

was 24.08.07.

1

2

3

4

1 2 3 4 5

Heig

ht

Length

Right Surface

26-28 28-30 30-32 32-34

1

2

3

4

1 2 3 4 5

Heig

ht

Length

Left Surface

24-26 26-28 28-30 30-32 32-34

58

The light intensity distribution on the surfaces of the 8 L indoor

photobioreactor run with Rb. capsulatus wild type on defined medium is shown in

Figure 4.7. The light intensity varied mostly 1000-2000 lux which corresponds for a

tungsten lamp to 57-114 W/m2

(Uyar, 2009) on the right surface of the

photobioreactor.

Figure 4.7 Light intensity distribution on the surfaces of the indoor photobioreactor

runs with Rb. capsulatus wild type on defined medium containing acetate (40-80

mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the experiment

was 24.08.07.

The light intensity on the surfaces of the photobioreactors was below the

saturation level for hydrogen production which was reported to be 5000 lux in a

malate glutamate system (Sasikala et al., 1991). Besides, it was also below 6500 lux

which was the saturation level both hydrogen production and growth in a lactate-

glutamate system. But the light intensity was in a range for the evolution of hydrogen

which was occurred during growth at light intensities as low as 540-1080 lux

(Hillmer and Gest, 1976). Light intensity was successfully adjusted to the proposed

range by Uyar et al., 2007.

1

2

3

4

1 2 3 4 5

Heig

ht

Length

Rigth Surface

0-1000 1000-2000 2000-3000

1

2

3

4

1 2 3 4 5

Heig

ht

Length

Left Surface

0-500 500-1000 1000-1500

1500-2000 2000-2500

59

4.2.1.2 Effect of Feed Composition on Long Term Application

In the experiment started at 24.08.2007 the effect of different feed

compositions on pH, growth and hydrogen production was investigated. The

experimental period was divided into 5 phases according to the composition of the

feeding medium. The feeding volume was the 12.5% by the volume of the

photobioreactor and the photobioreactor was fed every other day. The feed composed

of Biebl and Pfennig medium containing 40 or 80 mM acetate, and zero or 7.5 mM

of lactate and 2 or 10 mM of glutamate. The C/N ratio in the feed media varied

between 13 and 56.25.

The pH, cell concentration, cumulative hydrogen production, organic acid

consumption and gas composition were analyzed. The pH variation in the indoor 8 L

photobioreactor runs with Rb. capsulatus wild type on defined medium containing

acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM) started at

24.08.2007 is depicted in Figure 4.8. The pH was controlled around 6.4-8 which was

suitable for hydrogen production by Rhodobacter capsulatus (Van Niel et al., 1944).

Figure 4.8 The pH variation in the indoor photobioreactor runs with Rb. capsulatus

wild type on defined medium containing acetate (40-80 mM), lactate (7.5 mM) and

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

pH

Time (days)

I

40/7.5/2

A/L/G

II

40/7.5/2

A/L/G

III

40/7.5/10

A/L/G

IV

40/10

A/G

V

80/10

A/G

Batch Continuous

60

glutamate (2-10 mM). Starting date of the experiment was 24.08.07. Feeding was

started on 8th

day of the experiment.

The cumulative hydrogen production and biomass in Run240807 are shown

in Figure 4.9. Phase I is the start-up period and it was run in batch mode. The

photobioreactor was started with 2 liters of activated grown culture (corresponding to

25% of the reactor volume) and 6 liters of 40 mM acetate, 7.5 mM lactate and 2 mM

glutamate containing BP medium (C/N ratio was 56.25). The cell concentration was

lower than the critical cell concentration (0.3 gdcw/Lc) required for hydrogen

production (Eroglu et al., 2008). Hence the lowest molar hydrogen yield (5%), and

productivity (0.06 mmolesH2/Lc.h) were observed in this phase and light conversion

efficiency (0.09%) was also same with the light conversion efficiency obtained in

last phase. Also, no feeding was done in this phase.

Figure 4.9 Cumulative hydrogen production and biomass in the indoor


acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the



0

10

20

30

40

50

60

70

80

90

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

Cu

mu

lativ

e H2 P

rod

uctio

n (L

) Cel

l C

on

cen

rati

on

(g

dcw

/Lc)

Time (days)

Biomass Hydrogen Production (L)

I

40/7.5/2

A/L/G

II

40/7.5/2

A/L/G

III

40/7.5/10

A/L/G

IV

40/10

A/G

V

80/10

A/G

Batch

Continuous

61

In the phase II, continuous feeding of hydrogen production medium was

introduced to the photobioreactor. The feed contained acetate (40 mM) and lactate

(7.5 mM) and glutamate (2 mM) (C/N ratio was 56.25). The cell concentration was

between 0.2 gdcw/Lc – 0.4 gdcw/Lc in this phase and the hydrogen productivity,

molar hydrogen yield and light conversion efficiency were 0.11 mmolesH2/Lc.h,

17%, and 0.15%, respectively. The organic acid concentration in Run240807 is

shown in Figure 4.10. In this phase acetate concentration in the photobioreactor was

stabilized around 2 mM and lactate was utilized completely. The by-products of

photofermentation as propionic acid and butyric acid remained below 1 mM in this

phase.

Figure 4.10 Organic acids concentration in the indoor photobioreactor runs with Rb.

capsulatus wild type on defined medium containing acetate (40-80 mM), lactate (7.5

mM) and glutamate (2-10 mM). Starting date of the experiment was 24.08.07.

Feeding was started on 8th


In the phase III, acetate and lactate concentration were kept same in the feed

but the glutamate concentration was increased from 2 mM to 10 mM (C/N ratio was

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

Co

nce

ntr

ati

on

(m

M)

Time (days)

LA(mM) AA(mM) PA(mM) BA(mM)

II

40/7.5/2

A/L/G

III

40/7.5/10

A/L/G

IV

40/10

A/G

V

80/10

A/G

Continuous

62

15.25) to stimulate growth. The increase in glutamate amount in the feed led to an

increase in biomass from 0.3 gdcw/Lc to 1.0 gdcw/Lc while the hydrogen

productivity was low (0.07 mmolesH2/Lc.h), as shown in Figure 4.9. During this

phase, the acetate and lactate were utilized completely and used for biomass, rather

than hydrogen production. Therefore, the yield (16%) and light conversion efficiency

(0.10%) was lower than that was in the second phase.

In phase IV, the lactate was removed from the feed. Feeding medium

contained acetate (40 mM) and glutamate (10 mM) (C/N ratio was 13). The cell

concentration was stabilized in the range of 0.4 - 0.7 gdcw/Lc for 3 months and

continuous hydrogen production was observed during this period. Hydrogen

productivity was 0.12 mmolesH2/Lc.h and the yield was 32% which indicated that

1/3 of theoretical H2 that can be produced from acetate was achieved. The light

conversion efficiency was 0.16%. The productivity, yield, and light conversion

efficiency were the maximum in this phase. Till the last 15 days of this phase acetate

remained below 2 mM in the photobioreactor. Propionic, butyric and acetic acid

concentration in the photobioreactor started to increase since 125th

day. At the same

time biomass started to decrease in the photobioreactor. At the end of this phase,

acetic acid concentration in the photobioreactor effluent reached to 10 mM and

propionic and butyric acid concentration reached to 3.5 mM and 1 mM.

In the phase V, using the same dilution rate, the acetate composition in the

feed was increased from 40 mM to 80 mM while glutamate concentration remained

at 10 mM. This corresponds to C/N ratio of 21. The doubling of acetate

concentration led biomass to increase from 0.7 to 1.1 gdcw/Lc. Hydrogen

productivity, yield and light conversion efficiency dropped to 0.06 mmolesH2/Lc.h,

9%, and 0.09%, respectively. Acetic acid concentration increased by two fold (16

mM); propionic and butyric acid remained below 3 mM in this phase. In addition,

biofilm occurred on the surface of the photobioreactor which caused a reduction in

the light penetration. It could be a reason for accumulation of acetic acid. Although

substrate concentration increased, microorganism could not utilize light energy

sufficiently to produce hydrogen.

Gas chromatography analysis revealed that the average composition of the

collected gas was 95% H2 and 5% CO2 in all phases.

63

The maximum hydrogen production (0.12 mmolesH2/Lc.h) was obtained in

the fourth phase and it was lower than the productivity (0.6 mmolesH2/Lc.h) obtained

in an indoor batch study with Rb. capsulatus where acetate (40 mM) and glutamate

(2 mM) were used (Ozgur et al., 2009). The lower productivity might be attributed to

the scale differences, and the operational mode.

The productivity, molar yield and the light conversion efficiency of all phases

of Run240807 are tabulated in Table 4.4.

Table 4.4 Productivity, and molar yield values obtained during the fed batch

operation of the indoor 8 L photobioreactor runs with Rb. capsulatus wild type on

defined medium containing acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-

10 mM).

Phases Duration

(days)

Productivity

mmolesH2/Lc.h

Molar Yield

(%)

I 7 0.06 5

II 25 0.11 17

III 21 0.07 16

IV 91 0.12 32

V 23 0.06 9

Results indicate that continuous hydrogen production has been achieved with

fed batch operation.

4.2.1.3 Effect of Increased Glutamate Amount in the Feed on Hydrogen

Production

To determine effect of increased glutamate amount average biomass

concentration, productivity, and molar yield of Run240807 at Phase II and III in

which feeding medium containing 40 mM acetate, 7.5 mM lactate ad 2 mM

glutamate and 40 mM acetate, 7.5 mM lactate ad 10 mM glutamate was given to the

PBR, respectively are compared in Table 4.5.

64


yield values obtained in the indoor 8 L photobioreactor runs with Rb. capsulatus wild

type on defined medium containing acetate (40-80 mM), lactate (7.5 mM) and

glutamate (2-10 mM)in the Phase II and III.

Phases Feed

Content

Average

Biomass

Concentration

(gdcw/Lc)

Productivity

(mmolesH2/Lc.h)

Molar

Yield

(%)

II 40/7.5/2 0.363 0.11 17

III 40/7.5/10 0.576 0.07 16

In Phase II, productivity was higher than that obtained at the third phase.

Molar yield was similar with the yield in the third phase. This can be attributed to the

increased amount of glutamate in the feeding medium in the third phase because it

led to a decrease in evolution of hydrogen (Gest and Kamen, 1949). Adversely the

biomass concentration in the third phase was 1.58 fold of the average biomass

concentration in the second phase because increased glutamate amount led to an

increase in cell concentration (Meyer et al., 1978). In addition to that Miyake et al

(1982) stated that small amount of nitrogen sources lengthened the period of

hydrogen production in the case of Rhodosprillum rubrum. As a result increased

nitrogen sources in acceptable amounts in the feeding medium reduces hydrogen

production, increases biomass, provides prolonged hydrogen production.

4.2.1.4 Effect of Removing Lactate from the Feeding Media on Hydrogen

Production

For seeing the effect of lactate on hydrogen production and biomass growth,

lactate was removed from the feeding medium in the fourth phase. For comparison

average biomass concentration, productivity, and molar yield values obtained in

Run240807 in the Phase III and IV are tabulated in Table 4.6.

65


yield values obtained in the indoor 8 L photobioreactor runs with Rb. capsulatus wild

type on defined medium containing acetate (40-80 mM), lactate (7.5 mM) and

glutamate (2-10 mM) in the Phase III and IV.

Phases Feed

Content

Average

Biomass

Concentration

(gdcw/Lc)

Productivity

(mmolesH2/Lc.h)

Molar

Yield

(%)

III 40/7.5/10 0.576 0.07 16

IV 40/10 0.677 0.12 32

Removing lactate from the feeding media in the fourth phase did not decrease

the hydrogen productivity adversely in the fourth phase productivity was higher than

the previous phase. This can be attributed to the various kinetics of hydrogen

production with different carbon sources because of their differences in their

reduction states and patterns of metabolism (Hillmer and Gest, 1976). Average

biomass concentration was also higher than the third phase and molar yield in the

fourth phase was 2 fold of the third phase.

4.2.1.5 Effect of Increased Acetate Amount in the Feeding Media on Hydrogen

Production

To figure out the effect of increased amount of acetate on hydrogen

production acetate concentration in the feeding media increased two fold in the fifth

phase. Feed content, average biomass concentration, productivity and molar yield

values obtained in Run240807 in the Phase IV and V is tabulated in Table 4.7.

66

Table 4.7 Feed content, average biomass concentration, productivity and molar yield

values obtained in the indoor 8 L photobioreactor runs with Rb. capsulatus wild type

on defined medium containing acetate (40-80 mM), lactate (7.5 mM) and glutamate

(2-10 mM) in the Phase IV and V

Phases Feed

Content

Average

Biomass

Concentration

(gdcw/Lc)

Productivity

(mmolesH2/Lc.h)

Molar

Yield

(%)

IV 40/10 0.677 0.12 32

V 80/10 0.842 0.06 9

Increased acetate amount in the feeding media led to increase of 1.24 fold in

average biomass concentration but decrease in hydrogen production to its half in the

fifth phase. This can be attributed to the biofilm formation on the surface of the

photobioreactor in the last phase. Because in the biofilm formed PBRs light cannot

penetrate into the deeper part of the photobioreactors efficiently. So, light conversion

efficiency decreases (Zhang et al., 2010) on account of hydrogen productivity

decrease. Barbosa et al. (2001) also stated that at a certain biomass concentration and

light intensity light can become a limiting factor for hydrogen production due to self-

shading and light absorption by the cells close to the illuminated surface.

Lower molar yield considering increased acetate amount was very low in the

fifth phase. This can be attributed to inhibitory effect of acetate concentration on

hydrogen production. Asada et al. (2008) also studied hydrogen production by gel

immobilized Rb. sphaeroides RV on 21 mM, 42 mM, 84 mM and 168 mM acetate

containing medium and showed that maximum hydrogen production was obtained on

the medium containing 42 mM acetate and emphasized that higher concentrations of

acetate (84 or 168 mM) inhibited hydrogen production.

4.2.1.6 Effect of C/N Ratio on Hydrogen Production

For determining the effect of carbon to nitrogen ratio on hydrogen production

different feed compositions was fed to photobioreactor. For this purpose C/N ratio,

67

productivity and molar yield values obtained in Run240807 are tabulated in Table

4.8.

Table 4.8 C/N ratio, productivity and molar yield values obtained in the indoor 8 L


acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM) in the Phase II, III,

IV and V

Phases C/N Productivity

(mmolesH2/Lc.h)

Molar

Yield

(%)

II 56.25 0.11 17

III 15.25 0.07 16

IV 13 0.12 32

V 21 0.06 9

Maximum hydrogen production and molar yield was obtained in the fourth

phase. In this phase C/N ratio was the lowest and 13. Kuriaki et al (1998) stated that

maximum hydrogen production was observed as the C/N ratio was 22.8 and Androga

et al (2009) was also showed that maximum hydrogen production was obtained in the

experiment that the C/N value was 25.

4.2.2 Outdoor Lab-Scale Panel Photobioreactors

In outdoor experiments two 8 liters flat plate photobioreactors without

cooling were used. Rhodobacter capsulatus wild type was used as microbial strain.

The photobioreactors were placed on a chromium-nickel construction 25 cm apart

vertically in an east-west position for optimum heat input, light intensity utilization

and temperature control (Modigell and Hall 1998). After 12th

of November, air

temperature during days decreased below 10 0C and reactors were moved to indoor.

The experiment continued for 134 days in the photobioreactor started at 26.09.07. All

gas measurements were corrected by calculating permeability of hydrogen through

reactor surface and solubility of hydrogen in water and carryover amounts in inocula.

68

Methods for calculating hydrogen permeability through solids and solubility in water

and are given in Appendix G and H, respectively. Experimental data are given in

Appendix M.3. In experimental period feed composition was changed 2 times in

order to see the effect of feed composition on growth and hydrogen production. The

experiment continued for 160 days in the photobioreactor started at 310807.

Experimental data are given in Appendix M.4. In experimental period feed

composition was changed 3 times in order to see the effect of feed composition on

growth and hydrogen production. The pH, cell concentration, and cumulative

hydrogen production, organic acid consumption and gas composition were analyzed.

The photobioreactor temperature was recorded continuously. The total global solar

radiation data was taken from the National Meteorology Institute in Ankara. The

definition of total global solar radiation is given in Appendix J.

4.2.2.1 Variation in Photobioreactor Temperature

The variation in temperature in the outdoor 8 L photobioreactors runs with

Rb. capsulatus wild type on defined medium containing acetate (40-80 mM), lactate

(7.5 mM) and glutamate (2-10 mM) started at 26.09.07 and 31.08.07 are shown in

Figure4.11 and 4.12, respectively. The photobioreactor temperature decreased and

around 20 0C in daytime in the first week of November so the reactors were moved

in indoor during the December and January. The temperature of the photobioreactors

in indoor was kept higher than 25 0C with a heater placed into the room and they

were illuminated with tungsten lamps.

The photobioreactor temperature in Run260907 increased up to50 0C in

daytimes and decreased below 10 0C at nights in the first 15 days of the experiment

so the temperature difference between day and night reached up to 40 0C in these

days. After the batch period of Run260907 the photobioreactor temperature

decreased below 40 0C during days and 5

0C during nights. The photobioreactor

temperature continued decreasing down to 20 0C in the daytime till the

photobioreactor was carried out to indoor.

69






The photobioreactor temperature in Run310807 increased up to 50 0C in

daytimes in first 20 days of the experiment and decreased below 10 0C at nights at

the same period. The temperature difference between day and night also reached up

to 40 0C in this photobioreactor. The photobioreactor temperature decreased down to

20 0C in the daytime and below zero temperatures at nights till the photobioreactor

was carried out to indoor. Although there was temperature fluctuations bacteria could

survive and kept its hydrogen production ability in two photobioreactors because

bacteria were exposed to extreme high temperatures only for a short time during day.

The temperature variation was in tolerable range for the microbial activity.

Temperature of the photobioreactors were higher than the air temperature; probably

due to absorption of heat input from the sun and exothermic heat generation within

the photobioreactor depending on the energy balance in which the total bacterial light

energy absorption is split up and attributed to destinations such as biomass growth

-10

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Tem

per

atu

re (

0C

)

Time (days)

II

40/7.5/10

A/L/G

III*

40/7.5/10

A/L/G

IV*

80/10

A/G

I

40/7.5/2

A/L/G

Batch

Continuous

Indoor Outdoor

70

and maintenance, generation of hydrogen and heat dissipation (Hoekama et al.,

2006).






Daily global solar radiation energy of the first 74 days of the Run310807 and

first 48 days of the Run260907 is illustrated in Figure 4.13. Global solar radiation

energy during the start-up days of Run310807 and Run260907 was around 5700

Wh/m2

and 4900 Wh/m2, respectively. It decreased to 2600 Wh/m

2 before the

photobioreactors were carried out to the indoor.

-10

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Tem

per

atu

re(0

C)

Time (days)

I

40/7.5/2

A/L/G

II

effluent of

indoor reactor III

40/7.5/10

A/L/G

IV*

40/7.5/10

A/L/G

V*

80/10

A/G Batch

Continuous

Indoor Outdoor

71

Figure 4.13 Daily global solar radiation energy during outdoor period of the

photobioreactors (Run260907 and Run310807) run with Rb. capsulatus wild type on

defined medium containing acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-

10 mM). Starting date of the data is 31.08.07.

4.2.2.2 Effect of Feed Composition on Long Term Operation

The effect of feed composition on long term behavior of fed-batch

photobioreactors was investigated in outdoor conditions in two parallel runs. Figure

4.14 is the plot of the pH variation of Run260907. The pH was controlled around 7.0

in the first three phases but in the last phase pH increased to 8.0. Increase in acetate

concentration in the feeding media in the last phase might be a reason for the

increase in pH.

0

1000

2000

3000

4000

5000

6000

7000

0 10 20 30 40 50 60 70 80

Da

ily

Glo

ba

l S

ola

r R

ad

iati

on

(W

h/m

2)

Time (days)

Run310807

Run260907

72




started on 15th


The biomass and cumulative hydrogen production of Run260907 are shown

in Figure 4.15. In this run the photobioreactor was started with 4 liters of activated

fresh culture and 4 liters of hydrogen production medium (40 mM of acetate, 7.5 mM

of lactate and 2 mM of glutamate) (C/N ratio was 56.25). Fifty percent inoculation

was carried out to enhance the biological growth and to decrease the lag time. The

cell concentration increased to 0.4 gdcw/Lc in batch phase. The molar yield and the

productivity of hydrogen were 9% and 0.06 mmolesH2/Lc.h, respectively. The

organic acid consumption in Run260907 is shown in Figure 4.16. Lactic acid was

consumed fully in all phases and the acetic acid concentration was around 2.5 mM at

the end of phase I.

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

pH

Time (days)

II

40/7.5/10

A/L/G

III*

40/7.5/10

A/L/G

IV*

80/10

A/G

I

40/7.5/2

A/L/G

Batch Continuous

Indoor Outdoor

73

Figure 4.15 Growth and cumulative hydrogen production in the outdoor


acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the



After 14 days of batch period medium containing 40 mM acetate, 7.5 mM

lactate and 10 mM glutamate (corresponding to C/N ratio of 15.25) was fed to the

photobioreactor. The biomass increased to 0.9 gdcw/Lc due to high glutamate

concentration in the feed. The hydrogen productivity and the molar yield were 0.06

mmolesH2/Lc.h and 13%, respectively. In this phase, acetic acid concentration

remained around 2.5 mM and lactate was depleted, propionic and the butyric acid

concentrations were negligible.

In phase III, due to the onset of winter, temperature (especially at night) of the

photobioreactor decreased and the photobioreactor was carried out to indoor. Feed

rate and composition was kept the same with the previous phase, and continuous

illumination using two tungsten lamps was provided. Biomass stabilized at 0.9

gdcw/Lc and hydrogen productivity was about 0.04 mmolesH2/Lc.h. The molar yield

of hydrogen was 16% Acetate concentration was around 5 mM in the

0

5

10

15

20

25

30

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Cm

ula

tive H

2 Pro

du

ction

(L) C

ell

Co

nce

ntr

ati

on

(g

dcw

/Lc)

Time (days)


I

40/7.5/2

A/L/G

II

40/7.5/10

A/L/G

III*

40/7.5/10

A/L/G

IV*

80/10

A/G

Outdoor Indoor

Batch Continuous

74

photobioreactor effluent, lactate was consumed completely and the byproducts of

fermentation were remained below 2.5 mM in the entire phase.






In phase IV the feeding medium containing acetate (80 mM) and glutamate (10

mM) was given to the photobioreactor. The C/N ratio of the feed medium increased

to 21. Hydrogen productivity increased to 0.05 mmolesH2/Lc.h and yield was 14% in

this phase. The pH of photobioreactor increased to 8.0. The acetic acid was not

completely consumed. Therefore its concentration increased from 5 mM to 27 mM.

Lactate was not detected; propionic and the butyric acids remained below 3 mM. The

composition of the evolved gas was 95% H2 and 5% CO2.

The productivity and molar yield of all phases obtained in Run260907 are

summarized in a Table 4.9.

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Co

nce

ntr

ati

on

(m

M)

Time (days)


I

40/7.5/2

A/L/G

II

40/7.5/10

A/L/G

III*

40/7.5/10

A/L/G

IV*

80/10

A/G

Outdoor Indoor

Batch Continuous

75

Table 4.9 The productivity and molar yield of all phases obtained in the outdoor 8 L


acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM)

Phases Productivity

(mmolesH2/Lc.h)

Yield

(%)

I 0.06 9

II 0.06 13

III 0.04 16

IV 0.05 14

Maximum hydrogen productivity was obtained in the first two phases in which

the photobioreactor was operated in outdoor conditions and the cell concentration

was in the exponential phase. The maximum molar yield was obtained in the third

phase.

In Run310807 the effect of different feed compositions on pH, growth and

hydrogen production in outdoor and indoor conditions was also investigated. Figure

4.17 is the plot of the pH variation in the outdoor 8 L photobioreactor run with Rb.


mM) and glutamate (2-10 mM) started at 31.08.07. The pH was controlled around

7.0 in the entire processes.

76

Figure 4.17 pH variations in the outdoor photobioreactor runs with Rb. capsulatus



started on 6th


The biomass and the cumulative hydrogen production of Run310807 are shown

in Figure 4.18. Photobioreactor was started with 6 liters of hydrogen production

medium containing acetate (40 mM), lactate (7.5 mM) and glutamate (2 mM) (C/N

ratio was 56.25) and 2 liters of activated culture (25% inoculation by the volume of

the reactor). The cell concentration was 0.02 gdcw/Lc in the first 6 days. That’s why

hydrogen production was almost negligible.

In the second phase effluent of indoor photobioreactor containing biomass was

fed to the photobioreactor at every two days in order to increase cell concentration.

Average cell concentration was 0.2 gdcw/Lc in this phase. Feeding the reactor with

the effluent of indoor photobioreactor didn’t work well. It doesn’t contain enough

substrate to support bacterial growth at high temperature. The bacteria started to

produce hydrogen after 13th

day but it continued only a few days and then stopped

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

pH

Time (days)

I

40/7.5/2

A/L/G II

effluent of

indoor reactor

III

40/7.5/10

A/L/G

IV*

40/7.5/10

A/L/G

V*

80/10

A/G

Outdoor Indoor

Batch Continuous

77

again. Consequently the hydrogen productivity and yield were 0.05 mmolesH2/Lc.h

and 35%, respectively. The yield seemed to increase but it is because of giving low

substrate to the photobioreactor. The organic acid consumption in Run310807 is

shown in Figure 4.19. The acetic acid concentration was around 2 mM and the lactic

acid was fully consumed in this phase. There was no propionic and butyric acid

formation.

Figure 4.18 Growth and hydrogen production in the outdoor photobioreactor runs

with Rb. capsulatus wild type on defined medium containing acetate (40-80 mM),

lactate (7.5 mM) and glutamate (2-10 mM). Starting date of the experiment was

31.08.07. Feeding was started on 6th


In phase III, medium containing acetate (40 mM), lactate (7.5 mM) and

glutamate (10 mM) (C/N ratio was 15.25) was fed to the photobioreactor. Giving

feeding medium in an increased amount of glutamate led to an increase in biomass to

0.75 gdcw/Lc. Hydrogen productivity was 0.11 mmolesH2/Lc.h and yield was 22% in

this phase. The acetic acid concentration in the effluent increased to 2 to 6 mM. The

0

5

10

15

20

25

30

35

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Cu

mu

lativ

e H2 P

rod

uctio

n (L

) Cel

l C

on

cen

tra

tio

n (

gd

cw/L

c)

Time (days)


I

40/7.5/2

A/L/G

II

effluent of

indoor reactor

IV*

40/7.5/10

A/L/G

V*

80/10

A/G

III

40/7.5/10

A/L/G

Outdoor Indoor

Batch Continuous

negligible.

78

lactic acid concentration was around 1.5 mM. Propionic and butyric acid

concentrations were






In phase IV, the photobioreactor was taken to the indoor. Here continuous

illumination was provided using two tungsten lamps 15 cm away from one side of

the reactor. The same feed composition and dilution rate as in phase II was used. The

biomass stabilized at around 0.9 gdcw/Lc. The hydrogen productivity was 0.07

mmolesH2/Lc.h; molar yield was 23% in this phase. In this phase the acetic acid

concentration was around 6mM as the previous phase. Lactic acid in the system was

consumed almost fully. Propionic and butyric acid concentrations were also

negligible.

In phase V, the acetate concentration in the feed was increased to 80 mM and

glutamate concentration was remained in 10 mM. The C/N ratio of the feeding media

was 21. The same dilution rate was used in this phase. As a result of increase (two

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Co

nce

ntr

ati

on

(m

M)

Time (days)


II

effluent of

indoor reactor

III

40/7.5/10

A/L/G

IV*

40/7.5/10

A/L/G

V*

80/10

A/G

Outdoor Indoor

79

fold) acetate concentration hydrogen production rate increased. The biomass

increased to 1.1 gdcw/Lc and hydrogen productivity increased to 0.06

mmolesH2/Lc.h. The molar yield was 8%. The acetic acid accumulated rapidly in this

phase and at the end it reached to 22 mM. The lactic acid was consumed almost fully

and the propionic acid concentration increased to 5 mM at the end of the experiment.

Butyric acid concentration also increased but it remained under 2 mM during this

phase. The collected gas contained 95% H2 and 5% CO2 all throughout the

experiment.

The productivity and molar yield of all phases obtained in Run310807 are

summarized in Table 4.10.


L photobioreactor (Run310807) runs with Rb. capsulatus wild type on defined

medium containing acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM)

Phases Productivity

(mmolesH2/Lc.h)

Yield

(%)

I 0.02 1

II 0.05 35

III 0.11 22

IV 0.07 23

V 0.06 8

Maximum hydrogen productivity was obtained in the third phase in which 40

mM acetate, 7.5 mM lactate and 10 mM glutamate was fed to the photobioreactor in

outdoor conditions. Maximum molar yield (35%) was obtained in the second phase.

This can be attributed to hydrogen production due to the lower substrate

concentration.

4.2.3 Outdoor Pilot-Scale Panel Photobioreactor

In this study 20 liter flat plate photobioreactor with cooling coil made of

aluminum was placed on a construction oriented in east-west. Rhodobacter

80

capsulatus hup- mutant was used as microbial strain. The photobioreactor was

started with 32 liters of hydrogen production medium containing 30 mM of acetate

and 2 mM of glutamate and 8 liters of activated bacteria (20% inoculation) at

02.12.2008. Experimental data are given in Appendix M.5. The Plexiglas bulged and

working volume increased to 45 liters during the experimental period. A systematic

feeding procedure could not be applied because working volume of the

photobioreactor changed day by day. Hydrogen production medium (10% of the

initial volume of the photobioreactor) was given to the photobioreactor for 5 times

during the experiment. The experiment was run in a greenhouse heated with a heater.

By this way the photobioreactor temperature was kept between 20 and 30 0C. All gas




solubility in water and are given in Appendix G and H, respectively. The pH was

controlled around 7.0 during the process (Figure 4.20).

Figure 4.20 The pH variation in outdoor pilot-scale photobioreactor runs with Rb.

capsulatus hup- on defined medium containing 30 mM acetate and 2 mM glutamate

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

pH

Time (days)

Batch Continuous

81

at the start-up period. Starting date of the experiment was 02.12.08. Feeding was

started on 4th


The growth and hydrogen production in outdoor pilot-scale photobioreactor

are shown in Figure 4.21. In the first 6 days cell concentration increased to 0.2

gdcw/Lc than remained around this value in the next 9 days. After 15th

day, it

fluctuated between 0.2 – 0.7 gdcw/Lc but it didn’t stabilize. Unstable and low cell

concentration might be due to low temperature and light intensity at that period.

Besides, lack of the uptake hydrogenase enzyme, which utilizes hydrogen when there

is need for energy, might be prevented the microorganism recovering the biomass.

Figure 4.21 Growth and hydrogen production in outdoor pilot-scale photobioreactor

runs with Rb. capsulatus hup- on defined medium containing 30 mM acetate and 2

mM glutamate at the start-up period. Starting date of the experiment was 02.12.08.



0

5

10

15

20

25

30

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Cu

mu

lativ

e H2 P

rod

uctio

n (L

) Cel

l C

on

cen

tra

tio

n (

gd

cw/L

c)

Time (days)


Batch Continuous

82

In the first three days 6 liters hydrogen was produced but it stopped after third

day and then there was no hydrogen produced till the day 18. After the cell

concentration exceeded the critical threshold which was 0.3 gdcw/Lc hydrogen

production started again (Eroglu et al., 2008). This means that the critical cell

concentration for hydrogen production specified for wild type strains was also valid

for mutant strains. Hydrogen production was started again in the 19th

day and

continued till the end of the experiment.

Variation in the organic acid concentration in outdoor pilot-scale

photobioreactor is shown in Figure 4.22.

Figure 4.22 Organic acid consumption in outdoor pilot scale photobioreactor runs

with R. capsulatus hup- on defined medium containing 30 mM acetate and 2 mM

glutamate at the startup period. Starting date of the experiment was 02.12.08.



Acetic acid concentration in the photobioreactor was decreased to 10 mM at

the 18th

day. After 18th

day acetic acid concentration stabilized around 8 mM in the

photobioreactor effluent. There was no lactate in the system. Formic acid was

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Co

nce

ntr

ati

on

(m

M)

Time (days)

LA(mM) FA(mM) AA(mM) PA(mM) BA(mM)

Batch Continuous

83

produced as a fermentation product but its concentration remained under 4 mM.

Propionic and butyric acid were produced at negligible amounts.

Hydrogen production obtained in this study was 0.08 mmolesH2/Lc.h and

molar yield was 16% at the end of the experiment.

4.2.4 Parameters Affecting Prolonged Hydrogen Production

4.2.4.1 Effect of Inoculum Ratio at the Start-up

The amount of inoculation by freshly grown bacteria at the start-up affected

the duration of batch period, in outdoor experiments. Higher inoculation ratio

resulted in higher biomass concentration at the start-up and bacteria grew more

rapidly due to quorum sensing effect (Waters and Bassler, 2005). Feed content,

percentage of activated bacteria, duration of batch period, biomass concentration at

the end of batch period and hydrogen productivity at start-up in Run240807,

Run260907, Run310807 are given in Table 4.11.

Table 4.11 Feed content, percentage of activated bacteria, duration of batch period,

biomass concentration at the end of batch period and hydrogen productivity at start-

up in three photobioreactors run with Rb. capsulatus wild type on defined medium

containing acetate (40-80 mM), lactate (7.5 mM) and glutamate (2-10 mM) at start-

up, batch period

PBR

(Run

Time)

Feed

Content

Percentage

of

Activated

Bacteria

(v/v)

Duration

of

Batch

Period

(days)

Biomass

Concentration

at the

end of

Batch Period

(gdcw/Lc)

Productivity

(mmolesH2/Lc.h)

240807

(indoor) 40/7.5/2 25% 7 0.167 0.06

260907

(outdoor) 40/7.5/2 50% 14 0.295 0.06

310807

(outdoor) 40/7.5/2 25% 5 0.022 0.02

84

The start-up procedures were same in all of the three experiments but

percentage of activated bacteria given at start-up and operational conditions differed.

Run240807 was started with 75% hydrogen production medium and 25% activated

bacteria this was sufficient for Rhodobacter capsulatus to grow in indoor conditions.

Consequently hydrogen was produced in the startup period in run240807.

Run310807 was started before the Run260907 and it was started with 75% hydrogen

production medium and 25% activated bacteria as Run240807 in outdoor conditions

but this ratio was not sufficient for rapid biomass increase which would lead rapid

production of hydrogen under sunlight illumination in outdoor conditions. By the

help of this experience the Run260907 was started with 50% inoculum in outdoor

conditions. Eventually hydrogen productivities were same in the start-up phase in

Run260907 and Run240807; the lowest productivity was obtained in Run310807.

4.2.4.2 Determining the Feeding Strategy for Continuous Operation

Operational mode, feed content, average biomass concentration, hydrogen

productivity, molar yield of Run240807, Run260907 and Run310807at Phase II in

which feeding medium was started to be given to the PBRs are tabulated in Table

4.12.

Table 4.12 Operational mode, feed content, average biomass concentration,

hydrogen productivity, and yield of three photobioreactors run with Rb. capsulatus


glutamate (2-10 mM) at Phase II

PBR

(Run Time) Mode

Feed

Content

Average

Biomass

Concentration

(gdcw/Lc)

Productivity

(mmolesH2/Lc.h)

Yield

(%)

240807 indoor 40/7.5/2 0.363 0.11 17

260907 outdoor 40/7.5/10 0.717 0.06 13

310807 outdoor effluent 0.175 0.05 35

In Run240807 feeding medium contained 40 mM of acetate, 7.5 mM of

lactate and 2 mM of glutamate, in Run260907 feeding medium contained 40 mM of

85

acetate, 7.5 mM of lactate and 10 mM of glutamate and in Run310807 feeding

medium was the effluent of Run240807. Similar hydrogen productivities were

observed for Run260907 and Run310807, at phase II. Despite introducing poor

substrate into Run310807 photobioreactor, hydrogen productivity was as much as

Run260907. However, yields differed in three photobioreactors and maximum molar

yield was obtained in the Run310807 because hydrogen was evolved due to low

substrate concentration in the feeding medium. Besides, by this feeding strategy that

effluent of Run240807 with biomass was given to Run310807 the cell concentration

could not exceed threshold value (0.3 gdcw/Lc). As a result giving much more

substrate including both carbon and nitrogen source was necessary for continuous

operation and keeping biomass concentration in the critical range for hydrogen

production. There is a maximum threshold value for saturation of hydrogen

production as well as minimum threshold value. The threshold value of saturation of

hydrogen production is 0.7 gdcw/Lc for Rb. sphaeroides B5 (Kim et al., 1987b). On

the other hand feeding media containing 2 and 10 mM glutamate were enough for

biomass increase in indoor and outdoor conditions, respectively. In Run260907

which was a feeding media containing 10 mM glutamate increased biomass

concentration two times than Run240807 fed with 2 mM glutamate amount. In

outdoor conditions, because of fluctuation in temperature, microorganism needed

higher concentration of nitrogen source to support bacterial growth.

Feeding medium (12.5% by the volume of the photobioreactor) was

introduced every other day to the photobioreactor. This corresponds to the hydraulic

retention time of 16 days. This feeding rate caused a fluctuation in biomass,

especially in outdoor conditions. To stabilize the biomass for maximizing the

hydrogen productivity the feed rate increased to 10% daily in the molasses DFE

experiments.

4.3 Hydrogen Production on Dark Fermentor Effluent of Molasses

For the purpose of recovery of waste materials the real dark fermentor effluent

(DFE) of the molasses were used as substrate in the experiments. The composition of

the molasses DFE was given in Table 4.13. In order to obtain environmentally

86

friendlier photofermentation effluent, replacing potassium phosphate buffer with

sodium carbonate buffer was investigated. Hydrogen production on molasses DFE

with Rb. capsulatus wild type and hup- in batch and continuous mode was

researched in indoor and outdoor conditions in small and laboratory scale

photobioreactors was researched.

Table 4.13 Composition of the molasses dark fermentor effluent

Component Concentration Component Concentration

Ni (µM) 0.68 Acetic Acid(mM) 65

Co (µM) 3.39 Lactic Acid (mM) 5

Mn (µM) 12.38 Formic Acid(mM) 5

Fe (µM) 25.86 Propionic Acid (mM) 0

Cu (µM) 0.29 Butyric Acid (mM) 0

Zn (µM) 21.05 Ethanol (mM) 5

Ca (mM) 0.61 Sucrose (mM) 2

Mg (mM) 5.26 TOC (mg/L) 1870

Mo (µM) 5.92 TN (mg/L) 420

B (mM) 0.00 COD (mg/L) 6500

Na (mM) 239.24 Ammonia (mM) 1

4.3.1 Effect of Buffers

In this study indoor bottle photobioreactors with 50 ml working volume was

used under 2000 lux illumination at 30 0C. The experiments were carried out with

wild type Rb. capsulatus for 7 days. The buffer capacity of Na2CO3 and

KH2PO4buffers on the molasses DFE was tested. Different concentrations of buffers

(5, 10 and 15 mM) at pH 6.4 were used. Experimental data are given in Appendix

M.6. The pH, biomass and cumulative hydrogen production in the photobioreactors

(50 ml) run by Rb. capsulatus wild type on media containing the molasses DFE

supplemented with 5, 10 and 15 mM concentrations of Na2CO3 and KH2PO4 buffer

is illustrated in Figure 4.23.

Even though the initial pH was set to 6.4 after autoclaving the pH values of

the molasses DFE supplemented with Na2CO3 buffer increased to 8.0.This was

attributed to formation of CO2 gas due to the high temperature during autoclaving.

87

The highest initial and average pH was obtained in the photobioreactor buffered with

15 mM Na2CO3. In addition to that the minimum initial pH and average pH was

obtained in the photobioreactor supplemented with 15 mM KH2PO4. The pH of the

molasses DFE supplemented with 5, 10, 15 mM Na2CO3stabilized around 8.0 along

the experiment. However, the pH values of the photobioreactors that were

supplemented with 5, 10, 15 mM KH2PO4 increased to 8.0 at the end of the

experiment. As a result considering the pH increase at the start-up in the

photobioreactors supplemented with Na2CO3 buffer was tolerable because the pH

increase was up to 8.0 during the experiment. In a study that C. acetobutylicum

19012 was used for hydrogen production in a 120 ml glass vial; 1 ml 2% Na2CO3

solution was used for pH adjustment and the initial pH in the vial was 7.0-8.0 (Noike

et al., 2002).

The microorganism started to grow from the first day and its concentration

reached its maximum at the fifth day in all photobioreactors. After the fifth day the

microorganism in the photobioreactors stopped to grow and cell concentrations were

stabilized between 0.92 and 1.13 gdcw/Lc. The lowest average cell concentration was

obtained in the photobioreactor in which the concentration of the Na2CO3 buffer was

10 mM. The highest average cell concentration was in the photobioreactors that 5

and 10 mM KH2PO4 buffer was used. The trend in cell concentrations of all

experiments was very similar. There were no inhibition observed in the

photobioreactors that Na2CO3 buffer was used.

88

Figure 4.23 pH, biomass, and cumulative hydrogen production in the

photobioreactors run by Rb. capsulatus wild type on media containing the molasses

DFE supplemented with 5, 10 and 15 mM of Na2CO3 and KH2PO4 buffer

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

10,0

0 1 2 3 4 5 6 7 8

pH

5mM Na2CO3 10mM Na2CO315mM Na2CO3 5mM KH2PO410mM KH2PO4 15mM KH2PO4

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 1 2 3 4 5 6 7 8

Cel

l C

nce

ntr

ati

on

(g

dcw

/Lc)

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8

Cu

mu

lati

ve

H2 P

rod

uct

ion

(m

l)

Time (days)

89

The maximum hydrogen was produced in the photobioreactor supplemented

with 5 mM Na2CO3 buffer and the minimum was produced in the photobioreactor

supplemented with 10 mM KH2PO4 buffer. The hydrogen production in all

photobioreactors in which Na2CO3 buffer used was higher than the photobioreactors

that KH2PO4 was used. Na2CO3 buffer might have been positive effect on hydrogen

production mechanism.

The average biomass concentration, and average pH, total hydrogen

production in the photobioreactors are tabulated in Table 4.14.

Table 4.14 The average biomass concentration, and average pH, total hydrogen

production in the photobioreactors

Buffer

Buffer

Concentration

(mM)

Average

Biomass

(gdcw/Lc)

Average

pH

Total

H2 Production

(ml)

Na2CO3 5 0.77 7.56 58

Na2CO3 10 0.72 7.65 46

Na2CO3 15 0.78 7.71 37

KH2PO4 5 0.82 7.50 24

KH2PO4 10 0.82 7.38 17

KH2PO4 15 0.77 7.28 30

The hydrogen productivity, molar yield, light conversion efficiency, and the

composition of the evolved gas in the photobioreactors are summarized in Table

4.15. The maximum hydrogen productivity (0.30 mmolesH2/Lc.h) was obtained in

the photobioreactor supplemented with 5 mM Na2CO3 buffer; the gas composition

was 92.7% H2 and 7.3% CO2 in this photobioreactor. The minimum hydrogen

productivity (0.09 mmolesH2/Lc.h) was obtained in the photobioreactor

supplemented with 10 mM KH2PO4 buffer. This means that Na2CO3 buffer had

positive effect on hydrogen production. In all photobioreactors supplemented with

Na2CO3 the hydrogen productivity and yield were higher than the photobioreactors

90

supplemented with KH2PO4 but CO2 percentage of the gas phase was a little bit

higher when the Na2CO3 buffer used.

Table 4.15 Productivity, yield, light conversion efficiency and the composition of

evolved gas in indoor photobioreactors run on the molasses DFE supplemented with

different concentrations of phosphate and carbonate buffer, using Rb. capsulatus

wild type.

Buffer Concentration

(mM)

Productivity

(mmoles/Lc.h)

Yield

(%)

Light

Conversion

Efficiency

(%)

Gas

Composition

(%)

H2 CO2

Na2CO3 5 0.30 30 1 92.7 7.3

Na2CO3 10 0.24 24 1 92.8 7.2

Na2CO3 15 0.19 19 1 93.1 6.9

KH2PO4 5 0.13 13 0.4 95.6 4.4

KH2PO4 10 0.09 9 0.3 94.4 5.6

KH2PO4 15 0.15 15 1 93.9 6.1

In this study as Na2CO3 concentration in the media increased the productivity

and yield decreased. This indicates that the concentration of the Na2CO3 buffer

increased above 5 mM hydrogen productivity decreased. Most probably the excess

CO3 ions enhance the utilization of hydrogen by photosynthesis reaction. The

composition of the gas didn’t change significantly. Besides the Na2CO3 buffer is

environmentally acceptable and more economical than KH2PO4 buffer. So it can be

used as buffer in the systems that the molasses DFE was used as substrate.

4.3.2 Continuous Biohydrogen Production with Rb. capsulatus Wild Type

The small photobioreactors were operated in indoor conditions. Laboratory-

scale photobioreactors were operated in both indoor and outdoor conditions on the

molasses DFE with Rb. capsulatus wild type.

91

4.3.2.1 Indoor Experiments

In this study prolonged biohydrogen production in fed-batch mode was aimed

both in small and lab-scale photobioreactors.

In this study a 500 ml glass serum bottle was used as photobioreactor. The

photobioreactor was started with 250 ml the molasses DFE, 200 ml (10 mM) sodium

carbonate supplemented with 0.1 mM of iron and 0.16 µM of molybdenum and 50

ml activated bacteria. The light intensity was set to 2000 lux. The photobioreactor

was incubated at 30 0C. The feeding was started at the fourth day and the feeding

media (10% by the volume of the photobioreactor) was given to the photobioreactor

daily. The feeding medium composed of 50% (v/v) the molasses DFE and 50% (v/v)

(10 mM) buffer supplemented with 0.1 mM of iron and 0.16 µM of molybdenum.

The experiments run for 15 days. Experimental data are given in Appendix M.7. All

gas measurements were corrected by calculating permeability of hydrogen through

reactor surface and solubility of hydrogen in water and carryover amounts in inocula.

Methods for calculating hydrogen permeability through solids and solubility in water

and are given in Appendix G and H, respectively. The pH variation in the 500 ml

indoor continuous photobioreactor runs on the molasses DFE using Rb. capsulatus

wild type is illustrated in Figure 4.24. The initial pH in the photobioreactor was 6.8

but it increased very rapidly and reached to 8 in 3 days then it stabilized around 7.7

during the experiment.

92

Figure 4.24 pH variation in the 500 ml indoor continuous photobioreactor runs on

the molasses DFE using Rb. capsulatus wild type. Feeding was started at 4th

day.

The growth and daily hydrogen production in the 500 ml indoor continuous

photobioreactor is shown in Figure 4.25. The initial biomass in the photobioreactor

was 0.17 gdcw/Lc. The growth continued to increase till the 8th

day and the

maximum biomass at this point was 0.67 gdcw/Lc. After 8th

day there was a slight

decrease in biomass and then it stabilized around the 0.6 gdcw/Lc. Hydrogen

production started in the 3rd

day and it continued till the end of the experiment.

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

pH

Time (days)

Batch Continuous

93

Figure 4.25 Growth and daily hydrogen production in the 500 ml indoor continuous

photobioreactor runs on the molasses DFE using Rb. capsulatus wild type. Feeding

was started at 4th

day.

Organic acid consumption in the reactor is illustrated in Figure 4.26. In batch

period acetic acid introduced at the start up consumed down to 5 mM. After feeding

started its concentration increased to 10 mM. Between the 6th

and 9th

days acetate

was consumed completely in the photobioreactor. Then it accumulates in the system

up to 5 mM at the 10th

and 13th

days of the experiment. Lactate concentration in the

reactor at the startup was 2.6 mM. Then it consumed fully in the reactor till the end

of the experiment. Formic acid concentration in the reactor was below 1 mM till the

last day of the experiment. There was no propionic and butyric acid production in the

reactor during the experiment.

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Cell C

on

centra

tion

(gd

cw/L

c )

Da

ily

H2 P

rod

uct

ion

(m

l)

Time (days)

Hydrogen Production (ml) Biomass

Batch Continuous

94

Figure 4.26 Organic acid consumption in the 500 ml indoor continuous

photobioreactor runs on the molasses DFE using Rb. capsulatus wild type. Feeding

was started at 4th

day.

In this study the productivity, molar yield, light conversion efficiency and

overall acetate conversion efficiency was 0.14 mmolesH2/Lc.h, 8%, 0.97% and 69%,

respectively. The hydrogen productivity (0.14 mmolesH2/Lc.h) and yield (8%)

obtained in this study is lower than the hydrogen productivity (0.30 mmolesH2/Lc.h)

and yield (30%) obtained in the 50 ml photobioreactor runs in indoor condition with

the molasses DFE supplemented with 5 mM Na2CO3 in batch mode. This can be

attributed to the difference in biomass concentration in the photobioreactors. The

average biomass concentration in the 500 ml photobioreactor was 0.52 gdcw/Lc but

in the 50 ml photobioreactor average biomass concentration was 0.77 gdcw/Lc. Cell

concentration in the photobioreactors can be one of the reasons of the low hydrogen

productivity obtained in this study. The other reason can be the variation in the pH in

the photobioreactors. In 50 ml photobioreactor average pH was 7.56 and it had a

decreasing trend during the experiment and at the end of the experiment it decreased

down to 7.4. But in the 500 ml photobioreactor average pH was 7.66 and it increased

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Co

nce

ntr

ati

on

(m

M)

Time (days)


Batch Continuous

95

up to 8.17 at the end of the experiment. Scale differences between the

photobioreactors can also be another reason of lower hydrogen productivity obtained

in 500 ml indoor photobioreactor.

In this study an indoor continuous photobioreactor with 4 liter working volume

was used. The light intensity on the surface of the photobioreactor was set to 2000

lux. The photobioreactor temperature was kept around 32 0C by an air conditioner in

the room that the experiment took place. The photobioreactor was started with 45%

the molasses DFE, 45% (22 mM) KH2PO4 buffer (pH=6.4) and 10% activated Rb.

capsulatus wild type. After 4th

day, the photobioreactor was fed daily with the feeding

media with the volume of 10% of the photobioreactor. The hydraulic retention time

was 10 days. The composition of the feeding media was 50% (22 mM) KH2PO4

buffer and 50% the molasses DFE. The experiment continued 23 days and feed

composition was changed once during the experiment. Experimental data are given

in Appendix M.8.

The pH variation in indoor continuous photobioreactor (4 L) run by Rb.

capsulatus wild type on the molasses DFE is illustrated in Figure 4.27. The initial pH

was 6.57 in the photobioreactor. The pH remained around 6.6 till the end of the

experiment. This means that KH2PO4 had enough buffering capacity to arrange the

pH of the molasses DFE.

96

Figure 4.27 pH variations in indoor continuous photobioreactor runs by Rb.

capsulatus wild type on the molasses DFE. Feeding was started at the 3rd

day.

The biomass and daily hydrogen production in indoor continuous

photobioreactor (4 L) are shown in Figure 4.28. The initial biomass was 0.16

gdcw/Lc in the photobioreactor. The cell concentration increased to 1.05 gdcw/Lc at

the end of the batch operation, it continued to increase after feeding started and

reached to 2.05 gdcw/Lc at the 8th

day. Cell concentration increased very rapidly to

2.05 gdcw/Lc which was very high for effective hydrogen production because too

much cell concentration (>1.0 at 660 nm for Rb. sphaeroides) causes the difference

of light intensity which is distributed to the cells (Kuriaki et al., 1998). So the

composition of the feeding media was changed as 25% the molasses DFE and 75%

buffer for 3 days in order to decrease biomass. The cell concentration decreased after

3 days of feeding to 0.5 gdcw/Lc. Then feeding media containing 50% the molasses

DFE and 50% KH2PO4buffer (22 mM) was started to be given to the photobioreactor

again. This feeding strategy continued till the end of the experiment. The biomass

was stabilized around 0.5 gdcw/Lc after 11th

day.

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

pH

Time (days)

I

Start-up

Molasses:Buffer

1:1

II

Feeding

Molasses:Buffer

1:1

III

Feeding

Molasses:Buffer

1:3

IV

Feeding

Molasses:Buffer

1:1

Batch Continuous

97

Figure 4.28 Biomass and daily hydrogen production in indoor continuous

photobioreactor runs by Rb. capsulatus wild type on the molasses DFE. Feeding was

started at the 3rd

day.

The hydrogen production started in the second day and maximum hydrogen

production was obtained in the first 7 days. The maximum daily hydrogen production

reached to 1.2 L/day and daily average hydrogen production was 0.69 L/day in these

days. After 7th

day the evolved daily average hydrogen decreased to 0.39 L/day.

In Figure 4.29 organic acid concentration in the reactor is figured out. During

batch operation in the first 4 days 96% of acetate was consumed. Although feeding

started acetate concentration in the reactor remained below 1 mM till 9th day. Then it

started to accumulate and its concentration reached to 12 mM at the 17th

day in the

photobioreactor. After 17th

day its concentration started to decreased and at the end it

was 5 mM in the photobioreactor. Overall acetate conversion efficiency obtained in

this study was 83%. Formic acid production started in the 3rd

day of the experiment and

its concentration was 11.5 mM at that day. At the 6th

day its concentration reached its

maxima and it was16 mM. After 7th

day its concentration decreased and stabilized

around 12 mM till 18th

day of the experiment. At the end of the experiment formic acid

0,0

0,5

1,0

1,5

2,0

2,5

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cell C

on

centra

tion

(gd

cw/L

c )

Da

ily

H2 P

rod

uct

ion

(L

)

Time (days)

Hydrogen Production (L) Biomass

I

Start-up

Molasses:Buffer

1:1

II

Feeding

Molasses:Buffer

1:1

III

Feeding

Molasses:Buffer

1:3

IV

Feeding

Molasses:Buffer

1:1

Batch Continuous

98

concentration in the reactor was decreased to 5 mM. Lactic acid was completely

consumed throughout the experiment. Propionic and butyric acid concentration was

negligible during the experiment.

Figure 4.29 Organic acid consumption in indoor continuous photobioreactor runs by

Rb. capsulatus wild type on the molasses DFE. Feeding started at the 3rd

day.

The overall hydrogen productivity obtained at the end of the experiment was

0.17 mmolesH2/Lc.h, the molar yield was 29%, and the light conversion efficiency

was 0.12%. In the study that operated in batch mode in 55 ml glass bottles incubated

at 300C and illuminated under 150-200 W/m

2onthe molasses DFE the maximum

hydrogen productivity was 0.75 mmolesH2/Lc.h and the molar yield was 20% (Ozgur

et al., 2010). The productivity obtained in Ozgur et al. (2010) is higher than the

productivity of this study. This was attributed to the scale differences and the sudden

biomass increase in this study. In another study the overall hydrogen productivity

(0.13 mmolesH2/Lc.h) that 20% dilution of sugar refinery waste water with L-malic

acid was used as substrate for hydrogen production by Rb. sphaeroides O. U.001 in a

0.4 L column photobioreactor for 100 days in indoor conditions (Yetis et al., 2000) is

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Co

nce

ntr

ati

on

(m

M)

Time (days)


I

Start-up

Molasses:Buffer

1:1 II

Feeding

Molasses:Buffer

1:1

III

Feeding

Molasses:Buffer

1:3

IV

Feeding

Molasses:Buffer

1:1

Batch Continuous

99

lower than the productivity obtained in this study. Molasses DFE is more suitable

substrate than sugar refinery waste water for photobiological hydrogen production. The

results obtained in this study and the similar studies are compared in Table 4.16.

Table 4.16 Comparison of the hydrogen productivity values obtained in this study

with similar studies in the literature

Ref. Bacteria Substrate PBR Volume

(L) Mode

Productivity

(mmolesH2/Lc.h)

Yeti

et al.

2000

Rb.

Sphaeroides

O.U.001

20%

SRWW

L-Malic

Acid

Column 0.4 Semi

continuous 0.13

Ozgur

et al.

2010

Rb.

Capsulatus

DSM 1710

Molasses

DFE Bottle 0.055 Batch 0.75

This

Study

Rb.

capsulatus

DSM 1710

Molasses

DFE Panel 4

Semi

continuous 0.17

4.3.2.2.Continuous Hydrogen Production in Outdoor Experiment

In outdoor experiments continuous biohydrogen production was researched

both in short and long term applications with Rb. capsulatus wild type and mutant

strains.

In Run270709, continuous hydrogen production on the molasses DFE in panel

photobioreactor in outdoor conditions was aimed. The lab-scale photobioreactor with

4 liter working volume were used. The photobioreactor was started at 27th

of July in

2009 with 50% the molasses DFE, 25% (20 mM) Na2CO3 buffer (pH=6.4)

supplemented with 0.1 mM of iron and 0.16 µM of molybdenum and 25% activated

bacteria. The temperature of the photobioreactor was controlled with chilled water

circulating through a cooling coil inserted into the photobioreactor. The

photobioreactor was started to be fed daily at 3rd

day with the feeding media had a

volume of 10% of the photobioreactor. Hydraulic retention time was 10 days. The

composition of the feeding media was 50% (10 mM) Na2CO3 buffer supplemented

with 0.1 mM of iron and 0.16 µM of molybdenum and 50% the molasses DFE. The

100

experiment was run for16 days in outdoor conditions. The pH, cell concentration,

cumulative hydrogen production, and organic acid consumption were analyzed daily.

The gas composition was analyzed ones a week. The photobioreactor temperature

was recorded continuously. All gas measurements were corrected by calculating

permeability of hydrogen through reactor surface and solubility of hydrogen in water

and carryover amounts in inoculum. Methods for calculating hydrogen permeability

through solids and solubility in water and are given in Appendix G and H,

respectively. The total global solar radiation data were taken from the National

Institute of Meteorology in Turkey. Experimental data are given in Appendix M.9.

The definition of total global solar radiation is given in Appendix J.

The pH variation in outdoor continuous photobioreactor (4 L) with cooling coil

run by Rb. capsulatus wild type on the molasses DFE started at 27.07.09 is illustrated

in Figure 4.30. The pH stabilized around 7.5 along the process.

Figure 4.30 Variation in pH in outdoor continuous photobioreactor during hydrogen

production runs by Rb. capsulatus wild type on the molasses DFE. Starting date of

the experiment was 27.07.09. Feeding was started at 3rd

day.

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

10,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

pH

Time (days)

Batch Continuous

101

The variation in growth and hydrogen production of Run270709 is illustrated

in Figure 4.31. The initial cell concentration of the photobioreactor was 0.35

gdcw/Lc. After startup the biomass grew up to 0.67 gdcw/Lc in the batch period of the

experiment. Then it continued to increase and reached to its maxima at 1.05 gdcw/Lc

at 11th

day. After 6th

day it was almost around 0.96 gdcw/Lc in the photobioreactor.

Hydrogen production was started at the 3rd

day of the experiment and it continued till

the end of the experiment. The average daily hydrogen production was 0.51 LH2/day.

Figure 4.31 Variation in growth and hydrogen production in outdoor continuous

photobioreactor runs by Rb. capsulatus wild type on the molasses DFE. Starting date

of the experiment was 27.07.09. Feeding was started at 3rd

day.

The maximum and minimum temperatures attained in Run270709 are shown

in Figure 4.32. During the entire process the photobioreactor temperature was

controlled around 30 0C in day time and at nights it was around 15

0C. Hydrogen was

produced continuously during the experiment. This means that the photobioreactor

temperature was in the hydrogen production range along the experiment. This means

that controlling the temperature with chilled water passing throughout the cooling

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Cell C

on

centra

tion

(gd

w/L

c )

Da

ily

H2 P

rod

uct

ion

(L

)

Time (days)


Batch Continuous

102

coil inserted into the photobioreactor was successful. This conclusion would be the

leading purpose for long term operation.

Figure 4.32 The maximum and minimum temperatures attained in outdoor

continuous photobioreactor runs by Rb. capsulatus wild type on the molasses DFE.


day.

Figure 4.33 illustrates the variation in daily global solar radiation and daily hydrogen

production. The total global solar radiation varies between 7301 Wh/m2 and 5060

Wh/m2 during the experiment. A direct relation between daily hydrogen productivity

and daily global solar radiation energy was observed.

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Tem

per

atu

re (

0C

)

Time (days)

Tmax Tmin

Batch Continuous

103


outdoor continuous photobioreactor runs by Rb. capsulatus wild type on the

molasses DFE. Starting date of the experiment was 27.07.09. Feeding was started at

3rd

day.

In Figure 4.34 organic acid concentration in Run270709 is plotted. The initial

concentration of acetic acid was 30.7 mM. Acetate concentration gradually decreased

to 11.3mM whereas formate concentration gradually increased from 3.67 mM to 11

mM. The lactic acid concentration was initially 4.6 mM and it consumed almost

completely. After 7th

day its concentration decreased below 1 mM and remained

around this value till the end of the experiment. The propionic and butyric acid

concentrations were negligible during the entire process.

0

1000

2000

3000

4000

5000

6000

7000

8000

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Da

ily G

lob

al S

ola

r Ra

dia

tion

(Wh

/m2)

Da

ily

H2 P

od

uct

ion

(L

)

Time (days)

Hydrogen Production (L) Solar Radiation

Batch

Continuous

104

Figure 4.34 Organic acid consumption in outdoor continuous photobioreactor during

hydrogen production runs by Rb. capsulatus wild type on the molasses DFE. Starting

date of the experiment was 27.07.09. Feeding was started at 3rd

day.

The overall hydrogen productivity obtained at the end of the experiment was

0.34 mmolesH2/Lc.h and the molar yield was 32% of the theoretical maximum. The

overall acetate conversion efficiency was 70% in this experiment.

4.3.2.3. Long Term Stability of Continuous Hydrogen Production in Outdoor

Conditions

In Run170809 long term operation for hydrogen production was aimed. The

photobioreactor was started at 17th

of August with 50% the molasses DFE, 25% (20

mM) Na2CO3 buffer (pH=6.4) supplemented with 0.1 mM of iron and 0.16 µM of

molybdenum and 25% activated Rb. capsulatus wild type. The photobioreactor was

started to be fed daily at 6th

day with the feeding media with a volume of 10% of the

photobioreactor. The hydraulic retention time was 10 days. The composition of the

feeding media was 50% (10 mM) Na2CO3 buffer (pH=6.4) supplemented with 0.1

0

5

10

15

20

25

30

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Co

nce

ntr

ati

on

(m

M)

Time (days)


Batch Continuous

105

mM of iron and 0.16 µM of molybdenum and 50% the molasses DFE. The

experiment continued 131 days. The photobioreactor temperature, pH, cell

concentration, cumulative hydrogen production, organic acid consumption and gas

composition were measured daily. The, chemical oxygen demand, total organic

carbon and nitrogen and ammonia, and elemental analyzes were done once a week.

Daily global solar radiation energy data were taken from National Institute of

Meteorology. The definition of total global solar radiation is given in Appendix J.

The photobioreactor was carried into the green house at 79th

day. All gas




solubility in water and are given in Appendix G and H, respectively. Experimental

data are given in Appendix M.10.

The pH variation in outdoor continuous photobioreactor (4 L) with cooling coil

run by Rb. capsulatus wild type on the molasses DFE started at 17.08.09 is shown in

Figure 4.35. The pH was controlled around 7.0 till 55th

day. After that day the buffer

concentration in the feed was decreased which has led to increasing pH up to 8.7. Re-

adjustment of the buffer concentration was not sufficient to stabilize the pH at 7.0. A

sharp decrease in the pH value down to 6.0 has been observed at 105th

day. Then it

remained around that value. The pH is one of the most important factors affecting the

hydrogen productivity. Sufficient amount of buffer addition is absolutely necessary

for long term stable operation.

106

Figure 4.35 Long term stability of pH in outdoor continuous photobioreactor during

hydrogen production runs by Rb. capsulatus wild type on the molasses DFE. Starting

date of the experiment was 17.08.09. Feeding was started at 6th

day.

Growth and daily hydrogen production in Run170809 are illustrated in Figure

4.36. After the inoculation the biomass concentration was 0.28 gdcw/Lc. The

continuous feeding has been started on the 6th

day where the biomass concentration

reached 0.74 gdcw/Lc. The cell concentration in the photobioreactor increased till

16th

day. Then the biomass decreased to 0.7 gdcw/Lc and recovered itself to 0.86

gdcw/Lc at the 57th

day. This shows the stabilization of the biomass at the long term.

By changing the molasses DFE concentration in the feed the biomass

concentration further increased up to 1.15 gdcw/Lc. After that day the molasses DFE

concentration was decreased back to the first feeding conditions which caused a

decrease in biomass concentration. Addition of fresh bacteria (freshly grown culture

of Rb. capsulatus at an OD660 of 1.0) improved the growth and an increase in

biomass concentration was observed. After 99th

day biomass concentration

continuously decreased. Even fresh bacteria addition did not improve the biomass

concentration.

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130

pH

Time (days)

0.5L

Bacteria

0.4L

Bacteria

0.1L

Bacteria

0.1L

Bacteria

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch Continuous

107

The hydrogen production started on the 4th

day. Daily hydrogen production

varied between 1.0- 1.5 L/day for first 10 days then it decreased to 0.2-0.8 L/day and

continued around this value for 30 days then it continued between 0.2-0.6 L/day till

79th

day. After the photobioreactor was taken into the green house daily hydrogen

production varied between 0-0.2 L/day. The maximum hydrogen productivity (0.54

mmolesH2/Lc.h) was obtained in the first ten days and in this period the molar yield

was 34%. After 10th

day hydrogen productivity decreased and between 11th

and 41th

days hydrogen productivity was 0.43 mmolesH2/Lc.h. In this period molar yield

increased to 43% of theoretical maximum. Between 42nd

and 56th

day hydrogen

productivity and molar yield were 0.28 mmolesH2/Lc.h and 36% of the theoretical

maximum, respectively. Overall acetate conversion efficiency in the hydrogen

producing period which comprise the first 56 days was 77%.

Figure 4.36 Growth and daily hydrogen production in outdoor continuous


of the experiment was 17.08.09. Feeding was started at 6th

day.

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Da

ily H

2 Pro

du

ction

(L)

Cel

l C

on

cen

tra

tio

n (

gd

cw/L

c)

Time (days)


0.5L

Bacteria

0.4L

Bacteria 0.1L

Bacteria

0.1L

Bacteria

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

108

Hydrogen productivity was not influenced by increasing the acetate

concentration in the feed increased to 49 mM by increasing the molasses DFE

percentage in the feeding medium to 75%. However, molar yield decreased to 20%.

Hydrogen productivity between 79th

and 131st

days was 0.08 mmolesH2/Lc.h and

molar yield was 14% of the theoretical maximum. The overall acetate conversion

between 57th

and 131st

days was 43%.

For 34 days the photobioreactor temperature was controlled around 350C with

running the cooler for 9 hours in daytime. After the 34th

day the internal cooling

system has not been operated. However, that caused a fleeting temperature to

increase above 450C during the day and decreasing the temperature below 10

0C

during the night. This indicates that for long term stability of temperature the outdoor

reactor must be integrated with an internal cooling (heating) system, even in autumn.

However, this temperature increase had not a significant effect on the biomass

concentration and hydrogen productivity. Although the extreme temperature

variations were reported to decrease the hydrogen production significantly in batch

cultures of Rb. capsulatus (Ozgur et al., 2009) bacteria can tolerate temperature

fluctuations in autumn in outdoor conditions.

109


Rb. capsulatus wild type on the molasses DFE. Starting date of the experiment was

17.08.09. Feeding was started at 6th

day.

At the day 79, day and night temperature decreased to 14.70C and -0.3

0C,

respectively. So biomass decreased by 30% in the 79th

day. Therefore, freezing of the

bacteria must be avoided. Therefore, after the 80th

day, the photobioreactor was

moved into the green house.

There is a direct relation between the global total solar radiation energy per day

and hydrogen production per day (Figure 4.38). The daily global total solar radiation

energy decreases gradually from 6600 Wh/m2

(17 August 2009) to 680 Wh/m2 (21

December 2009). Total daily global solar radiation energy varies depending on the

daily illumination time and weather conditions. On cloudy days this energy drops even

half of its value.

-10

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Tem

per

atu

re (

0C

)

Time (days)

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up IV

Feeding

Molasses:Buffer

1:1 Batch Continuous

Green House

110




6th

day.

In Figure 4.39 organic acid concentration in Run170809 is plotted. During

batch operation (first 5 days) 91% of acetate that was initially fed was consumed. After

feeding started the acetate concentration in the photobioreactor remained under 5 mM

due to simultaneous hydrogen production and biomass growth in following 15 days.

After 15 days of operation, acetate started to accumulate due to decrease in biomass

concentration and hydrogen productivity.

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0

1000

2000

3000

4000

5000

6000

7000

8000

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Da

ily H

2 Pro

du

ction

(L)

To

tal

Glo

ba

l S

ola

r R

ad

iati

on

(W

h/m

2)

Time (days)


III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Continuous

Green House

Batch

111


by Rb. capsulatus wild type on the molasses DFE. Starting date of the experiment

was 17.08.09. Feeding was started at 6th

day.

Acetate consumption was observed between days 52 to 57 where biomass

concentration increased. When the feed was changed at the day 57, the acetate

accumulation restarted although biomass concentration increased. No improvement

was observed in hydrogen production in that period. Hence, the previous feed

composition was restored after day 68. The acetate concentration in the

photobioreactor continued to fluctuate and reached to 30 mM at the end of experiment.

Lactic acid was completely consumed till the 80th

day but after that day its

concentration remained at 2.5 mM. Propionic and butyric acid formation was

negligible during 105 days then propionic acid concentration increased to 17 mM but

after 113th

day it was consumed. Formic acid was produced in a considerable amount

in the system. It reached to its maximum concentration which was 26 mM at 18th

day

and remained around 20 mM till 62nd

day. After that day it was consumed and its

concentration decreased to 6 mM after 80th

day. At the end of the experiment its

concentration decreased to 3 mM. Formate production by Rb. capsulatus is

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Co

nce

ntr

ati

on

(m

M)

Time (days)


III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Batch Continuous

Green House

112

associated with NAD-dependent formate dehydrogenase which reversibly catalyses

the formate synthesis from CO2 and NADH, H. The use of carbonate buffer may

stimulate the formate production by increasing soluble CO2 concentration in the

reactor. In addition, formate synthesis at high hydrogen production periods where

CO2 also evolves may explain its high production rate. So formate synthesis may be

attributed to the bacterial photosynthetic activity whereby it fixes CO2 into formate

through NAD-dependent formate dehydrogenase. If formate is not produced it

depletes proportional to the hydraulic retention time. After the second feeding period,

where carbonate buffer concentration was decreased a gradual decrease in formate

concentration was observed.

Figure 4.40 illustrates the chemical oxygen demand (COD), total organic

carbon (TOC), total nitrogen (TN), NH4+concentration detected in the PBR effluent

before the feeding and Carbon to Nitrogen (C/N) ratio. The tests were carried out

once a week for practical purposes. The COD of the molasses DFE is 6500 mg/L.

After dilution with buffer it decreased to 3250 mg/L. the COD value decreased to

1930 mg/L with photofermentation, the minimum was obtained at the 8th

day

(feeding started at the 6th

day). During continuous feeding COD value increased up

to 4600 mg/L after 107th

day. The total organic carbon didn’t change significantly

during the entire process and it was around 1500-2000 mg/L.

Total nitrogen value was 40 mg/L at the 8th

day of the experiment. Its

concentration varied between 20-150 mg/L. After 107th

day, where most of the

biomass has died COD, TN and NH4+ concentration increased to the feed

concentration.

The C/N ratio of the molasses DFE was 15.5. After the experiment started

C/N ratio of the photofermentation effluent increased to 26.5 at 8th

day. Then it

remained around 20 till 50th

day. After 50th

day average C/N ratio was around 45 till

the end of the experiment.

113

Figure 4.40 Variation in COD, TOC, TN and NH4+

in outdoor continuous


of the experiment was 17.08.09. Feeding was started at 6th

day.

Figure 4.41 illustrates the variation in concentrations of Magnesium (Mg),

Zinc (Zn), Cobalt (Co), Manganese (Mn), Iron (Fe), Nickel (Ni), Copper (Cu),

Calcium (Ca), and Sodium (Na) ions during long term operation in Run170809.

Sodium (Na) concentration was 239 mM in the molasses DFE but in the

photofermentation effluent it was 86 mM at the 8th

day and its concentration changed

between 34 and 160 mM during the experiment. Magnesium (Mg) concentration was

5.26 mM in the molasses DFE and after dilution with buffer its concentration

decreased to 2.63 mM and its average concentration in the photofermentation

effluent was 0.73 mM. Iron (Fe) concentration in the molasses DFE was 0.025 mM.

To increase Iron (Fe) concentration in the media to 0.1 mM it was added to the

Na2CO3 buffer. Although Iron (Fe) addition average Iron (Fe) concentration was

around 0.025 mM in the photofermentation effluent during the experiment so more

Iron (Fe) can be added to the buffer. Concentrations of Zinc (Zn), Cobalt (Co),

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

0 10 20 30 40 50 60 70 80 90 100 110 120 130

CO

D, T

OC

(mg

/L) N

H4+, T

N (

mg

/L)

C/N

Ra

tio

Time (days)

COD TOC NH4+ TN C/N

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

114

Manganese (Mn), Nickel (Ni), Copper (Cu), and Calcium (Ca) were also below 0.1

mM during the experiment.


in outdoor continuous photobioreactor runs by Rb. capsulatus wild type on the


6th

day.

Figure 4.42 illustrates the hourly changes of hydrogen production, solar

radiation and temperature variation at 19-21.10.2009. It can be clearly seen from the

figure that significant hydrogen production occurred between 9:00 and 17:00 in

autumn, for 10 hours a day.

0

20

40

60

80

100

120

140

160

180

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Co

ncen

tratio

n (m

M)

(Na

)

Co

nce

ntr

ati

on

(m

M)

(Mg

, Z

n,C

o,

Mn

, F

e, N

i, C

u,

Ca

)

Time (days)

Mg(mM) Zn(mM) Co(mM) Mn(mM) Fe(mM)

Ni(mM) Cu(mM) Ca(mM) Na(mM)

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

115

Figure 4.42 The hourly hydrogen production, temperature and the solar radiation in


molasses DFE.

4.3.3 Long Term Stability of Continuous Hydrogen Production with Rb.

capsulatus hup- in Outdoor Conditions

In Run300709 the photobioreactor with 4 liter working volume was used. The

experiment was run in outdoor conditions; the photobioreactor temperature was

controlled with chilled water circulating through a cooling coil inserted into the

photobioreactor, till 56th

day. The reactor was moved into the green house after 97th

day. The photobioreactor was started with 50% the molasses DFE, 25% Na2CO3

buffer (20 mM, pH=6.4) supplemented with 0.1 mM of iron and 0.16 µM of

molybdenum and 25% activated Rb. capsulatus hup-. The photobioreactor was fed

daily with a feeding rate of 10% by the volume of the reactor, starting from 11th

day.

Hydraulic retention time was 10 days. The feed was composed of 50% (10 mM)

Na2CO3 buffer supplemented with 0.1 mM of iron and 0.16 µM of molybdenum and

50% the molasses DFE. Fresh bacteria (0.1 L, with OD660 of 1.0) were fed to the

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0

10

20

30

40

50

60

70

Ho

urly

H2 P

rod

uctio

n (L

)

Tre

act

or

(0C

),

Ho

url

y T

ota

l S

ola

r R

ad

iati

on

(1

03W

/m2)

Time (Days and Hours)

Hydrogen Production (L) Solar Radiation Treactor

116

reactor at 127th

and 142nd

days. The experiment continued 149 days. The

photobioreactor temperature, pH, cell concentration, cumulative hydrogen

production, organic acid consumption and gas composition were measured daily.

The, chemical oxygen demand, total organic carbon and nitrogen and ammonia, and

elemental analyzes were done once a week. Daily global solar radiation energy data

were taken from National Institute of Meteorology in Turkey. The definition of total

global solar radiation is given in Appendix J. All gas measurements were corrected

by calculating permeability of hydrogen through reactor surface and solubility of

hydrogen in water and carryover amounts in inocula. Methods for calculating

hydrogen permeability through solids and solubility in water and are given in

Appendix G and H, respectively. Experimental data are given in Appendix M.9.

Experimental data are given in Appendix M.11.

Figure 4.43 illustrates the pH variation. The pH stabilized around 7.0 till the

108th

day then it decreased to 6.2.

Figure 4.43 Long term stability of pH in outdoor continuous photobioreactor runs by

Rb. capsulatus hup- on the molasses DFE. Starting date of the experiment was


day.

0

1

2

3

4

5

6

7

8

9

10

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

pH

Time (days)

0.1L

Bacteria 0.1L

Bacteria

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch Continuous

117

Variations in the daily hydrogen production and biomass are illustrated in

Figure 4.44. Initial biomass concentration was 0.16 gdcw/Lc. The continuous feeding

was started on the 11th

day where the biomass concentration reached to 0.6 gdcw/Lc.

The cell concentration in the photobioreactor increased till 20th

day then it stabilized

around 0.8 gdcw/Lc. However, when the acetate concentration in the feeding medium

increased from 32.5 mM to 48 mM biomass concentration in the reactor stabilized

around 1.0 gdcw/Lc. This shows that biomass concentration can be increased by

changing the feeding medium. After acetate concentration in the feeding medium

decreased from 48 mM to 32.5 mM, the biomass has stabilized at 0.8 gdcw/Lc. After

115th

day, there is a gradual decrease in biomass concentration from 0.8 gdcw/Lc to

0.1 gdcw/Lc. Feeding the reactor with fresh bacteria did not result in biomass

recovery after that time. At the end of the experiment biomass concentration was

around 0.1 gdcw/Lc.

Figure 4.44 Growth and hydrogen production in outdoor continuous photobioreactor

runs by Rb. capsulatus hup- on the molasses DFE. Starting date of the experiment

was 30.07.09. Feeding was started at 11th

day.

0,0

0,5

1,0

1,5

2,0

2,5

0,0

0,2

0,4

0,6

0,8

1,0

1,2

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Da

ily H

yd

rog

en P

rod

uctio

n (L

) Cel

l C

on

cen

tra

tio

n (

gd

cw/L

c)

Time (days)


0.1L

Bacteria

0.1L

Bacteria

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

118

Hydrogen production started at the 10th

day. Daily hydrogen production

varied between 0.7- 2.1 L/day for 10 – 40th

days and between 0.4-0.8 L/day for 41–

74th

days. The maximum productivity (0.69 mmolesH2/Lc.h) and molar yield (76%)

were obtained in the first 41 days. Hydrogen productivity decreased to 0.38

mmolesH2/Lc.h and molar yield was 42% of the theoretical maximum between 41th

and 74th

days. The overall acetate conversion efficiency for the first 74 days was

77%. When the acetate concentration in the feeding media increased from 32.5 mM

to 49 mM both hydrogen productivity (0.25 mmolesH2/Lc.h) and molar yield (18% of

theoretical maximum) decreased. The molar yield was increased to 45% by

decreasing the acetate concentration in the feed from 49 to 32.5 mM while

productivity remained almost the same.

Variation in photobioreactor temperature is illustrated in Figure 4.45.


Rb. capsulatus hup- on the molasses DFE. Starting date of the experiment was


day.

-10

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Tem

per

atu

re (

oC

)

Time (days)

III Feeding

Molasses:Buffer 3:1

II Feeding

Molasses:Buffer 1:1

I

Start-up

IV Feeding

Molasses:Buffer 1:1

Batch Continuous

Green House

119

The temperature in the photobioreactor was controlled with chilled water

circulating through the internal coil. For 55 days the photobioreactor temperature

was controlled around 35 0C with running the cooler for 9 hours in day-time. At 97

th

day the reactor was moved into green house.

Figure 4.46 illustrates daily global solar radiation energy and daily hydrogen

production in Run300709 and it is similar to the Figure 4.38. There is a direct

relation between the hydrogen production and daily global solar radiation energy.

The daily global solar radiation energy varied between 7000 Wh/m2 and 660 Wh/m

2



outdoor continuous photobioreactor runs by Rb. capsulatus hup- on the molasses

DFE. Starting date of the experiment was 30.07.09. Feeding was started at 11th

day.

Figure 4.47 illustrates acetic, lactic, formic, propionic and butyric acids

variations during the experiment. During the batch operation 30% of acetate initially

fed was consumed. The acetate concentration in the photobioreactor continued to

0,0

0,5

1,0

1,5

2,0

2,5

0

1000

2000

3000

4000

5000

6000

7000

8000

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Da

ily H

yd

rog

en P

rod

uctio

n (L

)

Da

ily

Glo

ba

l S

ola

r R

ad

iati

on

(W

h/m

2)

Time (days)


III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

120

decrease even after the feeding started. It dropped to 4 mM at 40th

day. After 40th

day,

acetate started to accumulated and its concentration reached to the feed concentration

after 123rd

day. Lactic acid was completely consumed till 113rd

day. After that day it

accumulated slightly and its concentration reached to 5 mM between 113rd

and 133rd

day. Then it was consumed again and depleted at the end of the experiment. Propionic

acid formation was negligible till 96th

day. At 99th

day propionic acid concentration

increased to 10 mM. After that day its concentration remained below 5 mM till the end

of the experiment. In addition butyric acid concentration in the system was nearly zero

till 116th

day but after that day its concentration increased and reached to 12 mM. After

142nd

day its concentration gradually decreased at the end it was 7 mM. Formic acid

was produced in a considerable amount in the photobioreactor. It reached to its

maximum value which was 28 mM at 28th

day and decreased to 2.5 mM at the end of

the experiment, similar to the trend observed in Run170809.


by Rb. capsulatus hup- on the molasses DFE. Starting date of the experiment was


day.

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Co

nce

ntr

ati

on

(m

M)

Time (days)


III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1 Batch

Continuous

Green House

121

Figure 4.48 illustrates the chemical oxygen demand (COD), total organic

carbon (TOC), total nitrogen (TN), NH4+ concentration detected in the PBR effluent

before the feeding and carbon to nitrogen (C/N) ratio. During continuous feeding

average COD value was around 3400 mg/L till 125th

day. After that day COD value

reached up to 9400mg/L. The total organic carbon didn’t change significantly during

the entire process and it was around 1500-2000 mg/L.

Total nitrogen value was 126 mg/L at the 8th

day of the experiment. Its

concentration varied between 30-140 mg/L. After 110th

day, where most of the

biomass has died COD, TN and NH4+ concentration increased to the feed

concentration.

The average C/N ratio of the photofermentation effluent was almost 25


Figure 4.48 The COD, TOC, TN, and NH4+ concentrations in outdoor continuous

photobioreactor runs by Rb. capsulatus hup- on the molasses DFE. Starting date of

the experiment was 30.07.09. Feeding was started at 11th

day.

Figure 4.49 illustrates the variation in concentrations of Magnesium (Mg),

Zinc (Zn), Cobalt (Co), Manganese (Mn), Iron (Fe), Nickel (Ni), Copper (Cu),

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

CO

D, T

OC

(mg

/L) N

H4+, T

N (

mg

/L),

C/N

Ra

tio

Time (days)

COD TOC NH4+ TN C/N

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1

I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch

Continuous

122

Calcium (Ca), and Sodium (Na) ions during long term operation in Run300709. In

the photofermentation effluent sodium (Na) concentration was 70 mM at the 28th

day

and its concentration changed between 35 and 104 mM during the experiment.

Magnesium (Mg) concentration was 5.26 mM in the molasses DFE and in the

photofermentation effluent its concentration decreased to 0.78 mM at the 28th

day

and its average concentration in the photofermentation effluent was almost same with

its initial concentration in the effluent. Although Iron (Fe) addition average Iron (Fe)

concentration was around 0.03 mM in the photofermentation effluent during the

experiment. Concentrations of Zinc (Zn), Cobalt (Co), Manganese (Mn), Nickel (Ni),

Copper (Cu), and Calcium (Ca) were also below 0.1 mM and similar within the

Run170809.


in outdoor continuous photobioreactor runs by Rb. capsulatus wild type on the


11th

day.

0

20

40

60

80

100

120

140

160

180

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1,1

1,2

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

Co

ncen

tratio

n (m

M)

Na

Co

nce

ntr

ati

on

(m

M)

Mg

, Z

n,

Co

, M

n, F

e, N

i, C

u,

Ca

Time (days)

Mg(mM) Zn(mM) Co(mM) Mn(mM) Fe(mM)

Ni(mM) Cu(mM) Ca(mM) Na(mM)

III

Feeding

Molasses:Buffer

3:1

II

Feeding

Molasses:Buffer

1:1 I

Start-up

IV

Feeding

Molasses:Buffer

1:1

Green House

Batch Continuous

123

The hourly hydrogen production, temperature in Run300709 and the solar

radiation are plotted in Figure 4.50. Significant hydrogen was produced between 9:00

and 17:00 during a day in autumn.

Figure 4.50 The hourly hydrogen production, temperature in outdoor continuous

photobioreactor runs by Rb. capsulatus hup- on the molasses DFE. Starting date of

the experiment was 30.07.09. Feeding was started at 11th

day.

4.4 Logistic Growth Model

The logistic function model is increasingly being used to describe microbial

growth (Classen et al., 2000). The logistic function model describes the growth of

microbial populations as a function of initial population density, time, growth rate,

and final population density (Wachenheim et al., 2003).

By using logistic model it is possible to model bacterial growth at exponential

phase together with stationary phase. The predictive power of logistic model may be

limited since it does not involve a substrate term; however, for the purposes of batch

hydrogen production experiments, where the initial substrate concentrations and the

0,00

0,05

0,10

0,15

0,20

0,25

0

10

20

30

40

50

60

70

Ho

urly

Hy

dro

gen

Pro

du

ction

(L)

Tre

act

or

(0C

),

Ho

url

y T

ota

l S

ola

r R

ad

iati

on

(1

03 W

/m2)

Time (Days and Hours)

Hydrogen Production (L) Solar Radiation Treactor

124

inoculation volume are kept constant, the logistic model is a fair approximation of

the growth curve (Koku et al, 2003). Uyar et al (2008) showed that Logistic model

can be used for modeling growth of Rb. sphaeroides O.U.001 for malate, acetate,

propionate, lactate, and butyrate.

The cell growth can be explained in three phases; lag phase, exponential phase

and stationary phase. Lag phase is the adaptation period of the microorganism to a

new media so in this phase there is small growth observed. The Exponential phase is

the reproduction period of the microorganism so in this phase there is an abruptly

biomass increase. In the stationary phase microorganism has already accommodated

the new media and already utilized all of the substrate for growing so biomass is

stabilized in this phase.

The cell growth can be theoretically represented by exponential model:

d

dt µ (4.1)

Where X is the cell dry weight concentration (gdcw/Lc) and μ is the specific

growth rate (h−1

).

After the equation 4.1 was integrated the equation below was obtained

f iexp (μt) (4.2)

The growth rate for the logistic model is expressed as:

d

dt kc (

1-

max) (4.3)

Where kc is the apparent specific growth rate (h-1

), X is the dry cell weight

(gdcw/Lc), and Xmax is the maximum dry cell weight (gdcw/Lc).

Integrating, the equation becomes:

max

(1 ( max

i-1) exp(-k

c.t))

(4.4)

125

Where Xi is the initial bacterial concentration at the lag time (gdcw/Lc) and t

is the actual time minus lag time (h).

The experimental data collected for defining growth kinetics in this study

were fitted into the logistic model using a program for fitting curves, Curve Expert

1.4.

Figure 4.51 shows the logistic model for the growth of Rhodobacter

capsulatus (DSM 1710) studied in indoor continuous photobioreactor (8 L) on

defined medium started at 24.08.07. Experimental data used for modeling are

between 24.08.07 and 28.01.08. Correlation coefficient and standard error of the

system are 0.90 and 0.092, respectively. Although composition of the feeding media

was changed five times during the experiment the correlation coefficient of logistic

model is very high.

Figure 4.51 The logistic model for the growth of Rb. capsulatus (DSM 1710) studied

in indoor continuous photobioreactor (8 L) on defined medium (24.08.07-28.01.08)


capsulatus (DSM 1710) studied in outdoor continuous photobioreactors (8 L) on

S = 0.09235978

r = 0.90055359

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

0.0

0.2

0.3

0.4

0.6

0.7

0.9

126

defined medium started at 26.09.07. The experimental data used for growth modeling

are the data between 24.08.07 and 28.01.08. Last two weeks of experimental data

were not taken into account. So the correlation coefficient and standard error of the

model are 0.984 and 0.058, respectively.


in outdoor continuous photobioreactor (8 L) on defined medium (26.09.07-23.01.08)


capsulatus (DSM 1710) studied in outdoor continuous photobioreactor (8 L) on

defined medium started at 31.08.07. All of the experimental data is used for

modeling growth. The correlation coefficient and standard error of the model are

0.987 and 0.063, respectively.

S = 0.05800994

r = 0.98449691

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 10 20 30 40 50 60 70 80 90 100 110 120

0.0

0.2

0.4

0.6

0.8

1.0

1.2

127


in outdoor continuous photobioreactors (8 L) on defined medium (31.08.07)

Figure 4.54 gives the logistic model for the growth of Rhodobacter

capsulatus hup- studied in outdoor continuous pilot-scale photobioreactor on defined

medium. All of the experimental data is used for modeling of growth of the bacteria.

The correlation coefficient and standard error of the model are 0.914 and 0.072,

respectively.

S = 0.06329833

r = 0.98737573

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

0.0

0.2

0.4

0.6

0.8

1.1

1.3

128

Figure 4.54 The logistic model for the growth of Rb. capsulatus hup- studied in

outdoor continuous pilot-scale photobioreactor (20 L) on defined medium

Table 4.17 tabulates the parameters affecting modeling of growth of Rb.

capsulatus DSM 1710 and Rb. capsulatus hup- on defined media in indoor and

outdoor conditions in 8 L and 20 L panel photobioreactors. Parameters which are

obtained from logistic model and experiments are in accordance. If the parameters

affecting bacterial growth like temperature or light intensity kept strictly controlled

the fluctuations in bacterial growth can be minimized. By this way parameters

obtained by logistic model like initial bacterial concentration (Xi), maximum

bacterial concentration (Xmax) will mainly fit the experimental data.

S = 0.07172163

r = 0.91414179

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 5 10 15 20 25 30 35

0.1

0.2

0.3

0.4

0.5

0.6

0.7

129


hup- on defined medium in panel photobioreactors

Indoor

Continuous

PBR (8 L) on by

Rb. capsulatus

wild type

Defined

Medium

(24.08.07)

Outdoor

Continuous

PBR (8 L) by

Rb. capsulatus

wild type on

Defined

Medium

(26.09.07)

Outdoor

Continuous

PBR (8 L) by

Rb. capsulatus

wild type on

Defined

Medium

(31.08.07)

Outdoor

Continuous

Pilot-Scale PBR

(20 L) by Rb.

capsulatus hup-

on Defined

Medium

(02.12.08)

Xi,e 0.09 0.15 0.02 0.11

Xi,m 0.17 0.11 0.07 0.09

Xmax,e 0.82 1.10 1.15 0.62

Xmax,m 0.71 0.98 1.04 0.49

µmax 0.05 0.07 0.05 0.18

kc 0.05 0.12 0.04 0.23

Figure 4.55 gives the logistic model for the growth of Rhodobacter

capsulatus DSM 1710 studied in indoor continuous photobioreactor (500 ml) on the

molasses DFE. Correlation coefficient and standard error of the model are 0.974 and

0.044, respectively.

130

Figure 4.55 The logistic model for the growth of Rb. capsulatus DSM 1710 studied

in indoor continuous photobioreactor (500 ml) on the molasses DFE.


capsulatus DSM 1710 studied in indoor continuous photobioreactor (4 L) on the

molasses DFE. The experimental data used for growth modeling are the data between

24.04.09 and 03.05.09. Correlation coefficient and standard error of the model are

0.930 and 0.28, respectively.

S = 0.03909117

r = 0.96246964

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.1

0.2

0.3

0.4

0.5

0.6

0.7

131

Figure 4.56 The logistic model for the growth of Rb. capsulatus DSM 1710 studied

in indoor continuous photobioreactor (4 L) on the molasses DFE (24.04.09-03.05.09)

Figure 4.57 and 4.58 gives the logistic model for the growth of Rhodobacter

capsulatus DSM 1710 studied in outdoor continuous photobioreactor (4 L) with

cooling on the molasses DFE started at 27.07.09 and 17.08.09. The experimental data

used for growth modeling of the photobioreactor started at 17.08.09 are the data

between 17.08.09 and 11.10.09. As a result correlation coefficient and standard error

of the model are 0.88 and 0.088. Correlation coefficient and standard error of the

model are 0.965 and 0.059.

S = 0.28776472

r = 0.93071740

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 1 2 3 4 5 6 7 8 9 10 11

0.0

0.4

0.8

1.1

1.5

1.9

2.2

132

Figure 4.57 The logistic model for the growth of Rb. capsulatus (DSM1710) studied

in outdoor continuous photobioreactor (4 L) with cooling on the molasses DFE

(27.07.09)

Figure 4.58 The logistic model for the growth of Rb. capsulatus (DSM1710) studied

in outdoor continuous photobioreactor (4 L) on the molasses DFE (17.08.09-

11.10.09)

S = 0.05947720

r = 0.96507025

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0.3

0.4

0.6

0.7

0.8

1.0

1.1

S = 0.08883863

r = 0.88009400

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 10 20 30 40 50 60

0.1

0.3

0.4

0.6

0.8

0.9

1.1

133


capsulatus (DSM 1710) studied in outdoor continuous photobioreactor on the

molasses DFE started at 30.07.09. The experimental data used for growth modeling

are the data between 30.07.09 and 11.10.09. Correlation coefficient and standard

error of the model are 0.051 and 0.0974, respectively.

Figure 4.59 The logistic model for the growth of Rb. capsulatus hup- studied in

outdoor continuous photobioreactor (4 L) on the molasses DFE (30.07.09-11.10.09)

Table 4.18 tabulates the characteristics of growth modeling of Rb. capsulatus

DSM 1710 and Rb. capsulatus hup- on molasses dark fermentor effluent in indoor

and outdoor conditions in 4 L panel photobioreactors with cooling coil.

S = 0.05117345

r = 0.97469507

Time (days)

Ce

ll C

on

cen

tra

tio

n (

gd

cw

/Lc

)

0 10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.7

0.9

1.1

134


hup- on the molasses dark fermentor effluent

Indoor

Continuous

PBR

(500 ml)

by Rb.

capsulatus

wild

type

Indoor

Continuous

PBR

(4 L)

by Rb.

capsulatus

wild type

Outdoor

Continuous

PBR

(4 L)

with

Cooling

by Rb.

capsulatus

wild type

(27.07.09)

Outdoor

Continuous

PBR

(4 L)

With

Cooling

by Rb.

capsulatus

wild type

(17.08.09-

11.10.09)

Outdoor

Continuous

PBR

(4 L)

With

Cooling

by Rb.

capsulatus

hup-

(30.07.09-

11.10.09)

Xi,e 0.17 0.16 0.35 0.21 0.16

Xi,m 0.18 0.13 0.30 0.15 0.12

Xmax,e 0.67 2.05 1.05 0.99 1.00

Xmax,m 0.60 1.79 0.97 0.84 0.87

µmax 0.43 0.58 0.31 0.32 0.15

kc 0.79 1.09 0.69 0.58 0.23

4.5 Comparison of Photofermentation Efficiency of Define Medium with the

Molasses DFE

Table 4.19 tabulates the yields and productivities obtained on defined medium

and on the molasses DFE. Maximum hydrogen productivity and maximum molar

yield was obtained in indoor continuous photobioreactor (8 L) by Rb. capsulatus

wild type on defined medium started at 24.08.07 but its productivity was very similar

with the maximum productivity of Run310807. Among the molasses DFE

experiments maximum hydrogen productivity and yield obtained in Run300709 by

Rb. capsulatus hup-. Hydrogen productivity and molar yield of Run170809 is also

higher than the other experiments that the molasses DFE used as substrate. The

yields and productivities obtained in Run300709 and Run170809 are also higher than

the productivity and yields obtained in the hydrogen production experiments on

defined medium.

135

Tab

le 4

.19 C

om

par

ison o

f yie

lds

and p

roduct

ivit

ies

of

conti

nuous

hydro

gen

pro

duct

ion o

n d

efin

ed m

ediu

m a

nd t

he

mola

sses

DF

E e

xper

imen

ts

in p

anel

photo

bio

reac

tors

PB

R

(Ru

n T

ime)

B

act

eria

S

ub

stra

te

Volu

me

(L)

Op

erati

on

al

Con

dit

ion

Du

rati

on

(days)

Pro

du

ctiv

ity

(mm

ole

sH2/L

c.h

)

Yie

ld

(%)

240807

Rb. ca

psu

latu

s

DS

M 1

710

Def

ined

Med

ium

8

Indoo

r 167

55-1

44

0.1

2

32

260907

Rb. ca

psu

latu

s

DS

M 1

710

Def

ined

Med

ium

8

Outd

oor

134

15-4

8

0.0

6

13

310807

Rb. ca

psu

latu

s

DS

M 1

710

Def

ined

Med

ium

8

Outd

oor

160

36-7

4

0.1

1

23

021208

Rb. ca

psu

latu

s

hup-

Def

ined

Med

ium

20

Outd

oor

32

0.0

8

16

240310

Rb. ca

psu

latu

s

DS

M 1

710

Mola

sses

DF

E

0.5

In

doo

r 15

0.1

4

8

240409

Rb. ca

psu

latu

s

DS

M 1

710

Mola

sses

DF

E

4

Indoo

r 23

0.1

7

29

270709

Rb. ca

psu

latu

s

DS

M 1

710

Mola

sses

DF

E

4

Outd

oor

16

0.3

4

32

170809

Rb. ca

psu

latu

s

DS

M 1

710

Mola

sses

DF

E

4

Outd

oor

131

1-1

0

10-5

6

0.5

4

0.3

8

34

41

300709

Rb. ca

psu

latu

s

hup-

Mola

sses

DF

E

4

Outd

oor

149

1-4

0

41-7

4

0.6

9

0.3

8

76

42

136

CHAPTER 5

CONCLUSIONS

In this study, a stable operation of a continuous panel photobioreactor by

Rhodobacter capsulatus on acetate-lactate-glutamate containing feeding media has

been achieved for long term.

There exists a critical cell concentration (0.3 gdcw/Lc) for hydrogen production to

commence. Below this threshold value, hydrogen production is negligible.

Increase in glutamate concentration increases biomass concentration in the

photobioreactor.

The removal of lactate from feeding media did not influence hydrogen

productivity significantly. But acetate and glutamate are prior for hydrogen production

and bacterial growth. And if we increase acetate concentration the hydrogen

production increase but there is a limiting acetate level for hydrogen production.

C/N ratio is an important parameter for long term operation.

There are prior parameters for a stable and long term operation. These can be

simply designated for an indoor operation as light intensity, feed composition and

feeding rate.

Temperature can easily be controlled in indoor conditions but in outdoor

conditions temperature control get the most important parameter and it brings along

with a cooling or a heating requirement.

Feedings should be done daily to prevent fluctuations in bacterial growth.

In a long term operation bacterium can produce a thin film on the surface. The film

layer production is also a design and construction problem so the reason of production

of the film layer should be studied.

Dark fermentor effluent of molasses is a suitable feedstock for photofermentative

hydrogen production. It can be utilized for growth and hydrogen production by

Rhodobacter capsulatus. Besides it contains vitamins and minerals which is vital for

137

biological activity of microorganisms consequently there is no need to add additional

nutrients.

Sodium carbonate is a convenient buffer for controlling the pH in biological

systems. It is also cheap and environmentally friendly than potassium phosphate

buffer.

A continuous operation up to 131 days has been achieved on molasses the dark

fermentor effluent by Rhodobacter capsulatus DSM 1710 in a cooled panel

photobioreactor in outdoor conditions.

A continuous operation up to 149 days has been achieved on the molasses the dark

fermentor effluent by Rhodobacter capsulatus hup- in a cooled panel photobioreactor

in outdoor conditions.

During summer the maximum temperature should not be greater than 38 0C. The

temperature is effectively controlled by placing internal cooling coils into the panel

reactor. A cooling apparatus helped keeping the temperature around 35 0C so

continuous hydrogen production was obtained.

For stable operation controlling the maximum temperature during the day and the

minimum temperature during the night is strongly recommended. The minimum

temperature level for hydrogen production is 10 0C because the hydrogen productivity

decreased as the reactor temperature decreased under this value.

There is a close relation with global solar radiation energy and hydrogen

production. As daily global solar radiation energy decreases daily hydrogen production

decreases.

The long term experiments show that pH is an important parameter for long term

stability.

Addition of fresh bacteria is a promising method for increasing cell concentration

in the photobioreactor.

138

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Washington, DC

Adams, M.W.W., 1990, “The structure and mechanism of [Fe]-hydrogenase”,

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Androga, D. D., 2009, “Biological hydrogen production on acetate in continuous

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APPENDIX A

COMPOSITION OF THE MEDIA AND SOLUTIONS

A.1 Composition of the Minimal Medium

Table A.1 Composition of the minimal medium (Biebl and Phening, 1981)

Component Amount

KH2PO4 (3.67 mM) 0.5 g/L

MgSO4.7H2O 0.2 g/L

NaCl 0.4 g/L

Sodium glutamate (10 mM) 1.8 g/L

CaCl2.2H2O 0.05 g/L

L-Malic acid (7.5 mM) 1.0 g/L

Vitamin solution (1X) 1 ml/L

Trace element solution SL 7 (1X) 1 ml/L

Fe-citrate solution (1X) 5 ml/L

154

A.2 Composition of the Growth Medium

Table A.2 Composition of the growth medium

Component Amount

KH2PO4 (22 mM) 3 g/L

MgSO4.7H2O 0.5 g/L

CaCl2.2H2O 0.05 g/L


Trace element solution (10X) 0.1ml/L

Fe-citrate solution (50X) 0.1ml/L

Sodium L Glutamate(10 mM) 1.8 g/L

Acetic Acid (40 mM) 1.15 ml/L

A.3 Composition of the Hydrogen Production Medium

Table A.3 Composition of the hydrogen production medium (40 mM Acetate/2 mM

Glutamate)

Component Amount

KH2PO4 (22 mM) 0.5 g/L

MgSO4.7H2O 0.5 g/L

CaCl2.2H2O 0.05 g/L


Trace element solution (10X) 0.1 ml/L

Fe-citrate solution (50X) 0.1 ml/L

Sodium L Glutamate(2 mM) 0.36 g/L

Acetic Acid (40 mM) 2.29 ml/L

155

A.4 Composition of the Trace Elements Solution (1X)

Table A.4 The composition of trace element solution

Component Amount

HCl (25% v/v) 1 ml/L

ZnCl2 70 mg/L

MnCl2.4H2O 100 mg/L

H3BO3 60 mg/L

CoCl2.6H2O 200 mg/L

CuCl2.2H2O 20 mg/L

NiCl2.6H2O 20 mg/L

NaMoO4.2H2O 40 mg/L

H2O Complete to 1 Liter

A.5 Composition of the Vitamin Solution (1X)

Table A.5 The composition of vitamin solution

Component Amount

Thiamine 0.05 g

Niacin (Nicotinate) 0.05 g

Biotin 0.015 g

H2O Complete to 0.1 Liter

A.6 Composition of the Fe-Citrate Solution (50X)

5 g Fe-citrate was dissolved in 100 ml distilled water and sterilized by autoclaving.

156

APPENDIX B

TECHNICAL PROPERTIES of CONSTRUCTION MATERIALS

B.1 Properties of Plexiglas

The Plexiglas has no color change as exposed to UV light, hot water and also

has a resistance to hot water and chemicals.

Table B.1 Physical properties of Plexiglas

Characteristic Value

Density (g/cm3) 1.185±0.01

Rupture Strength (N/mm2) 70-80

Stiffness (N/mm2) 70-80

Impact Resistance (kJ/m2) 18-20

Modulus of Elasticity (N/mm2) 3000-3600

Thermal Conductivity (W/m.0C) 0.18-0.19

Surface Hardness (Rockwell M Scale) 90-100

Softening Temperature (0C) 126

B.2 Properties of PVC Rigid Sheet

PVC rigid sheet comprises of PVC resin, stabilizers, lubricants, plasticizers,

fillers, impact modifiers, pigments and other additives. Hydrogen permeability of

unplasticised PVC is 1.3E+3 cm3.mm/m

2.day.atm (Pauly et al., 1999).

157

Table B.2 Properties of PVC Rigid Sheet

Specifications

Resistant to chemicals and corrosion

Easy for machining and welding

Flame retarded

Impact resistant

Weather resistant

Excellent UV resistance

Good Insulation

Aging-resistance

B.3 Properties of Aluminum 6061-T6 Tubing

Table B.3 Properties of Aluminum 6061-T6 Tubing

Specifications

Heat treated

Artificially aged

High strength

Good workability

High resistance to corrosion

B.4 Properties of Ball Valves

The ball valves are shut off valves type ON-OFF. The opening and closing

functions are made by hand. The body of the valves are coated with Cr-Ni plate.

Tubes made in copper, metal in general and various fittings are suitable for this

product. It can be studied with compressed air, water and oils. Minimum-maximum

temperature range varies from -20 to 80 0C and minimum-maximum pressure range

varies from -0.99 bar to 20 bar.

158

B.5 Properties of Adaptors

The adaptors work in push-in principle. The body of the fittings are coated

with Cr-Ni plate. Polyurethane, polyethylene tubing etc. are suitable for these

fittings. It can be studied with compressed air, and vacuum. Minimum-maximum

temperature range varies from -20 to 80 0C and minimum-maximum pressure range

varies from -0.99 bar to 15 bar.

159

APPENDIX C

LIGHT ABSORBTION SPECTRA of Rhodobacter capsulatus

Figure C The light absorption spectrum of Rhodobacter capsulatus (Weaver et al.,

1975)

160

APPENDIX D

CALIBRATION CURVE OF DRY CELL WEIGHT VERSUS OPTICAL

DENSITY AT 660nm

D.1 Calibration Curve of Dry Cell Weight versus Optical Density of

Rhodobacter capsulatus wild type


line for Rhodobacter capsulatus (DSM 1710) (Uyar, 2008). An optical density of 1.0

at 660nm corresponds to a cell density of 0.54 gram dry cell weight/liter of culture of

Rhodobacter capsulatus (DSM 1710).

161

D.2 Calibration Curve of Dry Cell Weight versus Optical Density of

Rhodobacter capsulatus mutant


line for Rhodobacter capsulatus mutant (Ozturk, 2005). An optical density of 1.0 at

660nm corresponds to a cell density of 0.47 gram dry cell weight/liter of culture of

Rhodobacter capsulatus hup- mutant.

162

APPENDIX E

SAMPLE CHROMATOGRAM FOR GAS ANALYSIS

E.1 A Sample Chromatogram for Gas Analysis

Figure E.1 Sample chromatogram for gas analysis (Androga, 2009)

163

APPENDIX F

SAMPLE HPLC CHROMATOGRAM OF ORGANIC ACID ANALYSIS AND

CALIBRATION CURVE OF ACETIC ACID

F.1 Sample Chromatogram of Organic Acid Analysis

Figure F.1 Sample chromatogram of organic acid analysis. Retention times of lactic,

formic, acetic, propionic and butyric acid are 20.4, 21.7, 23.6, 27.6, 33.4,

respectively.

164

F.2 Sample HPLC Calibration Curve for Lactic Acid

Figure F.2 Sample HPLC calibration curve for lactic acid

F.3 Sample HPLC Calibration Curve for Formic Acid

Figure F.3 Sample HPLC calibration curve for formic acid

y = 1286,5x

R² = 0,9987

0

10000

20000

30000

40000

50000

60000

70000

0 10 20 30 40 50 60

Are

a

ppm

y = 2466,8x

R² = 0,999

0

500000

1000000

1500000

2000000

2500000

3000000

0 200 400 600 800 1000 1200

Are

a

ppm

165

F.4 Sample HPLC Calibration Curve for Acetic Acid

Figure F.4 Standard HPLC calibration curve of acetic acid

F.5 Sample HPLC Calibration Curve for Propionic Acid

Figure F.5 Standard HPLC calibration curve of propionic acid

y = 1756,2x

R² = 0,9998

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

0 200 400 600 800 1000 1200

Are

a

ppm

y = 801,88x

R² = 0,999

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

0 20 40 60 80 100 120

Are

a

ppm

166

F.6 Sample HPLC Calibration Curve for Butyric Acid

Figure F.6 Standard HPLC calibration curve of butyric acid

y = 976,75x

R² = 0,9999

0

20000

40000

60000

80000

100000

120000

0 20 40 60 80 100 120

Are

a

ppm

167

APPENDIX G

PERMEABILITY of HYDROGEN THROUGH SOLIDS

Permeability of hydrogen through solids is defined by a variation of Fick’s first law,

=

x (G.1)

whereJ is the flux of diffusing species, in units of mole/m2.s, diffusing out of the

material; Dis the diffusion coefficient in m2/s; and dC/dx is the concentration

gradient of hydrogen across the solid.

oncentration of the gases is defined by Henry’s law (Steward, 1983),

H = H2

H2

(G.2)

where SH, is a constant relating the vapor pressure of hydrogen gas to its

concentration in a solid. The unit of SH in Henry’s law is mole/m3.Pa and SH can also

be defined as kH in other sources. CH2 is the concentration of hydrogen gas in the

solvent in mole/m3, and PH2 is the pressure of the hydrogen gas over the solvent in

Pa.

Substitution of Eq. (2) into Eq. (1) yields,

= H2

x

H2

x (G.3)

168

PH2, pressure difference of the diffusing hydrogen across a solid surface, and

x is the thickness of the solid. The negative sign in Eq. (G.3) represents the

direction of diffusion.

Equation (G.3) can be rearranged to correspond to the usual representation of

the permeation data, where the total amount of hydrogen gas that has permeated the

material, Q (mole), is plotted as a function of time. Equation (G.3) then becomes,

= t =

x H2

t (G.4)

where A is the slab area (m2), and t is the time (s) since reaching steady state.

The slope of this line (dQ/dt), the steady-state flow rate, includes the constant

DS, which is specific for a given material. This product of D and S, which is called

as P, is defined as the permeability. The unit of permeability is mole/m.s.Pa. We

have, by differentiating Eq. (G.5) and substituting G.4,

d

dt =

x H2

t (G.5)

The permeability, like the diffusivity and solubility, is temperature-dependent

and may be represented by an Arrhenius equation,

= 0 exp (-

R ) (G.6)

Where Ep is the activation energy of the sum of diffusivity and solubility processes.

169

APPENDIX H

SOLUBILITY of HYDROGEN in WATER

Solubility of a hydrogen gas in water can be described by Henry’s law constant; kH is

defined as:

kH = H2

H2

(H.1)

Here, CH2 is the concentration of hydrogen gas in the aqueous phase and PH2 is the

partial pressure of hydrogen in the gas phase.

If kH refers to standard conditions (T0 = 298.15 K) it will be denoted as kH

0.

Temperature dependence of Henry’s law can be described as:

kH = kH0 exp(

- Hsoln

R(1

-

1

0)) (H.2)

where - Hsoln = enthalpy of solution. kH0 = 7.8*10

-4 mole/(L.atm).

170

APPENDIX I

MEASURING HYDROGEN PERMEABILITY THROUGH REACTOR

MATERIAL

Hydrogen permeability through reactor material was measured by water

displacement method. The cylindrical column made of the chosen reactor material

full of water submerged into a vessel full of water. Then the hydrogen gas was given

to the column in a rate that water can easily leave the column. After the pressure of

the hydrogen gas inside the column reached to desired value the gas entry was

stopped. The water level inside the column was recorded at that time. Every 1 h

increase in water level was recorded till the water level stabilized. Then the volume

of hydrogen gas permeated through reactor material was calculated by the water

level increase. By multiplying volume of the hydrogen by density of hydrogen and

dividing the result by molecular weight of hydrogen, mole of hydrogen was

calculated. Permeability coefficient was calculated by the equation G.2. In this

equation which is also given below, A is the effective material area that hydrogen

was filled initially; x is the thickness of reactor material; H2 is the pressure

difference of hydrogen inside the column and in the ambient; t is the duration of the

experiment till water level stabilized.

d

dt =

x H2

t (I.1)

171

APPENDIX J

DEFINITION of GLOBAL SOLAR RADIATION

As solar radiation passes through the earth's atmosphere, some of it is

absorbed or scattered by air molecules, water vapor, aerosols, and clouds. The solar

radiation that passes through directly to the earth's surface is called Direct Solar

Radiation. The radiation that has been scattered out of the direct beam is called

Diffuse Solar Radiation. Reflected Radiation is reflected from surface features.

Direct radiation id the largest component of solar radiation and diffuse radiation is

the second largest component. The direct component of sunlight and the diffuse

component of skylight falling together on a horizontal surface and reflected radiation

from surface features make up Global Solar Radiation. It is measured by a

pyranometer. Direct radiation is best measured by use of a pyrheliometer, which

measures radiation at normal incidence. Diffuse radiation can either be derived from

the direct radiation and the global radiation or measured by shading a pyranometer

from the direct radiation so that the thermopile is only receiving the diffuse radiation

(www.appleylab.com).

Figure J.1 Diffuse, Direct and Reflected Radiation (www.arcgisdesktop/9.3/)

http://www.appleylab.com/

172

APPENDIX K

SAMPLE CALCULATIONS FOR EVALUATION OF THE ANALYSIS

K.1 Sample Calculation for Dry Cell Weight

Bacterial cell concentration (Cdcw) is calculated from multiplying the cell

number (OD660) with the slope of the standard calibration curve (m=0.54) for DSM

1710. The formula is given below;

dcw = 660 m (K.1)

0.54gdcw

c = 1 0.54

gdcw

c (K.2)

K.2 Sample Calculation for Bacteriochlorophyll a Content

Bacteriochlorophyll a concentration of the culture (Cbchl a) is calculated by

dividing absorbance (OD770) at 770 nm with extinction coefficient (ε=75)and the

length of the liquid in the cuvette (L=1)than multiplying the result with molecular

weight of bacteriochlorophyll a (MWbchl a=911.5). The formula is given below;

bchl a = 770

ε bchl a (K.3)

1.21mgbchl a

c =

0.1

(75m -1cm-1) 1cm x 911.5

mg

mmole (K.4)

173

K.3 Sample Calculation for Acetic Acid Concentration

Acetic acid concentration in the photobioreactor (CAA) is calculated by

dividing the area (A) obtained from organic acid analyses in HPLC with the slope of

the acetic acid calibration curve (m) and molecular weight of acetic acid (MWAA).

The formula is given below;

=

m (K.5)

28.65mmole

=

3022265m2

1756.2m2

ppm 60.05

mg

mmole

(K.6)

174

APPENDIX L

SAMPLE CALCULATIONS FOR EVALUATION OF THE

EXPERIMENTAL DATA

L.1 Sample Calculation for Permeability Correction

For determining the exact value of produced hydrogen permeability

correction of hydrogen through the PBR surface should be included in the

calculations. For this purpose permeability coefficient of hydrogen (PH2) through

Plexiglas (1.24E-15 mole/(m.s.Pa)) in the literature is used in the calculations. The

unit of the permeability coefficient without abbreviation is mole.m/(m2.s.Pa). So,

molar hydrogen (NH2) permeated through solid surface is calculated by multiplication

of permeability coefficient of hydrogen (PH2) by surface area of the reactor (A),

duration of hydrogen production (t) and pressure difference of hydrogen in and out of

the PBR ( H2) and by dividing the result by the wall thickness of PBR material (є).

At the end molar hydrogen permeated through the reactor surface is calculated. We

can assume the pressure difference of hydrogen in and out of the photobioreactor

constant and 1atm (101325Pa).The equation and its unitary explanation are given

below:

H2=

H2 t H2

(L.1)

0.004mole = 8.09 10

-16mole.m

m2.s. a 0.472m2 604800s 101325 a

0.006m (L.2)

175

L.2 Sample Calculation for Solubility Correction

L.2.1 Sample Calculation for Solubility Correction in Batch Operation

For calculating the correct value of produced hydrogen moles of solute

hydrogen in the bacterial culture (NH2) should be included into the total hydrogen

amount. To calculate solute hydrogen in bacterial culture (NH2) solubility coefficient

of hydrogen in water at standard temperature (kH0) which is 7.8E-4 mole/L.atm is

multiplied by culture volume (Vc) and partial pressure of hydrogen (PH2) in the

headspace of the photobioreactor.

H2 = kH0 c H2

(L.3)

0.01mole = 7.8 10-4 mole

.atm 8 0.95atm (L.4)

Before calculating mole of solute hydrogen (NH2) partial pressure of hydrogen

(PH2) should be calculated. For this purpose alton’s aw is used because alton’s

Law defines the relation between total gas pressure (P) and partial pressure of

hydrogen (PH2) in the headspace. The equation is given below:

H2= xH2

(L.5)

0.95atm = 1atm 0.95 (L.6)

Where mole fraction of hydrogen (xH2) in the gas phase is 0.95 and total gas pressure

is 1atm partial pressure of hydrogen is found easily.

L.2.2 Sample Calculation for Solubility Correction in Continuous Operation

For continuous operation to calculate moles of solute hydrogen in the

bacterial culture feeding rate should be taken into account so equation J.3 is valid for

only batch period of the experiment. In the fed-batch operations mole of solute

hydrogen in the bacterial culture (NH2) is calculated for the total volume of fresh

media (VFM) given to the photobioreactor daily because as the photobioreactor fed by

176

new fresh media continuously more hydrogen dissolves in the new fresh media. The

equation for fed-batch operation is defined as below:

H2= kH

0 F H2

(L.7)

0.01mole = 7.8 10-4 mole

.atm 13.5 0.95atm (L.8)

L.3 Sample Calculation for Molar Productivity

To calculate the molar productivity of hydrogen ( H2

) first volume of

produced hydrogen ( H2)is converted to moles of hydrogen ( H2( .)

). The ideal gas

equation is used for this purpose. In this equation barometric pressure of hydrogen in

the hydrogen collection column ( H2) at the recording time is multiplied by volume

of produced hydrogen ( H2) and then divided by gas constant (R) and temperature

( H2) of the hydrogen at recording time.

H2 H2

= H2( .) R H2

(L.9)

90910 a 41.38dm3 = 1513.0mmole 8.314

dm3. a

. mmole 299.05 (L.10)

After calculating moles of produced hydrogen ( H2( .)) the molar productivity

( H2

is calculated as dividing cumulative actual molar hydrogen productivity

( H2( .)) by the culture volume (Vc) in the photobioreactor and the total hour (t)

that experiment took place. The formula is given below;

H2

= H2( .)

c t (L.11)

0.91mmole

c.h=

1513.0mmole

4 c 415.1h (L.12)

177

L.4 Sample Calculation for Molar Percentage Yield

The molar percentage yield ( ) of the process is calculated by

multiplying the division of moles of hydrogen ( H2( .)) that was actually produced

to the moles of theoretical hydrogen ( H2( H .)) that can be produced from utilized

organic acids (acetate and lactate) with 100. The formula is given below;

= H2( .)

H2( H .)

100 (L.13)

76 = 1513.0mmole

1978.9mmole 100 (L.14)

The stoichiometric equations for hydrogen production from acetate and lactate are

given below;

Acetate: C2H4O2 + 2H2O 4H2 + 2CO2 (L.15)

Lactate: C3H6O3 + 3H2O 6H2 + 3CO2 (L.16)

L.5 Sample Calculation for Acetate Conversion Efficiency

Acetate conversion efficiency (

) of the process is calculated from

dividing the difference between input ( in( )) and output ( out( )

) concentrations of

acetate with the concentration of the input acetate ( in( )) for the continuous period of

the system. The conversion equation is given below;

= in( )

- out( )

in( )

100 (L.17)

92 =32.5m -2.53m

32.5m 100 (L.18)

For long term continuous operations the overall acetate conversion efficiency

(

) is calculated. In this calculation input concentration of acetate ( in( )) is the

sum of the acetate concentration in the photobioreactor before feeding started and the

178

daily acetate fed into the photobioreactor. Output concentration of the acetate ( out( ))

is the acetate taken before feeding.

L.6 Sample Calculation for Light Conversion Efficiency

The light conversion efficiencies are calculated in different methods for

indoor and outdoor experiments because light source differs in indoor and outdoor

conditions.

L.6.1 Light Conversion Efficiency for Indoor Experiments

Light conversion efficiency (

) of a photobiological system in indoor

conditions is calculated by dividing the multiplication of combustion enthalpy of

hydrogen ( H ) and produced hydrogen ( H2) with absorbed light ( ) from the

illuminated area (A) in daytime (s).

= H H2

t 100 (L.19)

0.63 = 285670j mole . 1.52 mole

114

m2 . 0.4m2 . 1494360s

(L.20)

L.6.2 Light Conversion Efficiency for Outdoor Experiments

Light conversion efficiency (

) of a photobiological system in outdoor

conditions is calculated by dividing the multiplication of combustion enthalpy of

hydrogen ( H ) and produced hydrogen ( H2) with daily global solar radiation (R)

passing through the illumination area (A) in illumination hour (s). Daily global solar

radiation is the total energy in illumination hours so this value is converted to

seconds in the equation below;

= H H2

R 3600 100 (L.21)

4.3 =285670

j

mole.1.52mole

7000 h

m2 . 0.4m2.3600

(L.22)

179

L.7. Sample Calculation for COD Removal Efficiency

The COD removal efficiency (

( ) of a system is calculated by dividing

the difference between initial and final COD concentration to initial concentration as

the formula (Srikanth et al., 2009) giving below;

( ) = i- f

i 100 (L.23)

63.6 = 5500mg -2000mg

5500mg 100 (L.24)

180

APPENDIX M

EXPERIMENTAL DATA

M.1Experimental Data of Selection of Material Construction of Cooling Coil

Table M.1 pH, OD and cumulative H2 production values of selection of cooling coil

research

pH

Day Control PU Al PVC Cu SS 316 SS 304

1 6,502 6,568 6,538 6,542 6,534 6,582 6,521

2 6,763 6,913 6,824 6,801 6,683 6,923 6,812

4 7,196 7,388 7,287 7,279 7,187 7,141 7,151

5 7,162 7,316 7,262 7,303 7,208 7,197 7,136

6 7,079 7,298 7,220 7,234 7,243 7,130 7,127

7 6,989 7,146 7,059 7,141 7,066 7,049 6,965

8 6,969 7,025 7,095 7,061 7,047 6,969 7,026

11 7,079 6,993 7,024 6,963 7,005 6,943 6,995

OD


1 0,131 0,230 0,155 0,145 0,163 0,220 0,151

2 0,450 0,628 0,516 0,492 0,376 0,664 0,512

4 1,284 1,724 1,260 1,260 1,236 1,120 1,288

5 1,324 1,452 1,360 1,436 1,118 1,200 1,180

6 1,100 1,388 1,216 1,352 1,096 1,108 1,116

7 1,104 1,212 1,052 0,960 1,076 1,044 1,076

8 1,000 1,040 0,924 1,104 0,976 0,952 1,040

11 0,820 0,888 1,012 0,988 0,884 0,820 0,924

181

Table M.1 (Continued)

Cumulative H2 Production (ml)


1 0 0 0 0 0 0 0

2 0,5 1,5 0,4 2 2,8 5,2 3

4 28 37 40 39 38 46 46

5 49 45 57,5 57 58 69 70

6 54 68 77 79 59 93 93

7 61 84,5 95 99 59 118 113

8 70 102 116 117 64 133 134

11 84 121 156 157 76 156 163

M.2 Experimental Data of Indoor Continuous Photobioreactor (8 L) on Defined

Medium (24.08.07)

Table M.2 Temperature variation on the surfaces of indoor continuous

photobioreactor (8 L) on defined medium

Temperature (

0C)

Right Surface Left Surface

Height/Width 1 2 3 4 5 1 2 3 4 5

1 30 30 29,5 29 29 30 29,5 29 28,5 28,5

2 31 30,5 30 30 29,5 31 30 29,5 30 29

3 30,5 30,5 32,5 31 31 30 30 33 30,5 29,5

4 28,5 28,5 29 30 29,5 28 28 28,5 28,5 27,5

182

Table M.3 Light intensity variation on the surfaces of indoor continuous

photobioreactor (8 L) on defined medium

Light Intensity (lux)

Right Surface Left Surface

Height

/Width 1 2 3 4 5 1 2 3 4 5

1 1039 865 495 285 332 985 740 520 350 300

2 2200 1830 885 790 815 2030 1360 1080 970 710

3 2000 1650 1565 2180 1865 1765 1410 1695 1995 1285

4 1016 1110 1515 2670 2190 825 950 1335 1690 1290

Table M.4 Cumulative H2 production, pH, OD, and organic acid values of indoor

continuous photobioreactor (8 L) on defined medium

Day Cumulative H2

(L) pH OD

LA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,49 0,17

2 0,00 6,55 0,20

3 0,00

4 0,07 6,67 0,18

5 0,27 6,72 0,15

6 1,31

0,31

8 3,43 6,96 0,38

10 4,40 6,87 0,59

11 5,30 6,94 0,66

12 5,58 7,07 0,78

13 5,67 7,11 0,80

14 6,20 7,09 0,65

15 6,84 7,25 0,79

16 7,86 7,21 0,77 0,39 3,57 0,05 0,00

17 8,09

183


18 8,62 7,02 0,71 0,47 9,33 0,04 0,00

19 9,77 7,10 0,66

20 10,55 7,13 0,70 0,20 1,83 0,07 0,00

21 11,15 7,04 0,64 0,56 2,56 0,04 0,56

22 12,35 7,08 0,69 0,29 2,01 0,07 0,00

23 12,74 6,99 0,69 0,53 2,57 0,00 0,00

25 12,95 7,20 0,68 0,53 2,43 0,03 0,02

27 13,08 7,05 0,64 0,26 2,02 0,08 0,00

29 13,15 7,12 0,59 0,23 2,02 0,09 0,00

32

6,91 0,62 0,26 2,17 0,07 0,00

34

7,04 0,53 0,31 2,20 0,11 0,00

35 13,24

36 13,55 6,94 0,54 0,27 2,00 0,05 0,00

39 13,62 7,04 0,58 0,31 1,91 0,06 0,00

40 13,70

41 14,24 7,11 1,06 0,20 1,30 0,07 0,00

42 15,16

43 16,81 6,88 1,07 0,25 0,00 0,00 0,00

45 17,34

46 18,13 6,97 1,05 0,25 0,00 0,14 0,00

47 18,52

48 19,05 6,95 1,63 0,14 0,08 0,00 0,00

49 19,47 6,68 1,77 0,08 0,00 0,00 0,00

50 19,54

51 20,00

53 20,60 7,02 1,36 0,25 1,14 0,00 0,00

54 21,38

55 23,13 6,95 1,40 0,10 4,43 0,33 0,00

56 24,32

57 25,96 6,96 1,28

184


58 25,96

59 25,96

60 27,02 6,93

0,23 3,78 0,00 0,00

61 27,55

62 29,16 7,18 1,20 0,30 3,34 0,00 0,00

63 30,13

64 31,30 6,98 1,34 0,31 3,50 0,00 0,00

65 32,07

66 33,13

67 33,84

68 34,46 6,70 1,24 0,04 3,12 0,00 0,00

69 34,97

70 36,05 7,04 1,28

71 37,48 7,10 1,40

72 38,84

73 40,40

74 41,35 6,96 1,34 0,12 1,92 0,00 0,00

75 42,18

76 43,37 7,04 1,40 0,24 1,52 0,00 0,00

77 44,80

78 45,91 7,02 1,36 0,11 1,20 0,00 0,00

79 46,48

80 47,52

81 48,28 6,93 1,52 0,04 5,87 0,00 0,00

82 48,72

83 49,66 6,99 1,25 0,25 5,49 0,00 0,00

84 50,28 6,96 1,28

85 50,84 7,08 1,29 0,06 2,13 0,00 0,00

86 51,88

185


87 52,96

88 53,92 6,94 1,24 0,08 1,97 0,00 0,00

89 54,15

1,17

90 55,01 6,97 1,34 0,05 3,28 0,00 0,00

91 55,63

1,14

92 56,30 7,03 1,28

93 57,07

94 58,01

95 58,77 7,08 1,48

96 59,14

1,51

97 59,95

1,39

98 60,69

1,32 0,05 1,98 0,72 0,00

99 61,61 7,09 1,43 0,30 0,00 0,00 0,00

100 62,85

101 64,01

102 64,65

103 65,20

104

1,01 0,33 1,13 0,00 0,00

105 65,73

106 66,10

1,39 0,05 0,00 0,00 0,00

107 66,28

108 66,35

109 66,47

1,26 0,01 0,10 0,07 0,00

110 66,58

111 66,82

1,20 0,00 0,70 0,41 0,00

113 67,19

1,04 0,06 1,47 0,32 0,00

114 67,56

115 68,09

116 68,44 7,18 1,19 0,38 0,00 0,00 0,00

186


117 68,71

118 69,10

1,07 0,46 8,69 1,27 0,70

125 69,52 7,12 1,09 0,47 0,00 0,15 0,00

126 69,87

127 70,07

1,03 0,00 0,75 0,54 0,00

128 70,23

129 70,39

130 70,54

131 70,67

132 70,81

133 71,15 7,24 1,47 0,47 7,99 3,06 0,00

134 71,23

135 71,32

136 71,53

137 71,78 7,15 0,82 0,07 4,49 2,62 0,00

138 71,85

139 71,85

140 71,96 7,08 0,79 0,12 9,85 3,40 0,74

141 72,01

142 72,01

143 72,01

144 72,01 7,11 1,18 0,09 4,27 3,71 0,81

146 72,52 7,35 2,44 0,05 9,82 3,60 1,51

148 73,50 7,47 1,48

151 74,54 7,58 1,48 0,22 8,73 2,52 2,00

152 74,94

153 75,35 7,70 1,38

154 75,58

155 75,83 7,68 0,81 0,23 16,30 2,99 2,59

187


158 77,12 7,92 1,43

159 77,63

160 77,83 8,02 1,86 0,00 16,85 2,21 1,89

161 77,83

162

8,21 1,94

166 78,60

167 78,60 7,24 2,11

M.3 Experimental Data of Outdoor Continuous Photobioreactor (8 L) on

Defined Medium (26.09.07)



Day Cumulative H2

(L) pH OD

LA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,68 0,27 0,57 2,50 0,00 0,00

4 0,50

5 1,10

6

6,85 0,24 0,51 2,46 0,00 0,00

7 1,10

8 1,15 6,87 0,31 0,46 21,71 0,00 0,00

9 1,30

10 1,36 6,69 0,55 0,02 1,73 0,00 0,00

12 1,72

13 1,72

14 1,72

15 1,72 6,86

0,20 1,73 0,00 0,00

16 1,73 6,97 0,79 0,30 2,48 0,00 0,00

17 1,87

188


18 1,99

20 2,00 7,09 0,98

21 2,16

22 2,52

23 2,92

24 3,36 6,99 1,29

26 3,59

27 3,64

28 3,96

29 4,20 6,98 1,35 0,99 10,49 0,00 0,00

30 4,37

31 4,49

35 4,69 6,85 1,60 0,00 2,65 0,00 0,00

36 4,85

37 4,89

38 5,00 6,86 1,55 0,94 4,05 0,00 0,00

41 5,09

42 5,17

43 5,17

44 5,17 6,91 1,73

45 5,27

48 5,94

49 6,44 6,88 1,63

50 6,49

51 6,76 7,07 1,46

52 7,40 7,02 1,85 1,27 5,99 0,00 0,00

56 8,73

57 9,31 6,87 1,80 1,11 7,23 0,00 0,00

58 9,45

1,31

189


59 9,92 6,97 1,57 0,18 5,66 0,26 0,00

62 10,44

63 10,83

1,48 0,24 7,02 0,00 0,00

64 10,92

65 11,07

1,57 0,00 3,21 0,00 0,00

66 11,23 6,96 1,88 0,07 5,47 0,06 0,00

69 12,06

73 13,16

1,81 0,18 6,19 0,05 0,00

76 13,94

1,86 0,06 7,50 1,05 0,00

78 14,53

1,56

80

1,57 0,36 6,28 0,62 0,00

83

7,08 1,84 0,06 5,32 2,51 0,00

85

1,58 0,00 5,08 1,31 0,00

92 15,81 7,12 2,00 0,64 5,87 2,26 0,72

94 16,29

1,65

99 17,11

100 17,40 6,93 1,78 2,94 10,51 2,35 0,57

101 17,53

107 18,51 6,97 2,04 0,58 4,26 1,05 0,88

111 19,11 6,90 1,57 1,03 4,96 1,42 0,89

113 19,35 7,01 1,72 0,00 4,36 0,47 0,38

115 19,86

0,03 12,88 2,47 0,85

118 20,46 7,14 1,32 0,68 16,11 2,45 1,20

120 21,72 7,15 1,14 0,79 18,80 1,08 0,67

122 22,26 7,30 1,00 1,03 18,11 2,74 0,72

125 23,12 7,42 1,20 0,59 16,02 1,90 1,02

127 23,83 7,41 1,33 1,01 24,52 1,69 1,42

129 24,45 7,47 1,07 0,87 21,64 1,26 0,99

134 25,453 7,58 1,12 0,95 27,26 1,93 2,33

190

M.4 Experimental Data of Outdoor Continuous Photobioreactor (8 L) on

Defined Medium (31.08.07)



Day Cumulative H2

(L) pH OD

LA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,46 0,04

2 0,00

3 0,00

4 0,00

5 0,00 6,43 0,04

6 0,00 6,61 0,18

7 0,00 6,85 0,20

8 0,00 6,88 0,19 0,61 15,77 0,02 0,02

9 0,00 6,87 0,25

10 0,00

0,44 3,34 0,13 0,00

11 0,00 6,88 0,38

12 0,00 6,96 0,41 0,50 2,43 0,02 0,00

13 0,00 6,95 0,37 0,56 2,45 0,02 0,00

14 0,00 6,93 0,34 0,29 2,01 0,07 0,00

15 0,63 6,86 0,43 0,69 2,96 0,00 0,00

16 1,37 7,05 0,35

17

0,43 2,29 0,00 0,00

18

6,79 0,42

19

0,40 2,17 0,00 0,04

20 2,71 6,83 0,33

21 3,02

0,34 2,21 0,00 0,17

22

6,89 0,28

24 3,20

26

0,34 2,15 0,00 0,23

27

6,80 0,27 0,28 2,02 0,06 0,00

191


28

6,85 0,27

31

0,29 2,09 0,06 0,23

32

6,94 0,27 0,03 14,50 0,00 0,00

33

0,23 14,33 0,04 0,00

34 3,43 6,70 0,56 0,12 15,79 0,00 0,00

35 3,56

0,00 2,04 0,11 0,00

36 4,29 6,70 0,49 0,40 9,57 0,00 0,00

38 6,22

39 6,30

40

0,20 1,26 0,00 0,00

41 6,45 6,84 0,86 0,23 4,27 0,00 0,00

42 7,14

43 7,85

44 8,11

46 8,11 6,87 0,92 0,57 6,61 0,00 0,00

47 8,11

48 8,11

49 8,34

50 8,65 6,83 0,86

52 9,02

53 9,11

54 9,33

55 9,39 6,90 0,85 1,50 8,27 0,00 0,00

56 9,47

57 9,53

61 9,73 6,81 1,34 1,14 6,06 0,00 0,00

62 9,99

63 10,23

64 10,54 6,84 1,15 2,61 10,29 0,00 0,00

192


65 11,53

67 12,04

68 12,13

69 12,13

70 12,13 6,91 1,32 1,02 6,31 0,00 0,00

71 12,13

74 12,50

75 12,80 6,86 1,41 0,98 5,27 0,00 0,00

76 12,80

77 13,72 6,94 1,64

78 15,06 7,01 1,69

82 16,04

83 16,69 6,87 1,63 0,00 0,46 0,32 0,00

84 16,91

1,09

85 17,41 7,07 1,20 0,09 0,57 0,46 0,00

88 17,73

89 18,22

1,72

90 18,51

1,61 0,37 4,45 2,53 0,00

91 18,88

92 19,20 7,04 1,82 0,19 5,79 0,84 0,00

95 19,97

99 19,97

1,64 0,06 4,89 2,20 0,39

102 20,66

1,61 0,13 0,90 1,26 0,23

104 21,06

1,46 0,06 6,00 1,38 0,13

106

1,69 0,52 6,26 0,97 0,31

109

6,95 1,88 1,67 12,46 0,13 0,11

111

1,41 0,00 7,96 0,00 0,00

118 22,04 7,13 1,95 5,02 15,38 1,68 0,48

120 22,49

1,44 2,25 5,62 1,65 0,70

193


125 22,93

126 23,19 7,05 1,75 3,39 10,51 1,29 0,87

127 23,75

133 25,09 6,99 2,06 0,00 4,71 1,63 0,85

137 26,19 6,96 1,74 0,00 5,16 1,04 0,65

139 26,98 7,02 1,96 0,14 11,55 1,20 1,14

141 27,31

144 27,41 7,20 1,95 1,19 14,45 2,45 1,49

146 28,25 7,23 1,87 1,13 19,02 2,49 1,33

148 29,11 7,34 1,52 0,46 11,92 1,22 0,83

151 30,07 7,42 1,83 0,00 17,02 2,01 1,36

153 30,66 7,23 2,00 0,97 26,00 2,76 1,50

155 31,61 7,29 2,13 0,98 26,86 3,27 1,79

160 32,68 7,19 1,94 0,69 22,06 5,05 1,86

M.5 Experimental Data of Outdoor Continuous Pilot-Scale Photobioreactor (20

L) on Defined Medium


continuous pilot scale photobioreactor (20 L) on defined medium

Day Cumulative H2

(L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,62 0,23 0,02 0,69 20,15 0,00 0,00

2 4,68 6,61 0,23 0,00 0,44 21,52 0,00 0,00

3 6,19 6,63 0,29 0,00 0,29 21,68 0,00 0,00

4 6,34 6,68 0,35 0,00 1,19 20,80 0,00 0,00

6 6,66 6,86 0,50 0,00 0,83 18,67 0,00 0,00

8 6,98 6,91 0,53

194


10 7,30 7,03 0,56 0,06 0,70 15,46 0,00 0,00

15 7,39 7,03 0,68 0,00 1,27 12,11 0,00 0,00

16 7,53 7,04 0,98 0,00 1,48 10,57 0,00 0,00

17 7,68 7,00 1,35

18 7,83 7,07 1,12 0,00 1,74 10,82 0,00 0,00

19 12,95 7,06 1,02

21 16,84 7,20 0,88

23 18,78 7,03 1,16 0,00 2,25 7,86 0,00 0,00

25 20,11 6,87 0,92 0,00 3,08 8,10 0,74 0,25

29 24,35 7,00 1,63

32 25,32 6,97 0,91 0,00 3,94 9,33 0,51 0,29

M.6 Experimental Data on the Effect of Buffer on the Molasses DFE

Table M.8 pH, OD, and cumulative gas production values obtained at different

concentrations of Na2CO3 and KH2PO4.

pH

5mM 10mM 15mM 5mM 10mM 15mM

Day Na2CO3 Na2CO3 Na2CO3 KH2PO4 KH2PO4 KH2PO4

1 7,82 7,93 7,98 7,11 6,89 6,81

2 7,75 7,79 7,82 7,49 7,18 7,03

3 7,67 7,70 7,80 7,52 7,28 7,23

4 7,48 7,56 7,60 7,42 7,30 7,25

5 7,38 7,41 7,73 7,47 7,27 7,33

6 7,40 7,49 7,25 7,57 7,65 7,67

7 7,40 7,66 7,77 7,92 8,13 7,64

195


OD


Na2CO3 Na2CO3 Na2CO3 KH2PO4 KH2PO4 KH2PO4

1 0,50 0,47 0,43 0,38 0,35 0,37

2 0,93 0,95 0,96 0,94 0,72 0,64

3 1,47 1,28 1,43 1,48 1,46 1,42

4 1,78 1,70 1,74 1,88 1,88 1,87

5 1,88 1,70 1,86 1,96 2,09 2,08

6 1,78 1,64 1,91 1,98 2,06 1,82

7 1,68 1,59 1,73 1,96 2,10 1,82

Cumulative Gas Production


Na2CO3 Na2CO3 Na2CO3 KH2PO4 KH2PO4 KH2PO4

1 0,00 0,00 0,00 0,00 0,00 0,00

2 12,00 13,00 9,50 5,00 0,00 0,00

3 40,00 31,00 27,50 13,50 9,00 14,00

4 52,50 41,00 32,50 23,50 12,50 24,00

5 60,50 47,00 37,50 24,50 13,00 28,20

6 61,50 48,00 39,00 24,50 15,00 29,00

7 61,50 48,50 39,00 25,00 18,00 30,50

196

M.7 Experimental Data of Indoor Continuous Photobioreactor (500 ml) on

Molasses DFE

Table M.9 Cumulative H2 production pH, OD values of indoor photobioreactor (500

ml) on the molasses DFE

Day Cumulative H2

(L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,82 0,31 2,63 0,00 13,18 0,00 0,00

2 0,00 7,71 0,61 0,00 0,00 8,79 0,00 0,00

3 0,04 8,04 0,74 0,00 0,28 7,68 0,00 0,00

4 0,10 7,63 0,87 0,00 0,42 5,12 0,00 0,00

5 0,15 7,49 0,96 0,00 0,00 10,78 0,00 0,00

6 0,19 7,49 1,12 0,00 0,31 0,00 0,00 0,00

7 0,23 7,70 1,19 0,00 0,00 0,00 0,00 0,00

8 0,27 7,77 1,24 0,00 0,00 0,84 0,00 0,00

9 0,33 7,71 1,16 0,00 0,00 0,00 0,00 0,00

10 0,39 7,71 1,09 0,00 0,00 4,03 0,00 0,00

11 0,43 7,61 1,07 0,00 0,00 0,34 0,00 0,00

12 0,46 7,64 1,02 0,18 0,00 0,00 0,00 0,00

13 0,51 7,65 1,09 0,00 0,00 5,21 0,14 0,00

14 0,57 7,83 1,04 0,23 4,44 0,98 0,00 0,00

15 0,57 8,17 1,06

197

M.8 Experimental Data of Indoor Continuous Photobioreactor (4 L) on

Molasses DFE

Table M.10 Cumulative H2production pH, OD an organic acid values of indoor

continuous photobioreactor (4 L) on the molasses DFE

Day Cumulative H2

(L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 6,57 0,29 1,63 0,00 24,83 0,00 0,00

2 0,04 6,81 0,81 1,42 0,00 1,83 0,00 0,00

4 2,34 7,03 1,94 0,00 11,56 1,78 0,00 0,00

5 3,58 6,94 3,04 0,00 11,78 0,95 0,00 0,00

6 4,42 6,84 3,36 0,00 16,35 0,80 0,00 0,00

7 4,86 6,78 3,56 0,00 16,07 0,22 0,00 0,00

8 5,08 6,87 3,79 0,00 12,54 0,00 0,00 0,00

9 5,25 6,84 3,43 0,00 13,37 1,77 0,00 0,00

10 5,48 6,71 2,24 0,00 12,33 1,42 0,00 0,00

11 5,65 6,60 1,21 0,00 12,58 4,66 0,00 0,57

12 5,83 6,47 0,93 0,00 12,92 4,22 0,28 0,77

14 6,36 6,43 0,84 0,00 11,78 6,54 0,26 0,90

15 6,71 6,50 0,80 0,23 11,30 9,25 0,36 0,86

16 7,07 6,36 0,98 0,00 12,27 9,94 0,69 1,03

17 7,42 6,51 0,90 0,00 11,88 11,80 0,98 1,01

18 7,86 6,55 0,93 0,00 11,40 10,45 1,01 1,18

19 8,48 6,51 0,96 0,00 12,56 10,14 0,83 1,34

22 10,07 6,71 1,09 0,00 12,66 5,24 0,49 1,24

23 11,13 6,60 1,12 0,00 13,22 5,00 0,33 1,32

198

M.9 Experimental Data of Outdoor Continuous Photobioreactor (4 L) with

Cooling by Rb. capsulatus wild type on Molasses DFE (27.07.09)


continuous photobioreactor (4 L) with cooling by Rb. capsulatus wild type on the

molasses DFE (27.07.09)

Day Cumulative

H2(L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 7,31 0,63 4,61 12,48 30,66 0,00 0,00

2 0,00 6,60 0,71 3,50 3,11 25,48 0,00 0,00

3 0,15 7,57 1,22 5,09 4,55 28,91 0,00 0,00

4 0,22 7,54 1,33 2,10 4,25 22,56 0,00 0,00

5 0,80 7,21 1,53 2,40 5,84 25,78 0,00 0,00

6 1,39 7,22 1,72 1,81 6,73 21,80 0,42 0,42

7 1,83 7,23 1,70 1,28 9,11 20,49 0,56 0,56

8 2,56 7,40 1,69 0,85 9,03 18,27 0,51 0,51

9 3,36 7,60 1,81 0,59 10,07 15,91 0,55 0,55

10 4,31 7,30 1,83 1,08 12,07 13,82 0,66 0,66

11 4,97 7,36 1,92 0,53 9,73 12,56 0,46 0,46

12 5,48 7,29 1,83 0,52 10,14 11,16 0,47 0,47

13 5,92 7,40 1,60 0,77 12,67 10,94 0,64 0,64

14 6,22 7,35 1,63

15 6,95 7,33 1,87 0,58 14,26 10,56 0,68 0,68

16 7,39 7,06 1,61 0,70 11,48 11,38 0,75 0,75

199


Cooling by Rb. capsulatus wild type on Molasses DFE (17.08.09)


continuous photobioreactor (4 L) with Cooling by Rb. capsulatus wild type on the


Day Cumulative

H2 (L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 7,32 0,53 2,73 6,44 29,20 0,00 0,00

2 0,00 7,35 0,38 1,42 1,86 14,80 0,00 0,00

3 0,00 6,98 0,51 2,20 5,07 17,34 0,00 0,00

4 0,00 7,48 0,81 1,68 1,90 15,34 0,00 0,00

5 1,54 7,36 1,00 1,18 2,67 10,05 0,00 0,00

6 3,17 6,76 1,37 0,21 9,50 2,67 0,00 0,00

7 4,63 7,05 1,33

8 5,77 6,93 1,38 0,39 15,39 2,42 0,00 0,00

9 6,83 6,96 1,46

10 7,80 6,90 1,51 0,00 19,37 2,21 0,22 1,22

11 8,54 7,02 1,59

12 9,19 6,98 1,62 0,00 18,29 2,27 0,18 0,00

13 9,84 7,37 1,69

14 10,40 6,98 1,70 0,00 20,85 1,88 0,33 0,00

15 10,97 7,02 1,78

16 11,30 7,35 1,81 0,00 17,76 2,26 0,65 0,00

17 11,71 7,12 1,81

18 12,27 6,90 1,78 0,00 24,02 3,81 0,62 0,13

19 12,76 7,22 1,71 0,00 25,89 3,54 0,46 0,15

20 13,41 6,97 1,96 0,00 23,87 4,38 0,91 0,20

21 14,06 6,95 1,68 0,00 21,83 4,88 1,17 0,25

22 14,88 6,90 1,60 0,00 24,58 5,56 1,29 0,36

23 15,61 6,94 1,55 0,00 19,75 4,21 0,69 0,27

200


24 16,18 7,19 1,65 0,00 24,58 5,56 1,29 0,36

25 16,50 7,02 1,63 0,00 25,47 4,38 1,22 0,26

26 17,15 7,19 1,48 0,00 20,08 6,18 1,39 0,43

27 17,88 7,06 1,61 0,00 23,20 7,35 1,73 0,60

28 18,29 7,10 1,44 0,00 19,78 6,95 1,51 0,57

29 18,70 7,02 1,53 0,00 19,68 7,34 1,25 0,68

30 19,43 7,19 1,57 0,00 18,91 6,61 1,48 0,65

31 20,00 6,80 1,56 0,00 21,42 7,32 1,42 0,85

32 20,73 6,93 1,52 0,00 21,18 7,48 1,35 0,78

33 21,38 7,34 1,45 0,00 19,24 6,51 1,28 0,68

34 22,11 7,27 1,42 0,00 17,92 5,26 0,93 0,62

35 22,60 7,24 1,51 0,00 19,35 5,97 1,27 0,72

36 22,76 7,31 1,40 0,00 16,45 6,01 1,33 0,70

37 23,00 6,98 1,30 0,00 15,97 12,49 2,24 0,99

38 23,49 7,16 1,30 0,00 14,10 7,65 1,33 0,60

39 23,98 7,12 1,29 0,00 19,00 10,92 2,25 0,80

40 24,63 7,03 1,49 0,00 18,62 9,98 1,97 0,96

41 25,20 7,55 1,27 0,32 15,99 9,87 1,74 1,17

42 25,36 6,97 1,31 0,00 17,68 12,27 2,12 1,01

43 25,77 7,14 1,30 0,00 18,29 12,71 1,77 1,22

44 26,17 7,45 1,86 0,47 13,18 8,92 1,31 0,68

45 26,58 7,04 1,35 0,71 15,83 11,79 2,09 1,06

46 26,99 7,28 1,25 1,02 20,09 15,75 2,16 1,30

47 27,39 7,27 1,31 0,31 19,22 15,83 2,99 1,98

48 27,80 7,30 1,38 0,13 13,27 9,98 1,02 0,97

49 28,04 7,33 1,21 0,20 15,75 11,96 1,44 1,19

50 28,21 7,02 1,37 0,31 19,47 16,66 0,52 2,39

51 28,61 7,20 1,28 0,00 19,25 18,19 0,60 2,41

52 28,94 7,28 1,42 0,00 20,42 18,22 0,59 2,49

201


53 29,26 7,23 1,49 0,00 16,67 14,37 2,34 1,47

54 29,59 7,39 1,89 0,00 19,75 13,70 0,69 0,27

55 29,83 7,45 1,49 0,25 14,20 9,78 1,82 1,18

56 30,16 7,39 1,67 0,00 14,23 9,52 1,42 1,02

57 30,48 7,63 1,57 0,17 13,98 8,84 1,19 0,96

58 30,89 7,58 1,67 1,24 21,78 14,71 4,95 2,18

59 31,21 7,50 1,76 0,67 17,54 10,81 2,52 0,93

60 31,28 7,48 1,60 0,47 16,60 11,96 2,56 1,13

61 31,54 8,04 1,68 1,02 16,10 11,15 2,40 0,89

62 32,01 7,52 1,96 1,93 22,21 15,30 2,46 1,28

63 32,46 7,98 1,98 1,13 16,68 13,23 2,21 0,96

64 32,69 7,97 2,03 0,48 19,65 16,86 2,28 1,00

65 32,91 8,48 1,89 0,00 18,73 19,64 3,23 0,51

66 33,28 7,91 2,16 0,00 16,39 17,36 3,34 0,48

67 33,60 8,17 2,07 0,00 15,40 15,13 1,88 0,49

68 33,91 7,90 2,20 0,00 15,39 17,66 3,07 0,37

69 34,32 8,94 2,09 0,43 16,15 19,33 2,83 0,30

70 34,73 8,12 1,93 0,00 18,53 24,52 3,96 0,19

71 35,30 8,63 2,02 0,00 15,77 20,73 2,97 0,13

72 35,60 8,31 1,90 0,00 13,67 17,83 1,15 0,26

73 35,88 7,79 1,79 0,00 14,28 19,91 2,37 0,15

74 36,19 8,19 2,01 0,00 14,06 20,45 1,25 0,00

75 36,35 7,92 1,78 0,00 12,34 19,69 1,98 0,00

76 36,68 7,43 1,97 0,00 10,97 19,54 0,00 0,00

77 36,84 8,12 1,70 0,00 9,05 19,07 2,32 0,13

78 37,17 8,46 1,52 0,97 7,19 17,06 0,00 0,00

79 37,25 8,59 1,82 1,42 8,13 20,64 0,00 0,00

80 37,25 7,37 1,32 1,61 6,15 20,34 0,00 0,00

81 37,33 7,49 1,37 2,18 5,27 17,63 0,00 0,12

202


82 37,49 7,72 1,50 0,00 3,51 14,54 1,78 0,25

83 37,53 8,30 1,75 0,00 5,15 16,31 0,91 0,27

84 37,71 8,82 2,08 0,00 6,46 16,23 0,70 0,30

85 37,79 8,92 2,09 1,23 6,09 15,55 0,70 0,36

86 37,83 8,97 2,01 1,02 5,48 13,40 0,63 0,28

87 37,91 9,09 1,73 1,27 5,50 13,56 0,80 0,60

88 37,93 8,76 1,86 0,67 2,86 16,43 0,00 0,18

89 37,93 8,46 1,92 1,89 6,85 19,45 1,92 0,65

90 37,95 8,52 1,87 0,91 3,72 20,14 0,52 0,37

91 37,95 8,89 2,10 1,82 5,42 18,24 0,00 0,59

92 37,95 8,00 1,74 2,80 5,05 20,82 0,00 0,78

93 37,95 7,74 1,63 1,96 4,03 16,47 0,00 0,53

94 37,95 8,75 1,40 2,56 5,10 20,02 0,00 0,62

95 37,96 7,21 1,79 2,54 4,54 17,68 3,49 0,75

96 37,96 7,28 1,85 3,10 4,45 16,81 2,78 0,89

97 37,98 7,57 2,12 2,54 5,10 10,26 2,19 0,89

98 38,14 8,99 1,91 3,25 6,81 12,35 2,49 1,04

99 38,22 8,18 2,06 2,22 6,41 8,40 0,00 0,67

100 38,35 8,12 1,77 3,13 8,57 10,77 2,07 0,78

101 38,37 7,86 1,58 3,05 9,25 11,41 2,76 0,83

102 38,48 8,25 2,09 1,59 5,87 10,32 2,96 0,87

103 38,66 8,59 1,64 0,53 7,54 8,64 2,71 0,62

104 38,79 8,90 1,48 0,71 8,02 11,47 2,56 0,59

105 38,97 8,85 1,26 1,05 8,06 12,30 3,30 0,61

106 38,99 7,88 1,44 1,17 8,04 19,69 6,26 0,78

107 39,00 5,91 0,80 1,82 5,63 16,97 6,81 1,01

108 39,16 6,04 0,82 0,79 6,82 15,46 7,32 0,91

109 39,31 6,16 0,73 1,18 8,62 18,20 4,46 1,05

110 39,31 6,13 0,57 1,26 5,83 13,72 2,79 0,88

203


111 39,56 6,17 0,67 0,00 6,84 15,05 4,62 0,68

112 39,56 5,86 0,63 0,79 5,32 15,72 10,84 0,81

113 39,56 5,83 0,55 1,80 6,32 23,90 16,89 0,61

114 39,56 5,81 0,51 1,73 4,65 25,29 16,92 0,99

115 39,80 5,80 0,57 3,00 6,38 27,85 13,86 1,14

116 39,86 5,80 0,51 1,92 4,71 27,21 13,39 1,46

117 39,86 5,78 0,57 2,44 3,99 28,80 13,44 1,91

118 40,11 5,80 0,46 1,06 4,20 24,82 12,71 2,11

119 40,19 5,88 0,34 0,93 3,59 24,62 12,36 2,82

120 40,27 6,22 0,38 1,03 4,11 28,37 11,58 2,83

121 40,27 6,05 0,25 0,56 4,15 28,85 10,33 3,60

122 40,42 6,06 0,28 0,66 2,99 29,78 9,67 4,03

123 40,42 5,99 0,35 0,44 3,49 28,75 8,60 3,99

124 40,47 6,06 0,32 0,63 2,83 30,59 8,31 4,44

125 40,53 6,26 0,30 0,51 3,20 31,46 8,22 4,34

126 40,56 6,30 0,37 0,61 3,12 30,21 6,89 4,08

127 40,81 6,42 0,29 0,93 2,26 29,13 6,84 4,03

128 40,81 6,32 0,27 0,52 2,64 29,42 6,87 4,08

129 40,81 6,30 0,27 0,53 1,52 30,31 4,61 2,99

130 40,81 6,64 0,25 0,50 2,31 27,74 5,91 4,05

131 40,81 6,64 0,22 0,42 2,45 29,71 5,91 4,39

204


Cooling by Rb. capsulatus hup- on Molasses DFE (30.07.09)


continuous photobioreactor (4 L) with cooling by Rb. capsulatus hup- on the


Day Cumulative

H2(L) pH OD

LA

(mM)

FA

(mM)

AA

(mM)

PA

(mM)

BA

(mM)

1 0,00 7,24 0,34 0,00 1,78 28,47 1,33 0,56

2 0,00 7,02 0,43 3,01 2,55 28,66 0,00 0,29

3 0,00 6,83 0,58 2,73 5,66 22,79 0,00 0,66

4 0,00 6,86 0,59 1,05 2,57 26,23 0,00 1,99

5 0,00 6,88 0,54 0,85 2,46 23,36 0,00 2,38

6 0,08 6,99 0,56 0,75 2,69 21,75 0,00 2,28

7 0,12 6,88 0,61 0,85 2,76 19,75 1,59 3,45

8 0,17 7,06 0,80 0,52 2,18 21,07 0,43 3,03

9 0,17 7,30 0,85 0,54 3,72 17,45 0,73 3,94

10 0,92 7,35 0,95 0,47 4,09 20,47 1,88 2,82

11 1,43 7,33 1,22 0,85 5,76 20,79 0,09 4,26

12 1,68 7,41 1,37 0,74 6,42 17,99 0,64 3,46

13 2,43 7,14 1,22 0,90 7,07 19,11 0,87 3,25

14 3,27 7,04 1,32 0,00 5,19 20,17 0,48 1,62

15 4,45 7,16 1,47 0,00 8,67 15,76 1,08 1,88

16 5,79 7,11 1,47 0,00 10,45 17,87 1,87 2,32

17 7,13 7,28 1,59 0,00 12,40 14,00 1,79 1,72

18 9,32 7,18 1,83 0,00 13,36 9,64 1,03 1,45

19 11,16 7,16 1,73 0,00 16,97 10,40 1,09 1,30

20 13,01 7,07 1,94 0,00 19,12 8,94 0,91 1,39

21 14,86 7,14 1,91 0,00 21,61 9,82 1,06 1,27

22 17,04 7,26 1,86 0,00 21,41 7,38 0,91 1,13

23 18,72 7,73 1,96 0,00 28,04 7,84 0,62 1,43

205


24 20,57 7,13 1,86 0,00 25,20 4,04 0,42 0,96

25 22,33 7,07 1,76 0,00 28,10 2,69 0,26 0,60

26 24,01 6,91 1,80 0,00 23,41 3,43 0,10 0,74

27 25,69 6,96 1,69 0,00 27,22 3,71 0,51 0,65

28 27,28 6,93 1,79 0,00 29,25 3,62 0,48 0,85

29 28,71 7,07 1,84 0,00 27,22 3,32 0,44 0,51

30 30,13 7,03 1,78 0,00 24,74 2,88 0,00 0,47

31 31,48 7,58 1,87 0,00 22,52 2,59 0,36 0,45

32 32,82 7,05 1,81 0,00 21,95 2,65 0,31 0,34

33 34,25 7,10 1,77 0,00 22,75 2,53 0,36 0,49

34 35,09 7,41 1,90 0,00 23,20 3,65 0,39 0,30

35 36,18 7,01 1,76 0,00 20,42 3,44 0,47 0,26

36 37,35 6,67 1,73 0,00 25,29 4,57 0,55 0,44

37 38,53 7,19 1,82 0,00 22,06 3,46 0,37 0,33

38 39,62 6,99 2,07 0,00 22,85 3,28 0,57 0,00

39 40,63 6,93 1,85 0,00 24,18 4,29 0,31 0,37

40 41,38 6,88 1,75 0,00 21,45 4,10 0,60 0,32

41 42,31 7,00 1,76 0,00 22,56 5,23 0,71 0,48

42 42,98 7,20 1,72 0,00 21,96 4,65 0,59 0,45

43 43,65 7,03 1,82 0,00 22,19 3,03 0,54 0,36

44 44,57 7,33 1,64 0,00 21,27 5,53 0,74 0,42

45 45,24 7,07 1,79 0,00 17,93 4,90 0,80 0,37

46 45,66 7,25 1,78 0,00 21,75 7,03 1,23 0,80

47 46,08 7,07 1,69 0,00 20,86 6,92 1,55 0,55

48 46,84 7,32 1,83 0,00 19,62 5,90 1,24 0,81

49 47,34 7,06 1,92 0,00 20,15 7,03 0,86 0,67

50 48,10 7,05 1,88 0,00 22,34 8,48 1,17 0,67

51 48,85 7,46 1,85 0,00 20,16 5,75 1,14 0,77

52 49,86 7,48 1,90 0,00 23,55 6,92 2,41 0,69

206


53 50,36 7,39 1,87 0,00 19,55 4,14 1,30 0,79

54 50,62 7,38 1,74 0,00 20,79 8,01 1,74 0,67

55 51,20 6,96 1,76 0,00 19,54 7,56 1,87 0,64

56 51,79 7,37 1,72 0,00 16,32 5,60 1,21 0,64

57 52,46 7,35 1,66 0,00 17,68 8,33 2,31 0,54

58 53,22 7,34 2,05 0,00 19,18 6,37 1,96 0,81

59 53,72 7,70 1,87 0,00 18,54 7,58 1,08 0,65

60 53,89 7,25 1,86 0,07 17,28 8,35 2,01 0,70

61 54,31 7,33 1,88 0,00 18,51 8,24 1,24 0,81

62 54,73 7,63 1,36 0,00 17,97 8,69 1,92 0,75

63 55,06 7,26 1,91 0,00 17,82 9,92 1,91 0,83

64 55,32 7,40 1,80 0,00 17,84 9,50 1,33 0,56

65 55,65 7,45 1,97 0,00 18,75 10,32 1,91 0,81

66 56,07 7,47 1,97 0,29 16,00 9,72 1,50 0,77

67 56,32 7,66 1,84 0,00 16,05 9,83 1,72 0,69

68 56,66 7,49 1,88 0,00 15,01 10,07 2,28 0,79

69 56,99 7,44 1,92 0,00 17,03 12,97 2,20 0,61

70 57,25 7,54 1,98 0,00 14,59 11,85 2,99 1,09

71 57,50 7,56 1,93 0,73 18,96 16,58 4,36 0,87

72 57,83 7,72 1,94 0,00 18,14 15,96 3,84 0,89

73 58,09 7,30 2,06 0,00 15,85 10,94 2,24 0,94

74 58,34 7,87 2,14 0,00 16,14 11,84 1,50 1,13

75 58,59 7,86 2,06 0,00 16,21 10,77 1,94 0,79

76 58,96 7,68 2,02 0,00 14,94 12,18 2,33 0,80

77 59,38 7,80 2,09 0,00 15,44 12,66 2,21 0,75

78 59,46 7,47 1,89 0,00 15,62 3,23 1,85 0,78

79 59,73 7,94 1,98 0,00 13,91 9,36 1,86 0,71

80 60,05 7,45 2,17 0,00 16,45 13,32 2,23 0,64

81 60,43 7,90 2,12 0,00 16,24 13,32 2,32 0,80

207


82 60,55 7,81 2,23 0,00 16,94 14,29 2,85 0,80

83 60,81 8,24 1,93 0,00 16,58 13,02 2,05 0,63

84 61,23 7,77 2,14 0,00 12,17 10,32 3,07 0,69

85 61,65 7,87 2,05 0,00 15,85 14,63 3,18 0,70

86 61,88 7,99 2,14 0,00 15,01 11,68 2,71 0,80

87 62,26 8,91 1,94 0,00 17,19 13,92 3,17 0,72

88 62,68 7,80 2,12 0,00 14,90 16,87 3,97 1,03

89 63,18 8,52 2,29 0,00 16,93 16,19 3,49 0,76

90 63,47 8,29 2,21 0,00 14,93 14,20 2,87 0,61

91 63,73 7,70 2,13 0,00 14,20 12,18 2,96 0,68

92 63,94 7,58 2,22 0,00 17,79 19,48 4,98 0,83

93 64,02 7,44 2,15 0,00 18,08 19,01 3,19 0,62

94 64,36 8,04 2,32 0,00 1,55 15,85 2,94 0,81

95 64,52 7,64 1,99 0,00 16,71 15,99 3,16 0,73

96 64,86 7,91 1,79 0,67 13,13 13,33 3,87 0,69

97 64,94 7,84 1,88 0,00 15,76 18,91 3,77 0,66

98 65,01 7,70 1,60 0,00 17,60 18,10 1,84 1,05

99 65,01 7,62 1,68 0,00 18,97 17,68 3,38 0,96

100 65,11 7,54 1,70 0,00 16,21 20,88 6,55 0,72

101 65,30 7,96 1,90 0,00 14,11 14,01 2,30 0,72

102 65,51 7,87 1,88 0,00 16,76 18,17 2,20 0,69

103 65,71 7,87 1,79 0,00 16,44 15,90 2,89 0,53

104 65,92 7,81 1,78 0,84 11,53 13,06 3,07 0,64

105 66,17 8,13 1,67 0,00 16,06 19,40 3,63 0,73

106 66,21 8,06 1,85 0,00 19,42 22,48 3,26 0,91

107 66,21 7,86 1,78 0,00 19,33 24,93 4,03 0,73

108 66,34 7,85 1,82 0,00 16,54 20,98 2,85 0,96

109 66,34 8,03 1,87 0,00 17,65 19,76 3,07 0,85

110 66,34 7,46 1,81 0,00 15,12 18,81 3,61 0,62

208


111 66,34 7,40 1,99 0,00 19,76 21,87 3,62 1,35

112 66,38 8,47 1,69 0,00 20,03 25,85 4,77 0,89

113 66,48 6,81 1,73 0,00 19,32 25,35 4,21 1,00

114 66,48 6,77 1,61 0,00 18,03 19,61 3,92 1,00

115 66,48 6,58 1,74 0,00 18,11 22,61 4,40 0,81

116 66,82 8,40 1,33 1,00 14,83 18,00 3,76 0,74

117 66,95 6,20 1,25 0,00 17,01 22,87 3,85 0,73

118 66,95 6,28 1,16 0,00 16,15 21,55 2,57 0,79

119 66,95 6,24 1,09 0,00 15,13 17,33 2,52 0,79

120 66,95 6,25 1,18 0,00 16,44 22,32 3,29 0,68

121 67,10 6,50 1,12 0,00 15,63 17,12 2,64 0,75

122 67,12 6,29 1,66 0,00 19,92 26,49 5,02 0,86

123 67,12 6,36 0,79 0,00 17,55 24,10 4,06 0,87

124 67,12 6,17 0,84 0,00 17,10 19,08 3,71 0,96

125 67,12 7,34 1,40 0,00 17,15 23,18 4,63 0,76

126 67,30 7,52 1,65 0,00 13,53 14,40 2,73 0,62

127 67,50 7,36 1,97 0,00 18,15 20,49 4,26 0,89

128 67,50 7,32 1,74 0,00 18,04 26,84 4,70 0,75

129 67,67 7,18 1,54 0,00 12,22 13,65 2,08 0,61

130 67,67 6,77 1,57 0,00 11,03 15,13 2,26 0,42

131 67,67 6,60 1,55 0,00 15,78 17,95 3,01 0,70

132 67,67 6,57 1,37 0,74 14,28 22,74 4,46 0,43

133 67,72 6,36 1,71 0,00 14,77 22,70 3,44 0,59

134 67,72 6,35 1,44 0,00 16,33 20,13 4,40 0,71

135 67,72 6,23 1,45 0,00 12,57 19,33 4,63 0,43

136 67,89 6,24 1,35 1,22 15,34 19,02 2,95 1,12

137 67,97 6,33 1,17 0,00 16,54 20,76 5,06 0,37

138 68,06 6,61 1,31 0,00 16,06 19,92 3,99 0,62

139 68,14 6,25 1,19 0,00 16,08 21,72 3,91 0,70

209


140 68,39 6,17 1,04 0,00 12,49 21,14 3,73 0,20

141 68,39 6,33 1,13 0,00 14,21 20,16 3,33 0,49

142 68,43 6,25 0,99 0,00 13,22 16,97 7,82 0,26

143 68,43 6,42 1,00 0,56 9,36 16,74 6,28 0,36

144 68,46 6,36 1,06 1,49 8,23 20,69 8,54 0,35

145 68,63 6,43 1,20 0,41 9,52 22,92 10,25 0,29

146 68,68 6,34 0,87 0,00 8,89 19,85 4,07 0,34

147 68,68 6,19 0,71 0,00 9,45 19,94 3,73 0,21

148 68,68 6,46 0,68 0,00 10,36 21,80 3,02 0,21

149 68,68 6,45 0,77 0,00 9,54 20,82 3,42 0,42

SCALE UP OF PANEL PHOTOBIOREACTORS FOR HYDROGEN … · A THESIS SUBMITTED TO . THE GRADUATE SCHOOL...

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Transcript of SCALE UP OF PANEL PHOTOBIOREACTORS FOR HYDROGEN … · A THESIS SUBMITTED TO . THE GRADUATE SCHOOL...