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Bio-Electrochemical Studies for Harvesting Carbon

Dioxide to Organic Compounds

By

Gugan Jabeen

CIIT/FA11-PCHEM-001/LHR

PhD Thesis

In

Chemical Engineering

COMSATS Institute of Information Technology

Lahore Pakistan

Fall, 2016

ii

COMSATS Institute of Information Technology



A Thesis Presented to

COMSATS Institute of Information Technology, Lahore

In partial fulfillment

of the requirement for the degree of

PhD (Chemical Engineering) By

Gugan Jabeen


Fall, 2016

iii



A Post Graduate Thesis submitted to the Department of Chemical Engineering as

partial fulfillment of the requirement for the award of Degree of Ph.D in Chemical

Engineering.

Name Registration Number

Gugan Jabeen CIIT/FA11-PCHEM-001/LHR

Supervisor

Dr. Robina Farooq

Professor Department of Chemical Engineering

COMSATS Institute of Information Technology (CIIT)

Lahore Campus

iv

Certificate of Approval

This is to certify that the research work presented in this thesis, entitled “Bio

Electrochemical Studies for Harvesting Carbon Dioxide to Organic Compounds” was

conducted by Ms. Gugan Jabeen, CIIT/FA11-PCHEM-001/LHR, under the supervision of

Dr. Robina Farooq. No part of this thesis has been submitted anywhere else for any other

degree. This thesis is submitted to the department of Chemical Engineering, COMSATS

Institute of Information Technology Lahore, in the partial fulfillment of the requirement

for the degree of Doctor of Philosophy in the field of Chemical Engineering.

v

Author’s Declaration

I Gugan Jabeen CIIT/FA11-PCHEM-001/LHR hereby state that my PhD thesis titled “Bio-

Electrochemical Studies for Harvesting Carbon Dioxide to Organic Compounds” is my

own work and has not been submitted previously by me for taking any degree from this

University i.e. COMSATS Institute of Information Technology or anywhere else in the

country world.

At any time if my statement is found to be incorrect even after I graduate the University

has the right to withdraw my PhD Degree.

vi

Plagiarism Undertaking

I solemnly declare that the research work presented in the thesis titled “Bio-

Electrochemical Studies for Harvesting Carbon Dioxide to Organic Compounds” is solely

my research work with no significant contribution from any other person. Small

contribution help wherever taken has been duly acknowledged and that complete thesis has

been written by me.

I understand the zero tolerance policy of HEC and COMSATS Institute of Information

Technology towards plagiarism. Therefore, I as an author of the above titled thesis declare

that no portion of my thesis has been plagiarized and any material used as reference is

referred/cited.

I undertake if I am found guilty of any formal plagiarism in the above titled thesis even

after award of PhD Degree, the University reserves the right to withdraw/revoke my PhD

degree and that HEC and the University has the right to publish my name on

HEC/university website on which names of students are placed who submitted plagiarized

thesis.

vii

Certificate

It is certified that Gugan Jabeen CIIT/FA11-PCHEM-001/LHR has carried out all the work

related to this thesis under my supervision at the Department of Chemical Engineering

COMSATS Institute of Information Technology, Lahore and the work fulfills the

requirement for award of PhD degree.

.

viii

DEDICATION

I dedicate my dissertation to my elder sister Dr. Irfana Sheikh who has never left my side.

I also dedicate this dissertation to my elder brother M. Azam Sheikh who has supported

me throughout the PhD process.

ix

ACKNOWLEDGEMENTS

All praise for ALMIGHTY ALLAH Who is The Lord of the universe and very merciful.

Who guides us from darkness to light.

All and every respect is for Holy Prophet Muhammad (PBUH) enabled us to recognize our

Creator.

It has been a great experience and honor to have carried out this project at The University

of COMSATS Institute of Information Technology Lahore.

I would like to express my deep and sincere gratitude to my Research Supervisor, Prof. Dr.

Robina Farooq for her keen interest, expertise, skilled advices and for all her inspirational

mentorship in accomplishing this work.

I am thankful to Prof. Dr. Asad Ullah Khan Head of Chemical Engineering Department for

his kind and sympathetic behavior and providing me all the facilities to complete this

research work.

I offer my special thanks to Dr. Mazhar Gillani and Fawad Ashraf for his cooperation in

the Analytical lab for the use of equipment.

My special thanks to IRCBM department for their assistance in SEM and FT-IR analysis.

I extend my thanks to Lab Assistant Asif Rana who arranged and provided all necessary

chemicals and equipment for the completion of my research work.

My family deserves equal accolade. Their unwavering encouragement and reassurance that

they provided me has been integral in my completion of this degree.

Gugan Jabeen


x

Abstract

Bio-Electrochemical Studies for Harvesting Carbon Dioxide to

Organic Compounds

Powering microbes with electricity in bio-electrochemical synthesis (BES) to produce

plethora of organics like volatile fatty acids and alcohols is an attractive bio sustainable

strategy to minimize our dependence on fossil fuels. Bio-electrochemical synthesis, a

beneficial key technique in which electro autotrophic bacteria utilize electric current as a

sole electron source from cathode to reduce CO2 to extracellular multicarbon exquisite

products through metabolic conversion. The anaerobic and autotrophic acetogens like

Sporomusa ovata, Clostridium ljungdahlii and Cupriavidus necator have been focused in

current study to convert waste greenhouse gas like CO2 into volatile fatty acids, alcohols

and Polyhydroxyalkanoates (PHA). These electroactive catalysts were able to capture

electron directly from cathode without any mediator due to the presence of C-type

cytochrome and type IV pili in Sporomusa ovata, RnF complexes for electron and proton

translocation in Clostridium ljungdahlii and Fln type adhesion on the surface of

Cupriavidus necator. The bio-electrochemical reactor was simplified to avoid time

expenditure by eliminating potentiostat and by improving start-up process of autotrophic

biocathode. The two stage strategy was integrated in this system based on heterotrophic

pre-enrichment of electro autotrophic biocatalysts on glucose or fructose, afterwards

acclimation of pre-enriched culture to BES reactor where CO2 was sole carbon source to

switch bacteria from heterotrophic to autotrophic metabolism. The biocathode was poised

at -0.4V, (versus Ag/AgCl electrode) high enough to avoid hydrogen production by DC

power source other than potentiostat to retain the high coulombic recovery in electro fuel

production. The development of pure microbial biofilm at cathode rather than mixed

culture further simplified the BES reactor from complex metabolic activities. The BES

technique was practiced first time to synthesize high quality Polyhydroxyalkanoates (while

fermentation process was followed before) merely from cheap and low cost substrates like

molasses and waste greenhouse gas CO2. Implementation of simplified reactor and specific

strategy for both batch and continuous system under ambient conditions of pH, temperature

and pressure enhanced the electroactivity of cells to transform the electrons to spectrum of

xi

extracellular products in less time duration. The net outcome was the renewable energy

which was stored in covalent bonds synthesized from waste greenhouse gas. The specific

products included acetate, butyrate, ethanol, hexanoic acid, hexanol, heptanoic acid and

heptanol by Clostridium ljungdahlii and Sporomusa ovata. The formation of heptanoic acid

and heptanol in this specific technique never reported earlier. This feature has potential to

make these electrotrophs beneficial for biotech industry. The intracellular

Polyhydroxyalkanoates accumulated as granules inside the cells are environment friendly

thermoplastics. The novel extraction techniques for the recovery of granules of carbon and

energy reserves from cell dry mass has made PHA more attractive. Gas chromatography

and mass spectrometry (GC-MS) analysis employed for identification and quantification

of electro fuels and further electroactivity was characterized by cyclic voltammetry. The

ANOVA test performed for statistical analysis between the batch and continuous system

for all three strains. ANOVA test for Sporomusa ovata proved the significant difference

between batch and continuous system rather than other two strains. The overall coulombic

recovery was more than 90% due to the redox electroactivity of these autotrophs. The

concentrations for ethanoic acid, ethanol, ethyl butyrate, hexanoic acid, heptanoic acid,

hexanol and heptanol were 2.99, 3.19, 2.2, 2.18, 2.01, 2.11 and 0.85mM at 120 hours of

medium cultivation. The Coulombic recovery was more than 80% proved the BES a

promising and remarkable technology than other chemical and photosynthetic based

chemical production.

xii

TABLE OF CONTENTS

1 Introduction ............................................................................................. 2

1.1 Significance of study ....................................................................... 7

1.2 Objectives of the study .................................................................... 7

2 Literature Review .................................................................................... 9

2.1 Autotrophic bacteria ........................................................................ 9

2.2 Microorganisms for Biosynthesis of Organic Compounds ........... 11

2.3 Substrates for Biosynthesis ............................................................ 14

2.4 Anaerobic digestion for biosynthesis............................................. 18

2.5 Anaerobic bio-reactors ................................................................... 20

2.6 Metabolic engineering to enhance product yield ........................... 21

2.7 Synthesis of organics by greenhouse gases ................................... 24

2.8 Role of Metabolic Pathways in biochemical synthesis ................. 26

2.9 Bio-electrochemical synthesis ....................................................... 28

2.10 Extracellular electron transfer (EET) ............................................. 31

2.10.1 Electron transport by fermentation products ...................................... 31

2.10.2 Electron transport by mediator ........................................................... 32

2.10.3 Direct transfer of electron .................................................................. 34

2.11 Electron transfer from electrode to microorganisms ..................... 36

3 Materials and methods ..........................................................................42

3.1 Experimental Setup ........................................................................ 42

3.1.1 Chemicals and Reagents .................................................................... 42

3.1.2 Chemicals for vitamin solution .......................................................... 42

3.1.3 Trace element solution ....................................................................... 42

xiii

3.1.4 Standards ............................................................................................ 43

3.1.5 Gasses ................................................................................................. 43

3.2 Equipment and Instruments ........................................................... 43

3.3 Bio-electrochemical reactor configuration .................................... 45

3.3.1 General setup ...................................................................................... 45

3.3.2 Electrodes ........................................................................................... 46

3.3.3 Proton exchange membrane ............................................................... 46

3.4 Coulombic Recovery ..................................................................... 48

3.5 ANOVA Test ................................................................................. 49

3.6 Cupriavidus necator ...................................................................... 50

3.6.1 Media composition ............................................................................. 50

3.6.2 Stock Solution for Trace Element ...................................................... 50

3.6.3 Carbon sources ................................................................................... 51

3.6.4 Culture growth conditions with CO2 and H2 ...................................... 51

3.6.5 Medium preparation with Molasses ................................................... 51

3.7 Reactor operation ........................................................................... 52

3.8 Cathode biofilm development ........................................................ 52

3.9 Experimental conditions of batch system for Cupriavidus necator

………………………………………………………………….54

3.10 Control cell for Cupriavidus necator ............................................. 55

3.11 Experimental conditions of continuous system for Cupriavidus

necator …………………………………………………………………..55

3.12 Cyclic voltammetry for Cupriavidus necator ................................ 56

3.13 FT-IR analysis ................................................................................ 56

3.14 Analytical Method ......................................................................... 56

3.14.1 The Biopolymer Extraction for GC-MS Analysis ............................. 56

3.15 The Biopolymer Analysis .............................................................. 57

3.15.1 SPME Procedure for Sample Preparation .......................................... 57

3.15.2 GC-MS procedure .............................................................................. 58

xiv

3.15.3 Extraction of dry PHA ....................................................................... 58

3.15.4 Calibration curve for extracted PHA ................................................. 59

3.16 Sporomusa ovata ............................................................................ 61

3.16.1 Media and cultivation method ............................................................ 61

3.16.2 Vitamin solution (medium 141) ......................................................... 61

3.17 Trace element solution SL-10 (medium 320) ................................ 61

3.18 Stock solutions preparation ............................................................ 62

3.18.1 NaHSeO3.10-7 M solution ................................................................... 62

3.18.2 Resazurin solution 500mg/1000ml .................................................... 62

3.18.3 NaHCO3 solution ................................................................................ 62

3.18.4 Lysozyme solution ............................................................................. 62

3.18.5 Phosphate buffer solution ................................................................... 63

3.18.6 Method for preparation of anaerobic medium ................................... 63

3.18.7 Medium preparation with CO2 and H2 for Sporomusa ovata ............. 64

3.18.8 Cathode biofilm development ............................................................ 64

3.18.9 Experimental conditions of batch system for Sporomusa ovata ........ 64

3.18.10 Continuous system for Sporomusa ovata........................................... 65

3.18.11 Control cell without electroactive catalysts ....................................... 65

3.18.12 Cyclic Voltammetry (CV) for Sporomusa ovata ............................... 65

3.18.13 Control Cell without electroactive catalysts ...................................... 66

3.18.14 Analytical Method for Sporomusa ovata ........................................... 66

3.18.15 Preparation of samples for GC-MS analysis ...................................... 66

3.18.16 SPME Procedure ................................................................................ 66

3.19 Calibration for volatile acids and alcohols .................................... 67

3.19.1 Calibration for volatile fatty acids ..................................................... 68

3.19.2 Calibration for volatile alcohols ......................................................... 69

3.20 Clostridium ljungdahlii .................................................................. 70

3.21 Medium and cultivation method for Clostridium ljungdahlii ....... 70

3.21.1 Vitamin solution (medium 141) ......................................................... 70

xv

3.21.2 NaHCO3 solution per 10.0ml ............................................................. 70

3.21.3 Fructose solution per 50ml. ................................................................ 71

3.21.4 Na2S.9H2O solution per 10.0ml. ........................................................ 71

3.21.5 Trace Element Solution per liter ........................................................ 71

3.22 Method for Preparation of anaerobic medium for C. ljungdahlii .. 71

3.22.1 Medium preparation with CO2 and H2 for Clostridium ljungdahlii ... 72

3.23 Cathode biofilm development ........................................................ 73

3.24 Experimental conditions for batch system for Clostridium

ljungdahlii ...................................................................................................73

3.25 Continuous system for Clostridium ljungdahlii ............................ 74

3.25.1 Control cell without electroactive catalysts ....................................... 74

3.26 Cyclic Voltammetry (CV) for Clostridium ljungdahlii ................. 74

3.26.1 Cyclic voltammetry of Control Cell without electroactive catalysts . 75

3.27 Analytical Method for volatile fatty acids and alcohols ................ 75

4 Results and discussion ...........................................................................77

4.1 Cupriavidus necator ...................................................................... 77

4.2 Gas Chromatography Mass Spectrometry analysis ....................... 79

4.3 Bio-electrochemical synthesis of PHA during batch system ........ 80

4.4 Bio-electrochemical synthesis of PHA during continuous system 81

4.4.1 Decrease in Acetic acid concentration during batch and continuous

system ………………………………………………………………………82

4.5 Coulombic Recovery ..................................................................... 85

4.6 Electrons recovery against the current consumption ..................... 86

4.6.1 Electron recovery during batch system .............................................. 86

4.6.2 Percent cathode recovery during batch system .................................. 87

4.6.3 Electron recovery during continuous system ..................................... 87

4.6.4 Percent cathode recovery during continuous system ......................... 88

4.7 Gas Chromatograms and Mass Spectrums for Cupriavidus necator

………………………………………………………………….. 89

xvi

4.8 Characterization of PHA in the presence of nitrogen supply ........ 91

4.9 Cyclic voltammetry results for Cupriavidus necator .................... 92

4.9.1 Cyclic Voltammetry analysis for Control Cell without electroactive

catalysts ............................................................................................................ 93

4.10 FT-IR Analysis .............................................................................. 94

4.11 Sporomusa ovata ............................................................................ 97

4.12 Characterization of Bio electrochemically synthesized organic

compounds ..................................................................................................98

4.13 Characterization of organic products synthesized by Sporomusa

ovata …………………………………………………………………..99

4.14 Characterization of organic compounds in Control cell .............. 100

4.14.1 Bio-electrochemical synthesis of organic acids during Batch system

……………………………………………………………………..101

4.14.2 Bio-electrochemical synthesis of alcohols during batch system...... 103

4.14.3 Bio-electrochemical synthesis of organic acids during continuous

system ……………………………………………………………………..104

4.14.4 Bio-electrochemical synthesis of alcohols during continuous system

……………………………………………………………………..105

4.15 Cyclic voltammetry (CV) analysis of biofilm ............................. 109

4.15.1 Cyclic voltammetry analysis for Control cell without electroactive

catalysts .......................................................................................................... 110

4.16 Electrons recovery against the current consumption ................... 111

4.16.1 Electron recovery during batch system ............................................ 112

4.16.2 Percent cathode recovery during batch system ................................ 113

4.16.3 Electron recovery during continuous system ................................... 114

4.16.4 Percent cathode Recovery during continuous system ..................... 115

4.17 Clostridium ljungdahlii ................................................................ 118

4.18 Characterization of Bio-electrochemically synthesized organic

compounds ................................................................................................ 119

xvii

4.19 Gas Chromatogram and Mass Spectrum for Clostridium ljungdahlii

………………………………………………………………….120

4.20 GC-MS Results for control cell without electro-autotrophs ........ 122

4.21 Bio-electrochemical synthesis of organic acids during batch system

………………………………………………………………….122

4.22 Bio-electrochemical synthesis of alcohols during batch system . 124

4.23 Bio-electrochemical synthesis of organic acids in continuous system

………………………………………………………………….124

4.24 Bio-electrochemical synthesis of Alcohols during continuous

system ………………………………………………………………….126

4.25 Cyclic voltammetry ...................................................................... 129

4.26 Electron recovery against the current consumption .................... 132

4.26.1 Electron recovery during batch system ............................................ 133

4.26.2 Percent cathode recovery during batch system ................................ 134

4.26.3 Electron recovery during continuous system ................................... 134

4.26.4 Percent cathode recovery in continuous system .............................. 136

4.27 Conclusions .................................................................................. 139

4.28 Highlights ..................................................................................... 143

4.29 Future perspective and challenges ............................................... 144

5 References .........................................................................................146

6 List of Publications ..............................................................................162

7 Appendix I ............................................................................................163

xviii

LIST OF FIGURES

Figure 2.1 Acetyl Co-A pathway for Polyhydroxyalkanoates by Cupriavidus necator ..11

Figure 2.2 Role of CO in the formation of acetyl-CoA ...................................................28

Figure 2.3 Electron transfer mechanism by Cytochrome and mediator where Cytochrome

directly transfer electron from cathode to cells and mediators in oxidized form

capture electrons from cathode and transfer to cells ......................................34

Figure 2.4 4 Mtr respiratory pathway and schematic electron flow from inner membrane

proteins CymA to outer membrane proteins OmcA and MtrC and then to

electrode ……………………………………………………………………36

Figure 2.5 Transfer of electron from cathode to electroactive cells for fumarate, nitrate

and carbon dioxide reduction to succinate, nitrite and acetate respectively ..37

Figure 2.6 Bio-electrochemical cell with bio-cathode and power supply for the synthesis

of bio-fuels .....................................................................................................39

Figure 3.1 Bio-electrochemical reactor......................................................................47

Figure 3.2 Relationship between cell dry weight concentration and absorbance at 620nm

…………………………………………………………………………..53

Figure 3.3 The optical density measured at 620nm exhibits the decrease in absorbance

per day …………………………………………………………………....54

Figure 3.4 Wet biomass on pre-weighed watch glass ....................................................59

Figure 3.5 White precipitate of extracted PHA with ethanol ..........................................59

Figure 3.6 Calibration curve between concentration and peak area for polyhydroxy

alkanoates .......................................................................................................60

Figure 3.7 Calibration curve between concentration and peak area for volatile fatty acid

mixture ……………………………………………………………………68

Figure 3.8 Calibration curve between concentration and peak area for alcohols ............69

Figure 4.1 Bio-electrochemical synthesis of 3-hydroxy dimethyl butyrate, ethyl 3-hydroxy

hexanoate, 2, 4 dimethyl heptanoate concentration in ppm against time

duration for batch system ...............................................................................81

xix


hexanoate, 2, 4 dimethyl heptanoate concentration in ppm against time for

continuous system ..........................................................................................82

Figure 4.3 Comparison in Acetic acid concentration in both batch and continuous system

…………………………………………………………………………..83

Figure 4.4 Acetyl Co-A pathway for Polyhydroxyalkanoates (Estelle et al., 2014 and

Razaad et al., 2014) ........................................................................................85

Figure 4.5 Total current consumed by Cupriavidus necator and current recovery in

polymers during batch system .......................................................................86

Figure 4.6 Percent cathode recovery for organic polymers by Cupriavidus necator during

batch system ...................................................................................................87


polymers in continuous system ......................................................................88

Figure 4.8 Percent cathode recovery for organic polymers by Cupriavidus necator in

continuous system ..........................................................................................88

Figure 4.9 Gas Chromatogram of PHA polymer ...........................................................90

Figure 4.10 Mass spectrum of poly hydroxyalkanoates ................................................90

Figure 4.11 Mass spectrum of acetic acid ...................................................................90

Figure 4.12 Gas chromatogram under nitrogen supply ..................................................91

Figure 4.13 Mass spectrum with nitrogen supply .......................................................91

Figure 4.14 Cyclic Voltammetry of Cupriavidus necator for biotic, abiotic cathode and

fresh medium against Ag/AgCl reference electrode at scan rate of 10mV/s .93

Figure 4.15 Scanning electronic micrographs of rod shaped PHB rich Cupriavidus

necator ………………………………………………………………........94

Figure 4.16 FT-IR spectroscopy of PHB biopolymer by Cupriavidus necator ...............95

Figure 4.17 Gas Chromatogram of various acids and alcohols from Sporomusa ovata...99

Figure 4.18 Mass spectrum of ethanoic acid ................................................................99

Figure 4.19 Mass spectrum of Butanoic acid...............................................................100

Figure 4.20 Mass spectrum of 2-ethylbutyric acid ........................................................100

Figure 4.21 Gas Chromatogram without Sporomusa ovata ..........................................101

Figure 4.22 Mass spectrum of medium without Sporomusa ovata ..............................101

xx

Figure 4.23 Bio-electrochemical synthesis of ethanoic acid, butanoic acid and 2-ethyl

butyrate by Sporomusa ovata in batch system ............................................102

Figure 4.24 Bio-electrochemical synthesis of ethanol, Pentanol and hexanol

concentration in mM by Sporomusa ovata in batch system ........................103


butyrate concentration in mM by Sporomusa ovata in continuous system ..104

Figure 4.26 Bio-electrochemical synthesis of ethanol, pentanol and hexanol in

continuous system by Sporomusa ovata ......................................................105

Figure 4.27 Schematic diagram of Acetyl Co-A pathway for Acetoacetyl and Butyryl-Co-

A …………………………………………………………………………108

Figure 4.28 Cyclic Voltammetry for Sporomusa ovata for biotic, abiotic and fresh medium

against standard Ag/AgCl reference electrode at scan rate of 10mV/s ........109

Figure 4.29 Scanning electron micrographs of electrosynthetic cathode biofilms for

Sporomusa ovata ..........................................................................................111

Figure 4.30 Scanninng electron micrograph of abiotic cathode without electroactive

catalyst …………………………………………………………………..111

Figure 4.31 Current recovery in organic acids and alcohols by Sporomusa ovata in batch

system against the total electrons consumed by biofilm ..............................112

Figure 4.32 Percent Cathode Recovery for organic acids and alcohols by Sporomusa

ovata in batch system ...................................................................................113

Figure 4.33 Current recovery in organic acids and alcohols by Sporomusa ovata in

continuous system against total current consumed by biofilm .....................114

Figure 4.34 Percent cathode recovery for organic acids and alcohols by Sporomusa ovata

in continuous system ....................................................................................115

Figure 4.35 Gas chromatogram for Clostridium ljungdahlii ......................................120

Figure 4.36 Mass spectrum for ethanoic acid ............................................................120

Figure 4.37 Mass spectrum for ethyl butyrate ..............................................................121

Figure 4.38 Mass spectrum for heptanoic acid ............................................................121

Figure 4.39 Mass spectrum for heptanol.....................................................................121

Figure 4.40 Control cell without electro-autotrophs ...................................................122

Figure 4.41 Mass spectrum of Clostridium ljungdahlii without electro-autotrophs .....122

xxi

Figure 4.42 Bio-electrochemical synthesis of volatile fatty acids against time duration of

Clostridium ljungdahlii for batch system .....................................................123

Figure 4.43 Bio-electrochemical synthesis of alcohols against time duration of

Clostridium ljungdahlii for batch system .....................................................124

Figure 4.44 Bio-electrochemical synthesis of volatile fatty acids against time duration

of Clostridium ljungdahlii for continuous system ........................................125

Figure 4.45 Bio-electrochemical synthesis of ethanol, hexanol and heptanol against time

duration for continuous system by Clostridium ljungdahlii .........................126

Figure 4.46 Overview of Wood–Ljungdahl metabolic pathways involved in the synthesis

of various metabolites phases (Ramio et al., 2015). .....................................129

Figure 4.47 Cyclic Voltammetry of Clostridium ljungdahlii for biotic, abiotic cathode

and fresh medium against Ag/AgCl reference electrode at scan rate of 10mV/s

…………………………………………………………………………130

Figure 4.48 Scanning electron micrographs of electrosynthetic cathode biofilms after 24

and 72 hours .................................................................................................131

Figure 4.49 Scanning electron micrograph of abiotic cathode without electroactive

cells ……………………………………………………………………….132

Figure 4.50 Current recovery in organic acids and alcohols by Clostridium ljungdahlii

in batch system against the total electrons consumed by biofilm ................133

Figure 4.51 Percent cathode recovery for organic acids and alcohols by Clostridium

ljungdahlii in batch system ...........................................................................134

Figure 4.52 Current recovery in organic acids and alcohols by Clostridium ljungdahlii in

continuous system against the total electrons consumed by biofilm ............135

Figure 4.53 Percent Cathode Recovery for organic acids and alcohols by Clostridium

ljungdahlii in continuous system ..................................................................136

xxii

LIST OF TABLES

Table 3.1 Chemical composition of molasses....................................................................51

Table 4.1 Synthesis of PHA from dry cell mass after 48 hours .......................................79

Table 4.2 GC-MS analysis of biodegradable polymer and their chemical composition

(RT; retention time in minutes, CN; compound name, MW; molecular weight,

MF; molecular formula). ................................................................................80

Table 4.3 Comparison (ANOVA) of continuous and batch process for Cupriavidus

necator 96

Table 4.4 Determination of Sum of Squares, df, Mean Square, F and p values for

continuous and batch process for harvesting waste (molasses and carbon

dioxide) to biopolymer (PHB) using Cupriavidus necator ...........................96

Table 4.5 Chemical composition of volatile fatty acids and alcohols revealed by GC-MS

analysis 98

Table 4.6 Comparison (ANOVA) of continuous and batch process for Sporomusa ovata .

…..................................................................................................................116


continuous and batch process for Sporomusa ovata ....................................116

Table 4.8 Chemical composition of volatile fatty acids and alcohols revealed by GCMS

analysis (RT; retention time in minutes, CN; compound name, MW; molecular

weight, MF; molecular formula, DF; displayed formula). ...........................119

Table 4.9 Comparison (ANOVA) of continuous and batch process for Clostridium

ljungdahlii ....................................................................................................137


continuous and batch process for Clostridium ljungdahlii ...........................138

Table 7.1 Cell dry weight concentration and absorbance ...............................................164

Table 7.2 Optical density between days and absorbance ..............................................164

Table 7.3 Calibration between concentration and standard peak area for polymers .....165

Table 7.4 Standard concentration of standard volatile acid mixtures in mM and peak

area from GC-MS analysis ...........................................................................165

xxiii

Table 7.5 Standard concentration of alcohols in mM and peak area from GC-MS analysis

……………………………………………………………………………..166

Table 7.6 3-hydroxy dimethyl butyrate concentration of Cupriavidus necator for batch

system…………………………………………………………………… 166

Table 7.7 Ethyl 3-hydroxy hexanoate concentration of Cupriavidus necator for batch

system……………………………………………………………………. 167

Table 7.8 2, 4 dimethyl heptanoate concentration of Cupriavidus necator for batch system

……...167

Table 7.9 3-hydroxy dimethyl butyrate concentration of Cupriavidus necator for

continuous system ........................................................................................167

Table 7.10 Ethyl 3-hydroxy hexanoate concentration of Cupriavidus necator for


Table 7.11 2, 4 dimethyl heptanoate concentration of Cupriavidus necator for continuous

system …………………………………………………………………..168

Table 7.12 Acetic acid concentration of Cupriavidus necator for batch system ..........168

Table 7.13 Acetic acid concentration of Cupriavidus necator for continuous system

…..................................................................................................................169

Table 7.14 Total current consumed by Cupriavidus necator and current recovery in

polymers in batch system .............................................................................169

Table 7.15 Percent Cathode Recovery for organic polymers by Cupriavidus necator in

batch system .................................................................................................170

Table 7.16 Total current consumed by Cupriavidus necator and current recovery in

polymers in continuous system ....................................................................170

Table 7.17 Percent Cathode Recovery for organic polymers by Cupriavidus necator in


Table 7.18 Ethanoic acid concentration of Sporomusa ovata for batch system .........171

Table 7.19 Butanoic acid concentration of Sporomusa ovata for batch system .........171

Table 7.20 2-ethyl butyrate concentration of Sporomusa ovata for batch system ......172

Table 7.21 Ethanol concentration of Sporomusa ovata for batch system ..................172

Table 7.22 Pentanol concentration of Sporomusa ovata for batch system ...................172

Table 7.23 Hexanol concentration of Sporomusa ovata for batch system ...................173

xxiv

Table 7.24 Ethanoic acid concentration of Sporomusa ovata for continuous system 173

Table 7.25 Butanoic acid concentration of Sporomusa ovata for continuous system .173

Table 7.26 2-ethyl butyrate concentration of Sporomusa ovata for continuous system ....

……………………………………………………………………………..174

Table 7.27 Ethanol concentration of Sporomusa ovata for continuous system ...........174

Table 7.28 Pentanol concentration of Sporomusa ovata for continuous system ...........174

Table 7.29 Hexanol concentration of Sporomusa ovata for continuous system..........175

Table 7.30 Total current consumed by Sporomusa ovata and current recovery in organic

acids and alcohols in batch system ...............................................................175

Table 7.31 Percent cathode recovery of organic products of Sporomusa ovata vs. time

duration in batch system ...............................................................................176

Table 7.32 Total current consumed by biofilm and electrons recovered in organic

compounds in continuous system by Sporomusa ovata ...............................176

Table 7.33 Percent cathode recovery of organic products of Sporomusa ovata vs. time

duration in continuous system ......................................................................177

Table 7.34 Ethanoic acid concentration for batch system by Clostridium ljungdahlii .177

Table 7.35 Ethyl butyrate concentration for batch system by Clostridium ljungdahlii .178

Table 7.36 Hexanoic acid concentration for batch system by Clostridium ljungdahlii.178

Table 7.37 Heptanoic acid concentration for batch system by Clostridium ljungdahlii 178

Table 7.38 Ethanol concentration for batch system by Clostridium ljungdahlii .........179

Table 7.39 Hexanol concentration for batch system by Clostridium ljungdahlii .........179

Table 7.40 Heptanol concentration for batch system by Clostridium ljungdahlii ........179

Table 7.41 Ethanoic acid concentration for continuous system by Clostridium ljungdahlii

…………………………………………………………………………180

Table 7.42 Ethyl butyrate concentration for continuous system by Clostridium ljungdahlii

180

Table 7.43 Hexanoic acid concentration for continuous system by Clostridium ljungdahlii

…………………………………………………………………………180

Table 7.44 Heptanoic acid concentration for continuous system by Clostridium

ljungdahlii ....................................................................................................181

Table 7.45 Ethanol concentration for continuous system by Clostridium ljungdahlii ..181

xxv

Table 7.46 Hexanol concentration for continuous system by Clostridium ljungdahlii .181

Table 7.47 Heptanol concentration for continuous system by Clostridium ljungdahlii .182

Table 7.48 Total current consumed by Clostridium ljungdahlii and recovered in volatile

fatty acids and alcohols in batch system ......................................................182

Table 7.49 Percent cathode recovery of organic products of Clostridium ljungdahlii vs.

time duration in batch system .......................................................................183

Table 7.50 Total current consumed by biofilm and electrons recovered in organic

compounds in continuous system by Clostridium ljungdahlii .....................183

Table 7.51 Percent cathode recovery of organic products of Clostridium ljungdahlii vs.

time duration in continuous system ..............................................................184

xxvi

List of acronyms

µM Micro meter

1,3PD propane Diol

ABE Acetone-butanol-ethanol

Acetyl-CoA Acetyl coenzyme A

ADH Alcohol dehydrogenase

ASBR Anaerobic Sequencing Batch Reactor

ATP Adenosine triphosphate

BES Bio-electrochemical synthesis

CV Cyclic voltammetry

DC Direct Current

DMAP Dimethyl allyl Pyrophosphate

DRB De oiled rice bran

EET Extracellular electron transfer

ET Electron Transfer

EU Endotoxin unit

FAD Flavins adenine dinucleotide

FT-IR Fourier Transform Infra-Red Spectroscopy

GC-MS Gas Chromatography Mass spectrometry

HS-SPME A Headspace Solid Phase Micro extraction

IPPP Isoprenyl Pyrophosphate

KDC 2-keto acid decarboxylase

m/z Mass per charge ratio

MEC Microbial electrolytic cell

MES Microbial electrochemical synthesis

MFC Microbial fuel cell

mM Millie mole

mV Millie volts

NADH Nicotinamide adenine dinucleotide

xxvii

NADPH Nicotinamide adenine dinucleotide phosphate

OD Optical Density

P3HB poly (3-hydroxybutyrate)

PEM Proton exchange membrane

PHA Polyhydroxyalkanoates

PTFE polytetrafluoroethylene

SEM Scanning electron microscopy

SS Stainless Steel

UASB Up flow anaerobic sludge blanket reactor

1

Chapter 1

Introduction

2

1 Introduction

Conversion of greenhouse gases to dense organic molecules is an attractive strategy.

Different processes to harvest greenhouse gas CO2 are electro-catalytic reduction (Shen et

al., 2015), enzymatic catalysis (Shi et al., 2015) and genetically modifications (Chirkov

2013). The electro-catalytically reduction of CO2 by using a range of different inorganic

and organometallic catalysts are to fix atmospheric carbon dioxide to low chain organic

compounds. The process is plagued with poor thermodynamic efficiency, low current

efficiency, low selectivity, slow kinetics, poor stability and high cost of metal catalysts.

The electrochemical fixing of CO2 is also inundated due to poisoning of electrodes, high

cost of electrodes, and use of hazardous solvents for concentrating CO2 and fouling of

electrodes with byproducts (Appel et al., 2013). Use of enzymes as catalyst in

electrochemical synthesis has limitations due to denaturing of enzymes. Therefore,

synthesis of renewable bio-chemicals is presently fueling the debate on the sustainable

synthesis of biofuels and bio-chemicals. The synthesis of biochemicals by microbial

electrochemical process offer many advantages over previous techniques. It can utilize low

cost living autotrophic biocatalysts in the form of acetogenic bacterial bio-film (Du, et al.,

2007). These autotrophic bacteria have ability to auto activate themselves. Therefore, bio-

electrochemical process in the presence of media or wastewater is a low cost reduction of

greenhouse gases to multicarbon organic compounds.

The autotrophic bacteria utilize carbon dioxide or carbon monoxide during the synthesis of

different organic compounds. For fatty acids and alcohols synthesis, Acetyl CoA pathway

is utilized for converting C1 carbon into multicarbon compounds. (Lovley 2011).

Microbial electrocatalysis denotes to direct catalysis of bio-electrode by electroactive

microorganisms. It totally relies on innate capability of microbes for electron transport. It

can be exploited in bio-electrochemical system for current generation or to provide

electricity to microorganisms for biofuels and biochemical synthesis. Bio-electrochemical

system consists of anode for oxidation and cathode for reduction. Exoelectrogens in

bioanode oxidize organic matter anaerobically and discharged electrons transferred

through electron transport chain to electrode. In biocathode electrotrophs capture electrons

from cathode for the reduction of CO2, sulfate or nitrates to multicarbon organic

3

compounds, the precursors of liquid transportation fuel (Sharma et al., 2014 and Fan et

al., 2012). Technical breakthrough has extended the bio-electrochemical synthesis of

volatile fatty acids and alcohols at low cost as compared to other electrochemical processes

(Rabaey et al., 2010).

The enrichment of biocathode for autotrophic electrotrophs for biofuel and polymer

synthesis provides a simplified method to isolate biochemicals from different inoculum

sources. Bacteria were heterotrophically grown first on glucose, fructose or glycerol and

after pre-enrichment and acclimation of culture carbon dioxide was delivered as sole

electron acceptor. (Zaybak et al., 2013). Biocathodes catalysis proved to be less expensive

as compared to traditional cathodes which are causing toxicity, corrosion and denatured

material ( Lovley 2011). Selection of microorganisms must be specific which would be

able to switch from heterotrophic to autotrophic metabolism. This mechanism may also

provide generalized approach along with metabolic activities of microorganisms for

various electron donors or acceptors during the development of anaerobic specialized

biocathode. In order to produce valuable fuels and other organic commodities, pure culture

was employed because the diversity of autotrophic acetogens accept electrons from

negatively poised cathode for the reduction of carbon dioxide. However, columbic

efficiencies remain low.

Majority of acetogens are mesophilic bacteria which work efficiently at temperature ranges

from 20 to 450C while 37 0C is the optimal growth temperature for most of the strains.

Synthesis rates are explained by higher solubility of greenhouse gas at this optimal

temperature. Synthesis of volatile fatty acids occur in acidogenesis phase and synthesis of

alcohols occur usually when metabolism shifts from acidogenesis to solventogenesis.

Bacteria have ability to reduce CO2 and H2 to volatile fatty acids and alcohols by utilizing

reductive acetyl-CoA pathway and have recently been investigated for the conversion of

CO2 to various organic products in bio-electrochemical synthesis (BES) system. Supplying

hydrogen was unlikely to be practical because of its energy input and requirement for

extensive catalysts. Recently H2 has been replaced by bio cathode with electron supply as

4

energy and electron source in BES system. The establishment of biocathode utilize the set

potential approaches along with the addition of hydrogen and chemicals.

Along with alcohols and acids, polyhydroxyalkanoates (PHA) have received considerable

attention in recent years due to its biodegradable nature and possible uses in industrial and

biomedical fields. Polyhydroxyalkanoates (PHA) polymers are of biological origin and

commercially more important among them are Polyhydroxybutyrate (PHB). These bio-

plastics are biodegradable polymers which accumulate energy and carbon sources inside

the cell structure. Though these polymers have potential to substitute the conventional

plastic based on fossil fuel, but PHB is still commercially far behind because of its high

cost for raw materials and downstream process. To acquire the commercial viability and

sustainability, CO2 and low cost substrate like molasses, glycerol and other carbon sources

are being used as feedstock for the synthesis of PHB.

Carbon dioxide is main greenhouse gas emitted by fossil fuel consumption. The modern

technologies for PHB synthesis from CO2 or other low cost substrates could stand among

the future sustainable technologies. Although fermentation of low cost substrates is in

practice over last many years, however little attention has been paid to harvest CO2 to PHB

using bio-electrochemical studies.

Though variety of microorganisms are gaining attention for PHB synthesis like Bacillus

cereus, Pseudomonas putida, Metylobacterium and Serratia sp., but Cupriavidus necator

is significantly and comprehensively studied microorganism capable of accumulating PHB

up to 80% of cell dry weight. Cupriavidus necator accumulates PHB in the presence of

excess carbon and limited supply of nitrogen, oxygen along with some essential nutrients

in the medium. Cupriavidus necator contains fln-like adhesion gene cluster which is used

for tight, non-specific adhesion to surfaces for direct transfer of electrons. It is important

to note that Cupriavidus necator specie autotrophically synthesize both short chain and

medium chain length monomers in the presence of carbon dioxide. Cupriavidus necator

have ability to reduce CO2 and H2 to polymers by utilizing reductive acetyl-CoA pathway.

Recent studies showed the replacement of H2 with biocathode as energy and electron source

5

in BES system. However, bio-electrochemical harvesting of carbon dioxide to monomers

using biocathode is not reported before. The enrichment of bio cathode for autotrophic

electrotrophs for biofuel synthesis provides a simplified method to isolate biochemicals.

The bio-electrochemical synthesis of organic compounds by different exoelectrogens

Sporomusa, Clostridium and Moorella species are reported (Giddings, et al., 2015, Nevin

et al., 2011, and Nevin et al., 2010). The initial study shows the synthesis of organic acids

including acetate using H type bio-electrochemical cell and recovery of electrons in the

form of organic compounds or hydrogen (Nevin et al., 2010). However, it was found that

the product yield and electrons recovery in organic compounds remained low.

Another important factor effecting bio-electrochemical harvesting of carbon dioxide is

cathodic material. Cathode in BES where biofilm is developed plays a crucial role on the

performance of bio-electrochemical cells. Graphite plate/rod, carbon cloth, carbon felt,

carbon paper and reticulated vitrified carbon graphite brush are being used as cathodic and

anodic material. Carbon fiber type electrodes have gained importance due to their highly

porous architecture. Recently several new fiber electrodes were developed such as carbon

nanotube-textile, conductive nanowires network and electrospun carbon fiber mat and had

delivered a high current density (Pocaznoi et al., 2012). However, the direct connection of

such electrodes to external circuit remained a big challenge.

Metal materials such as stainless steel (SS) materials show excellent mechanical and

electrical properties, low-cost, environmental stability and is easy to be shaped and

connected. SS materials had been widely used as cathode or current collector of cathode in

microbial electrochemical cells (Dumas et al., 2007).

Therefore, current study was undertaken to bio-electrochemically harvest carbon dioxide

using biocathode; design a simplified method by modified cathode; and to enhance product

yield and electrons recovery in organic compounds. Three autotrophic exoelectrogens

selected were Cupriavidus necator, Sporomusa ovata and Clostridium ljungdahlii. Fixing

inorganic CO2 to organic compounds was investigated using carbon cloth along with

stainless steel, as a cathode material by providing highly porous architecture of carbon

cloth and stainless steel for being excellent mechanical and electrical properties. Bio-

6

cathodes catalysis proved to be less expensive as compared to traditional cathodes which

are causing toxicity, corrosion and denaturation of material. Selection of microorganisms

must be specific which would be able to shift from heterotrophic to autotrophic

metabolism. It is reported in previous studies that electron transportation from cathode to

cells involves the use of or the production of electron shuttles. These electron shuttles are

produced as secondary metabolites in BES system by various organisms like flavins and

phenazines (Choi et al., 2016). These chemicals directly or indirectly effect the bio-

electrochemical synthesis of commodities of chemicals. The major drawback of shuttles

are their toxicity, loss in flow through system and limited stability. The present research

was based on the selection of electrotrophs that can directly capture electron, without

exploiting intermediate electron shuttles. Previous studies were conducted by employing

mixed population of microbial communities. BES in this context tackling the complexity

of organics and basic impediment is the selectivity toward desirable end product. Second

drawback related to mixed communities was deficiency of operational growth over

extended time periods. The lack of knowledge about the mode of electron transfer and the

products synthesized are being utilized by other communities in their complex metabolic

pathways are major drawbacks which cannot be ignored. In various bio-electrochemical

cells the metabolism was driven by hydrogen a good approach toward electricity driven

bio production. However hydrogen has shortcomings for microbial metabolism. It has less

solubility due to that microbial environment has to be pressurized to get concentrations of

the products. So cathodic bio production needs to circumvent hydrogen (Rabaey et al.,

2010). Thus high quality biocathode process was aimed to provide electrons to pure

electrotrophs for specific products generation.

The designing of BES cell relied totally on bio-cathode and its potential was carefully

controlled by Potentiostat as reported in previous studies. Implementation of Potentiostat

was to control the cathode potential and to avoid potential fluctuations that could damage

the cells. It proved to be impractical because of its limited control in large scale systems.

In current study, direct current (DC) power source was utilized to provide potential

difference between electrodes. Potentiostat was used only to examine the electroactivity of

7

bio-film of pure culture, developed at cathode. These specific implementations were

developed to simplify the reactor design and maintenance of energy efficiencies. All

explicit modifications in this technique has provided general approach and additional

potential to electro fuel synthesis.

1.1 Significance of study

Energy consumed worldwide is approximately 90% derived from fossil fuels. Combustion

of fossil fuel causing considerable environmental effects like global warming and climate

change. It has been estimated that fossil fuel reserves are going to be depleted by the year

2050 due to world’s rapidly increasing population. Hence studying the process for the

synthesis of renewable bio-chemicals from greenhouse gas is the aim of this study.

1.2 Objectives of the study

Objective of current study are to

design and fabricate simplified bioelectrochemical reactor for the synthesis of

renewable chemicals from waste greenhouse gas, carbon dioxide

study of nontoxic acetogens capable of capturing electrons from electrodes for

organic compounds synthesis

investigate the effect of batch and continuous BES system for products synthesis

identify and quantify organic compounds synthesized during BES systems

analyze redox activity of exoelectrogens through cyclic voltammetry

study coulombic efficiency for reducing carbon dioxide to organic compounds

determine cathodic current recovery of compounds in both systems

8

Chapter 2

Literature review

9

2 Literature Review

World’s energy consumption is enhanced due to the progression in human population and

increase in prosperity. Amplified economic growth and social development is reason for

the large gap between availability of fossil fuels and energy demands. Human social and

ecological activities are consuming these natural energy resources and causing depletion

of fossil fuels. Current methodologies for energy synthesis are not sustainable, other

environmental pollution problems and global warming require new methodologies by

using carbon- neutral sources. The use of renewable powers like geothermal energy,

hydropower, wind and solar energy has achieved greater attention due to the restrained

reserves of non-renewable traditional energy sources like fossil fuels. Recently the biomass

is one of the valued renewable energy source. Use of biomass decreases the use of toxic

fuel additives, the reliance on imported fuels, reduces the air, water pollution and

greenhouse gas emissions. Organic solvents and chemicals can be produced petro

chemically but recently attempts have been tried to produce chemicals by bacteria. Current

review is focused on literature survey for the bio-chemical synthesis of different solvents

like ethanol, heptanoic acid, hexanol, butanol, acetate, polyhydroxyalkanoates and other

products. Various techniques, bacterial strains and factors affect biochemical synthesis.

Biochemical synthesis is mainly carried out by autotrophic bacteria.

2.1 Autotrophic bacteria

Autotrophic bacteria are able to utilize raw materials to make their own energy essential

for life and every day functioning. Two major classes of Autotrophs are Chemoautotroph

and Photoautotroph. Chemoautotroph utilizes inorganic substances like carbon dioxide,

carbon monoxide, water and hydrogen and converts them to carbohydrates and sugar.

While Photoautotroph obtain the energy from sunlight and turns light energy to chemical

energy. Electrosynthesis is a procedure where bacteria utilize electrons from cathode for

carbon dioxide reduction to extra cellular organic compounds. Sugars, maize, corn, yeast,

glucose, xylose, cellulose and fructose etc. are consumed as energy and carbon source and

nonpathogenic bacteria like Clostridium autoethanogenum, C. ljungdahlii, and C.

10

ragsdalei, Sporomusa ovata and Cupriavidus necator etc. use waste industrial gases or

syngas as the sole carbon and energy source. The anaerobic acetogens are focused which

are helpful to convert greenhouse gases to chemicals. Autotrophic bacteria have the

potential to convert raw materials into valuable products like acetate, ethanol, acetone and

butanol. Metabolic pathways and genetic mutations are helpful for the synthesis of variety

of organic solvents.

Organisms have the ability to reduce CO2 to acetate through the acetyl coenzyme A (acetyl-

CoA) or Wood-Ljungdahl pathway are termed as acetogens. This property differentiates

the acetogens from other organisms which produce acetate by following any other pathway.

Acetyl-CoA is the central point in autotrophic bacteria for the metabolism and to produce

industrially relevant products. The oxidation of electrodes poised at reducing potential will

circumvent the need of hydrogen or other costly chemicals which can serve as electron

(Nevin et al., 2010) donor. Electrons can be derived from solar and wind power to store

energy in carbon-carbon bonds and possible liquid fuels. Acetogens can survive in diverse

ecosystem ranging from soil to termite gut and can survive under various temperature and

pH ranges. The new challenge is how to bring them in use and increase their synthesis

efficiency. Acetogens can grow on variety of substrate like hexose, glucose and glycerol.

Hexose exclusively converts to acetate (Muller 2003) as shown in the equation 2.1.

C6H12O6 3 CH3COOH ………….Equation 2.1

Among clostridium species C. Ljungdahlii utilizes different substrates for alcohol and

acetic acid synthesis. The autotrophic acetogens have potential to fix CO2 and H2 in their

acetyl Co-A pathway equation 2.2.

2CO2 + 4 H2 + n ADP + nPi CH3COOH + 2 H2O + n ATP…… Equation 2.2.

Acetogens are microorganisms which use sugars and other substrates like CO2, H2 and CO

gases. The anaerobic acetogens utilize the acetyl-CoA pathway to produce acetyl-CoA

from CO2. Acetyl-CoA is a precursor for the synthesis of other compounds like lipids,

nucleotides, amino acids and carbohydrates. As acetogens are anaerobic, H2 usually serves

as the electron donor and CO2 serves as the electron acceptor. The acetyl-CoA pathway

comprised of two components, the carbonyl and the methyl branch. The synthesis of acetyl-

11

CoA from CO2 and H2 involves the formation of the methyl precursor of acetyl-CoA and

the carbonyl precursor of acetyl-CoA. The condensation of the above two precursors can

be used to make products such as acetic acid, butyric acid, ethanol, butanol and cell mass

The bio-electrochemical synthesis also involves the conversion of carbon sources to poly

hydroxyalkanoates (PHA) by Cupriavidus necator. The metabolic pathway for PHA

synthesis is exhibited in the figure 2.1 (Verlinden et al., 2007).

Figure 2.1 Acetyl Co-A pathway for Polyhydroxyalkanoates by Cupriavidus necator

2.2 Microorganisms for Biosynthesis of Organic Compounds

Numbers of microorganisms are able to switch monosaccharide and oligosaccharides to

ethanol. Yeast and Saccharomyces cerevisiae are most commonly used microorganism

(Claasen et al., 1999). Some microorganisms are more useful than yeasts because of its

capability to grow under strict anoxic conditions, high substrate concentrations and high

ethanol tolerance. S. cerevisiae converts hexose into pyruvate via glycolysis then

decarboxylate pyruvate to acetaldehyde and further reduced to ethanol. Another promising

12

bacterium is Zymomonas mobilis, which yields 0.51g ethanol /g glucose the highest yield

than reported in literature. Z. mobilis can tolerate high temperature so decreases the expense

of cooling during fermentation. The main disadvantage of these two yeasts is that they are

unable to utilize pentose. Other microorganisms like yeasts Candida shehatae, Pichia

stipitis and Pachysolen tannophilus and bacteria can ferment xylose to ethanol but their

ethanol synthesis rate is less than S. cerevisiae. Adapted strains of S. cerevisiae have been

established (Jeppsson et al., 2003) to overcome the disadvantages. Enzymes can convert

xylose via xylitol to xylulose as it was a pentose therefore can be fermented by S.

cerevisiae. The main disadvantage of these recombinant strains is that it costs so high for

the safe disposal of waste water. Thermophilic bacteria like Clostridium thermocellum, C.

thermohydrosulfuricum, C. thermosaccharolyticum and Thermoanaerobacter mathranii

have saccharolytic activities which are useful for the conversion of lignocellulosic

materials into ethanol. There are some advantages of fermentation at high temperature, the

high productivity, low threat of contamination, boosted reactor efficiency and maximum

consumption of wide range of substrate. One disadvantage of thermophilic clostridia is its

low tolerance to ethanol than conventional ethanol producer, S. cerevisiae. The conversion

of lignocellulosic substrates by thermophilic bacteria needs further improvement.

Acetone-butanol-ethanol (ABE) fermentation utilizes the carbohydrate substrate and

produces the approximate ratio 3:6:1of solvent concentration. Butanol, ethanol and acetone

are chemicals used in great diversity like fuel additives and solvents. The three major

factors that reduce the feasibility of ABE fermentation are the low concentration of the

products (due to solvent toxicity), the cost of the substrates and the high product retrieval

costs. (Derk 1995). Microorganisms included in ABE fermentation are solventogenic

clostridia. The best recognized groups are the clostridium species like C. acetobutylicum,

mesophiles and C. beijerinckii. C. acetobutylicum produces acetate, butyrate, H2 and CO2

from glucose at pH values greater than about 5.6. Low pH, growth limiting factors, high

concentrations of acids and under fermentation solventogenic conditions clostridia can

produce propanol, Isopropanol.

13

Another approach in ABE fermentation is genetically modified bacteria which reutilize the

carboxylic acid during carbohydrate degradation. Overexpression of these genes increased

the solvent synthesis (Nair et al., 1999) and decrease in the carboxylic acid concentration.

recently synthesis of butanol at pilot scale by C. beijerinckii by butanol hyper-producing

mutant strain in glucose/corn steep water medium is described. C. beijerinckii mutant

proved the economic effectiveness of ABE fermentation. (Wang et al., 2012) studied the

two pathways based on syngas and sugar for the synthesis of ethanol by lignocelluloses

biomass. The ethanol synthesis is from syngas by using bacteria converting lignin,

cellulose and hemicelluloses biomass to ethanol. Regarding to Clostridium ljungdahlii first

patent was published in 1992. The maximum concentration of ethanol 48g/L was obtained

in continuous stirred tank. Bacterium were discovered like (Abrini et al., 1994)

Clostridium autoethanogenum, Butyribacterium methylotrophicum, Clostridium

ragsdalei, Butyribacterium methylotrophicum (Grethlein et al., 1991) and Alkalibaculum

bacchi (Allen et al., 2010) that can converse syngas to ethanol and acetic acid. This

bacterium can work under similar conditions of temperature and pH. It can grow on sugar

substrate at much higher rates than syngas substrate (Cotter et al., 2009). High yeast

extract promote the acetate growth and low yeast extract increase ethanol (Klasson et al.,

1991) concentration. The ethanol synthesis was 0.063 to 12 g/L and acetate synthesis was

0.0095 to 27 g/L by Clostridium ljungdahlii through syngas fermentation. It was studied

(Younesi et al., 2005 and Phillips et al., 1993) that the synthesis of acetate and ethanol

was possible by Clostridium species. Catalytical process can convert syngas into fuels and

like methanol, methane, ethanol, acetic acid and hydrogen (Klasson et al., 1992).

Anaerobic bacteria can grow autotrophically on syngas to produce chemicals and fuels

(Braun et al., 1981). It was found that CO2, CO and H2 can be converted to acetate with

the help of several acetogens like Clostridium aceticum, Clostridium ljungdahlii,

Acetobacterium woodii and Clostridium thermoacetica. Formation of acetate was same for

all pressures of syngas at 0.8–1.8 atm as well as cell concentrations. With hydrogen and

carbon dioxide in the culture media ethanol concentration could be increased. At 1.4 atm

pressure the maximum acetate synthesis was obtained. An attractive way to convert raw

materials to 1, 3-Propane Diol (PD) with the help of microbes without any toxic byproducts

(Nakumura et al., 2003).

14

Recently more attention is given to microbial conversion. Conversion of glycerol to 1, 3-

PD K.pneumoniae has shown wide productivity. Glycerol can be dissimilated to the

product under aerobic or anaerobic conditions (Chen et al., 2003). The Clostridium species

have been used for the synthesis of 1, 3-PD (Abbad et al., 1995). The different species

used are Klebsiella species, Lactobacillus species, Citrobacter species, Enterobacter

species, and Clostridium species. C. butyricum is important strain for the conversion of

glycerol to 1, 3 propanediol without the dependence of vitamin B12. It was proved that

there was no difference between raw and commercial grade glycerol fermentation. 1, 3 PD

volumetric productivity were same. From economical point of view (Gonzalez et al.,

2004), the glycerol is an appreciated substrate for 1, 3 PD synthesis. (Himmi et al., 1999)

worked on the important nutrients required for C. butyricum for glycerol fermentation. The

aim of this work was high synthesis yield from low nutrient medium. However there are

numerous difficulties in microbial synthesis, mainly limited yield, and product separation.

These drawbacks can be overcome through the metabolic engineering (Mukhopadhyay et

al., 2008).

2.3 Substrates for Biosynthesis

Microbial synthesis of chemical organics requires numerous substrates which are specific

for different biofuels. (Michael et al., 2011) through the C. Ljungdahlii, s analysis (Tanner

et al., 1993) genome proposed the complete pathway starting from carbon monoxide to 2,3

Butane Diol. By using steel mill waste gas the metabolic end product was ethanol, small

amount of 2,3BD, traces of lactate and acetate confirmed by GC-MS (Ji et al., 2011). For

the synthesis of 2, 3 BD from CO enzymes were located from Enterobacteriaceae, Bacillus

and Clostridium acetobutylicum species (Xiao et al., 2007) C. Ljungdahlii revealed that

homologues of enzymes Pyruvate, acetolactate decarboxylase, acetolactate synthase and

2,3BD dehydrogenase in 2,3BD synthesis from the central intermediate. These genes were

also present in C. autoethanogenum and C. ragsdalei and are usually clustered in

Enterobacteriaceae and Bacillus species (Blomqvist 1993).

15

A study of Wood-Ljungdahl pathway genes provides one of the oldest existing biochemical

pathways. The fermentation process with C.ljungdahlii ethanol concentration was over 50

g/L so proved a viable industrial process. (Al-Shorgani et al., 2012 and Mariano et al.,

2011), treated the de oiled rice bran (DRB) and rice bran (RB) by using Clostridium

saccharoperbutylacetonicum N1-4 for the acetate, butanol and ethanol (ABE) synthesis.

Results revealed that pretreated DRB produced more ABE than pretreated RB. Butanol has

the ability to tolerate water contamination and blend with gasoline increase the efficiency

of gasoline, so butanol can be used in conventional engines (Durre et al., 2007). Synthesis

of butanol with clostridium species has some problems of toxicity, low productivity but

high recovery costs. Many efforts were made to enhance the separation at low cost (Lee et

al., 2008). various raw materials or agricultural crops like starch (Madihah et al., 2001)

corn and molasses (Qureshi et al., 2001) and whey can produce butanol (Ennis BM (1985),

but rice bran proved the best raw material because it contains carbohydrates and cellulosic

polysaccharides (Tanaka et al., 2006), high amount of carbohydrates and less amount of

lignin. So it proved the best feed stock for the various valuable products like single cell

protein and ethanol (Chandel et al., 2009). Dilute acid hydrolysis was employed for the

conversion of biomass into fermentable sugars. High temperature was producing

fermentation inhibitors like furan, weak carboxylic acids and phenolic which affect the

performance of bio-butanol and bio-ethanol (Palmqvist et al., 2000) synthesis in

fermenting microorganisms. They studied the Clostridium saccharoperbutylacetonicum

N1-4 can produce butanol from rice bran hydrolysates because it is also known as (Kosaka

et al., 2007) hyper-butanol-producing strain. (Qureshi 2011) reviewed the variety of

agricultural residues and energy crops by biochemical or fermentation processes for the

synthesis of acetone, butanol and ethanol (ABE). Numerous organisms are available for

this bioconversion including Clostridium beijerinckii P 260, C. beijerinckii BA101,

Clostridium acetobutylicum and Clostridium saccharobutylicum P 262. Some of these

strains (P 260 and P 262) were used in an industrial setting in South Africa. One of the

major limitations of these cultures is that none of them produce greater than 30 g/l. He

utilized both hexose and pentose from lignocellulosic hydrolysates such as maize (corn)

fiber, wheat straw (one of the novel substrates) and barley straw, maize Stover and switch

grass. He reviewed the additional carbohydrates like maize, rye, millet, molasses, potato,

16

soya molasses and agricultural wastes too. Additional carbohydrates that can be used

include dextrins, fructose, sucrose and lactose. Technologies have been developed that

integrate lignocellulosic biomass hydrolysis, fermentation and simultaneous product

recovery.

In continuous process where a product was recovered simultaneously, 461.3 g/L ABE was

produced by using 1125 g sugar in one liter culture volume. In addition to ABE CO2 and

H2 were also produced which can be sold as dry ice and energy source respectively. He

reported that beside these 30,000m3 of biogas was also produced from fermentation sludge

per day. Additional by product separated was cobalamin. (Halan, et al., 2012 and Zhang

et al., 2009) studied about the (ABE) fermentation by Clostridia beijerinckii. The effect

of dilution rate was studied on solvent synthesis for 20 days in continuous flow operation.

The solvent productivity was enhanced by increase in the dilution rate. Continuous culture

with immobilized cells is a promising technique for solvent synthesis. Cell immobilization

common techniques are entrapment and adsorption. Entrapment include carrageenan

(Davison et al., 1993) chitosan (Frick et al., 1986) calcium alginate (Krouwel et al., 1980)

and polyvinyl alcohol (Lee et al., 2008a) as matrix material. But adsorption includes bone

char (Friedl et al., 1991) and brick. Some lignocellulosic materials include wood, rice

husks and straw (Forberg et al., 1985). Many species of microorganisms can grow on corn

stalk without any additional chemicals (Shukla et al., 1989 and Arne et al., 2000)

reviewed that biofilms have been used for bioremediation. These biocatalysts have been

used for the synthesis of chemicals, bio-fuels, and bio-hydrogen along with electricity

synthesis. In his review he highlighted the advancement in biofilm characterization, biofilm

reactor developments, fine chemicals and current challenges and future of these scenarios.

Biofilms can develop on all kind of interfaces and can nourish themselves by polymeric

substances produced by them. Catalytic biofilms are dynamic but integration of the

analytical tools into bioreactor to get real data is the biggest challenge in near future

(Katarzyna et al., 2011) reviewed that 1, 3-Propanediol is an essential chemical to

synthesize monomer for polycondensations of polyesters, polyether and polyurethanes.

Recently, 1, 3-PD is also used as a monomer for the preparation of a new type of a

polyester– polytrimethylene terephthalate (Biebl et al., 1999). This polyester can be

17

produced chemically too but it is expensive due to expensive chemicals, high temperature,

pressure and catalyst. (Kajan et al., 2012) focused the assimilation of different

biochemical and bioprocessing technologies for future biomass energy programs. The dry

biomass is a polymer of carbohydrate containing carbon, hydrogen and oxygen in a ratio

of 1:1.4:0.6 approximately. During the energy constrained the CO2 is recycled to form new

biomass. So its utilization is alternative for renewable source of energy. These fuels are

derived by using conventional technologies. The residues and byproduct from sugarcane

milling are useful for the synthesis of ethanol. Oil seed crops like rapeseed, soybean,

sunflower, coconut are valuable feed stock for the biofuel synthesis (Murphy 2012).

Microorganisms use different metabolic pathways with enzymes and co enzymes to

convert feedstock substrate to fuels. Biochemical conversions are usually slow so

alterations are done in the metabolic pathways to produce environmental friendly

chemicals.

Anaerobic digestion mediates the decomposition of organic matters under anoxic

conditions via bacterial species. Agricultural waste, animal manure, industrial waste,

waste, pulp and paper residues, even microalgae are utilized in the anaerobic chambers.

The clean energy carriers are bio-methane, ethanol, bio-hydrogen, bio-butanol and

biodiesel (Ranjan et al., 2013). Bio butanol is considered as the alternative for the gasoline

and diesel. The use of rice straw followed the typical trend of acidogenesis and

solventogenesis. This paper explored the utilization of rice straw for the bio-butanol

synthesis through ABE fermentation. Acid hydrolysis releases the sugar which depicts the

effectiveness of pretreatment. Acetobutylicum species were used for batch fermentation

which converts acids to solvents. Utilization of reducing sugars is more than glucose.

Acetone was produces on the second day of fermentation and ethanol was observed during

the stationary phase of fermentation. The acid hydrolysis of rice straw at high temperature

synthesizes the hydrolysates which could be a substrate for the fermentation. (Lakaniemi

et al., 2013) has discussed effective microalgal biomass anaerobic synthesis of methane,

hydrogen, ethanol, butanol and electricity was possible. Biomass from microalgae can be

an aerobically processed to biofuels. Lipids can be extracted from the cells for the further

synthesis of biodiesel. The deficiency of lignin along with high contents of lipids, starch

18

and proteins make microalgal biomass a promising substrate for anaerobic

microorganisms. Anaerobic biodegradation is split into four main phases: hydrolysis,

acidogenesis, acetogenesis and methanogenesis. Anaerobic digestion is applicable in

digestion of sewage sludge, manure and municipal solid waste. Synthesis of CH4 differ due

to variation in protein, carbohydrate and lipid content and cell wall structure. Retention

times are relatively long to obtain maximum CH4 yields from untreated microalgal

biomass. Anaerobic digestion of microalgal biomass has been conducted both in batch and

in continuously stirred tank reactors. Ethanol can also be produced from microalgae and

microbes especially are Saccharomyces yeast, especially S. cerevisiae and Wild-type S.

cerevisiae. The green microalgae have relatively high content of carbohydrates and are

good candidates for ethanol fermentation.

2.4 Anaerobic digestion for biosynthesis

Anaerobic digestion is also known as fermentation. It is a metabolic process converting

sugars to acids and alcohols by using bacterial and yeast species. Fermentation in the

absence of electron transport chain takes the reduced carbon source like glucose,

carbohydrates and make products like acetate, ethanol etc. Fermentation is anaerobic

digestion that can generates ATP by substrate-level phosphorylation. (Ni et al., 2009)

reviewed the ABE fermentation plants. They observed the capacity of ten plants for the

fermentation of acetone, butanol and ethanol. Number of ABE plants and strains are

available for the synthesis of these solvents but mainly clostridium strains were for

industrial preparation of these solvents. C. acetobutylicum, Clostridium beijerinckii,

Clostridium saccharobutylicum, and Clostridium Saccharoperbutylacetonicum .C.

acetobutylicum and C. beijerinckii are best for acetone and butanol fermentation but C.

saccharobutylicum and C. saccharoperbutylacetonicum are suitable for making these

solvents by utilizing molasses as substrate.

Continuous fermentation system consists of 6 to 8 tanks and fresh substrate is continuously

added into first and second tank and fermentation mash is discharged from bottom. In this

continuous process three stages are generally observed. In the first stage bacteria’s grows

19

quickly and produce butyric acid and acetic acid and hydrogen gas. In second stage acetone

and butanol synthesis is decreasing the acidity and acids are taken up by the cells spores

formation is increased and carbon dioxide formation is increased. In the third stage acetone

and butanol synthesis is decreased along with gas formation. Total time period is 7 to 20

days. A series of distilleries are used to separate different solvents. Evans et al., 1988)

proposed the extractive fermentation to enhance the productivity. It was good extractant

for butanol. Supplementation of glucose and butyrate in the extractive fermentation yielded

a 47% increase in butanol. (Berezina et al., 2011) studied that butanol, Ethanol, acetic

acid and butyric acid can be produced from anaerobic bacteria like clostridium

acetobutylicum. Alcohols and acetone was produced 12.7 to 15 g/ l with 6% wheat flour

medium and synthesis of butanol was 51 to 55%. The cheapest carbon containing substrate

like domestic and agricultural waste or plant biomass is profitable to use. Optimization of

fermentation medium is future goal to achieve larger amount of solvents. Angenent et al.,

2004) reviewed that present society need is less polluted world and low dependency on

fossil fuels. Waste water treatment addresses both these goals. There is prototype shift from

disposing of waste water to utilize it. Charles Mayer studied in 1982 that when clostridium

acetobutylicum grown in a continuous culture with neutral conditions the fermentation

product were acetate, butyrate hydrogen and carbon dioxide. When pH was decreased

below 5.0 the products were acetone and butanol. If 20 to 80 mM of butyric acid was added

at pH 4.3 it shifts the fermentation to solvent formation. The butanol and acetone produced

are consumed at butanol accumulation. Batch cultures were used but the continuous culture

gave better results. Medium was kept anaerobic by passing nitrogen and culture was stirred

at 100rpm. Constant passing of medium was to prevent the bacterial growth in medium

reservoir. Dry weight was measured by weighing the cell suspension on membrane filter.

Glucose was determined by glucose dehydrogenase. Products like ethanol, butanol,

butyrate, acetone and acetate were determined by gas chromatography. Glucose was taken

as a limiting factor clostridium species were grown in continuous culture. The fermentation

results were determined. Acetate and butyrate were in the ratio of 2 to 3. Butanol and

acetone were not detected among the products. Solvents could not be produced at neutral

pH because pH was decreased step wise. The butanol and acetone synthesis can be done in

a continuous way or not. Several cultures reduce the ability of solvent synthesis. Solvent

20

synthesis is not observed when bacteria’s were in continuous culture with pH level 6.5 or

5.7. In addition cultures were unable to sporulate and were weak solvent producers in a

batch process. So solvent synthesis was not supported in chemo stat. chemo stat cultures at

neutral pH forms acids and there is a possibility to change then to solvents by decreasing

the pH and by adding butyrate.

2.5 Anaerobic bio-reactors

Bioreactors are engineered devices to support biologically active environment. Recovery

of energy and appreciated materials somewhat reduce our dependence on fossil fuels and

the cost of wastewater treatment. These strategies produce bioenergy, methane, hydrogen

and small amount of current. Hydrogen can be produced in phototrophic processes but now

they are focusing on chemotrophic processes. Biological methane synthesis from organic

material is useful because of its organic removal with up flow anaerobic sludge blanket

reactor. Round 60% of the treatment facilities worldwide are based on the same reactor

treating the industrial and food processing waste water. Recently continuously- fed

horizontal flow bioreactor have been proved to be best for organic removal rates than the

previous one. The up flow anaerobic sludge blanket reactor (UASB) reactors have some

limitations of suspended solids, granulation and reactor performance (Kalogo et al., 1999)

but it efficiently convert organic compounds in waste water to methane so it can treat

diverse range of industrial waste water (Karim et al., 2003). Anaerobic Sequencing

Batch Reactor (ASBR) reactors were developed to control high suspended solids in waste

water. It was single vessel bioreactor, waste water fed into the reactor with biomass when

both are mixed the biomass settles down and effluent is withdrawn from the reactor. This

type of reactor is useful for the treatment of agricultural waste and swine waste (Angenent

2002). The methane produced by this has been used as a fuel source usually for heating or

electricity synthesis. Methane is now also used for the synthesis of syngas and biodiesel

too. Very interesting synthesis by dark fermentation is biological hydrogen synthesis.

Efforts have been made to get considerable amount of hydrogen. Bioconversion can be

increased by coupling of two bioreactors, fermentation, and aerobic conversion of fatty

21

acids. The conversion of food and agricultural waste to the biochemicals and its separation

and purification costs are so high.

Recently advanced purification technologies short separation routs like lactic acid

separation from fermentation broths has been employed by anionic fluidized bed columns.

Supercritical fluid technology is used for separation of compounds from cultures. Usually

separation is done by using nontoxic, nonflammable solvents and achieving product

crystallization. Hence post treatment facilities should be integrated to bioprocessing

operations. (Ezeji et al., 2010) studied about anaerobic bacteria with wide range of carbon

sources like glucose, galactose, cellobiose, xylose and mannose for the synthesis of

carboxylic acid and solvents like butanol, acetone and ethanol. Solvent response

mechanisms involve energy dependent efflux pumps which can export the toxic chemicals

from these microorganisms (Ramos et al., 2002 and Ingram 1990). These efflux pumps

response to the stress of toluene in E.coli, Pseudomonas aeruginosa. Scientists

investigating the improved gene expression in ethanol tolerance and the yield of ATP, over

250 genes are implicated in solvent tolerance.

2.6 Metabolic engineering to enhance product yield

Metabolic engineering is optimizing genetic and regulatory processes within the cells for

the synthesis of certain substances. A strain development method in C. acetobutylicum was

introduced in 1994 by (Aguilera et al., 2006 and Mermelstein et al., 1994) utilizing a

plasmid vector system for introducing foreign DNA into clostridium by electro

transformation. The genetic material transfer by this technique is widely used to observe

the solvent tolerance in clostridia species. (Muller 2003) reviewed that anaerobes have

respiratory system uses oxygen but they use NO3-, NO2

- and Fe (III) in the absence of

oxygen. This is totally different in anaerobes because they obtain energy only by

fermentation. So energy gained by anaerobe is small than by aerobes. These organisms rely

on phosphorylation and one mole of hexose yields 4 moles of ATP. Other organisms can

increase (Atsumi et al., 2008) studied the biofuel synthesis from Escherichia coli.

22

According to the ‘Renewable Fuels Association’ ethanol is the important biofuel. Its

synthesis capacity and the demand for bio ethanol are increasing gradually. Ethanol has

low energy density 30% and its incompatibility with existing fuel infrastructure. Other

biofuels aims to circumvent the problems of ethanol. Ethanol cannot be distributed in the

existing conduit because of its high percentage blends with gasoline due to high tendency

of absorbing water. The second problem is higher vapor pressure which is threat to air

quality. Higher carbon chains can circumvent these problems so advance fuels involve

longer chains. Higher chain fuel compounds are not produced biologically in large

quantities except n-butanol by Clostridium species.

It is believed that with metabolic engineering Escherichia coli or Saccharomyces

cerevisiae can produce such type of fuels. Isopropanol can also be used to esterify with

various fats and oils in place of methanol. Its addition lessen the tendency of biodiesel to

crystallize at low temperatures. Isopropanol is usually made from petroleum is widely used

for plastics formation after dehydration to propylene. Isopropanol is produced by following

the acetone pathway. Acetone synthesis in E.coli has been accomplished by introducing

four genes from clostridium acetobutylicum ATCC 824, thl, ctfAB, adc coding acetyl-CoA

acetyltransferase, acetoacetyl-CoA transferase, and acetoacetate decarboxylase. This strain

was helpful in producing 5.4g/L acetone. So this proved that E.coli could be best host for

Isopropanol and acetone synthesis. In the synthesis of Isopropanol a secondary alcohol

dehydrogenase (ADH) is required for the conversion of acetone to Isopropanol in an

NADPH-dependent reaction. Clostridium beijerinckii was compared to Clostridium

brockii. The combination of these genes produced 4.91 g/L with 0.41 g/ (L h) products

which is higher than the native Clostridium strains. Butanol is hydrophobic and quite

similar to that of gasoline. It can completely take the place gasoline or by mixing with

gasoline at any ratio. Vapor pressure of butanol is quite less than ethanol. Butanol is

produced by C. acetobutylicum with pathway similar to acetone and butyrate pathways as

(Zheng et al., 2009) produced butanol by engineered pathway in microbes. Genes involved

in butanol synthesis was cloned with E.coli. With this process they produced 139g/l of

butanol under anaerobic conditions.

23

Further work is required to optimize culture conditions and coordination of multigene

expression. (Charles et al., 1986 and Hubert et al., 1982) investigated about the

metabolic engineering of E.coli for the synthesis of biofuels The host bacteria A. baylyi

were lacking of the ability to produce ethanol because they are aerobic bacteria.

Introduction of some genes to produce FAEE proved best for other bacteria to exhibit this

ability. (Woods et al., 1986) C. acetobutylicum also produces by products such as ethanol,

butyrate and acetone. A redox reaction which release CO2 during the synthesis of butanol

from glucose. M. elsdenii and S. coelicolor genes gives much lower synthesis of 1-butanol.

In this investigation they found that host pathways that compete with n-butanol pathways

for NADH and acetyl-CoA were deleted. E. coli produce threefold butanol more than wild

type and by reducing the amount of acetate, ethanol and succinate. These results were not

as high as were expected but they demonstrated the feasibility of metabolic engineering

approach. ( Atsumi et al,) diverted the metabolic intermediates to higher alcohols by taking

advantage of this capacity in the host E. coli by inserting the last two steps in the Ehrlich

pathway.2-ketoacids are intermediates in amino acids formation and he converted it to

aldehyde by 2-ketoacid decarboxylase and then to alcohols by ADH. So the 2-ketoacids

improve the productivity of alcohols.

Biodiesel is a substitute for diesel fuel extracted from palm, soybean and converted to

biodiesel by transesterification. The availability of cheap vegetable oil is insufficient so

E.coli has been engineered to produce biodiesel oil. To produce biodiesel from E.coli,

esterification of ethanol with acyl moieties was done. The engineered E.coli produced

FAEE concentration of 1.30 g/L by fed-batch method with glucose and oleic acid.

Isoprenoids are metabolites synthesized from isoprenyl pyrophosphate (IPPP) and

dimethyl allyl Pyrophosphate (DMAP) in animals, plants and bacteria (Sacchettini et al.,

1997). These molecules are synthesized either by mevalonate pathway or by

methylerythritol pathway. Only trace amount of isoprenoids are produced from natural

organisms. But engineered E.coli produced isopentenol concentration of 110 mg/L. of fuel.

(Bermejo et al., 1998) studied that a synthetic acetone is composed of four Acetobutylicum

genes coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase was

introduced into E.coli.

24

Acetone synthesis represented that there is a reduction of acetic acid level. Different strains

are different in their growth and acetate metabolism and resulted in different concentration

of acetone but glucose fed cultures produced 150% increased acetone. With the addition of

sodium acetate there was further increase in acetone synthesis. (Finch et al., 2011) studied

the metabolic changes in clostridium acetobutylicum. They produce two voltage peaks.

Analysis designated that these dual voltage peaks were with glucose metabolism. The first

peak was related with acidogenic metabolism along with acetate and butyrate synthesis.

And other peak was related with solventogenic metabolism with acetone and butanol

synthesis. Since MFCs can process waste and generate power, they can increase the

efficiency of municipal or military systems while preventing environmental contamination

from untreated waste (Logan et al., 2008) the growth and hydrogen formation from 50 to

100% but it enhance the glucose uptake by 300%. So solvent formation is triggered to

another electron flow route.

2.7 Synthesis of organics by greenhouse gases

Clostridium ljungdahlii was able to convert CO to acetate and ethanol by following the

acetogenic pathway (Vega et al., 1989). The significant aspect of this fermentation was to

study the bioconversion of syngas to commercial fuel. A strictly anaerobic bacterium, C.

Ljungdahlii was utilized for the fermentation. It was observed that ethanol concentration

was four times greater for pressurized bioreactor. However (Chang et al., 2001) produced

ethanol by Eubacteriumlimosum KIST612 in a bubble column bio reactor. The working

volume was 0.21and gas volume was 80ml/ min. the maximum conversion of CO was 60%.

Acetate synthesis by Acetobacterium BR-446 was increased at 1.6 atm. pressure. Acetic

acid yield was 86% for CO2 consumption and 90% for H2 consumption (Morinaga et al.,

1990). Vega et al used Peptostreptococcus products U-1in a batch process for

bioconversion of carbon monoxide to acetate. The acetate yields at the lowest CO partial

pressure of 0.22 atm and at the highest CO partial pressure of 1.72 atm were 0.25 and 0.19

g acetate per gram CO. batch system is a closed system can maintain the cell viability for

25

limited time and growth cycle changes from one phase to another in the remaining medium

and environment. (Nielsen et al., 1994) studied the effects of initial total pressure of syngas

on microbial cell, substrate and products inhibition in the culture media were observed too.

The acetate and ethanol concentration were measured by gas chromatography. The dry cell

weight measurement was done by using UV spectrophotometer. (Ahmed et al., 2007),

focused on syngas composition, microbial cell population and pressure effect on ethanol

synthesis. It was observed that high ethanol synthesis was under 1.6 and 1.8 atm in a batch

reactor. High ethanol yield with CO was at 1.6 atm. pH is an important parameter for

bacterium growth. Clostridium ljungdahlii is a gram positive rod shaped strict anaerobe.

Its growth occurs in anoxic conditions at 37 0Cand the pH is between 5.8 and 6.0. It has

the ability to convert hydrogen, carbon mono and dioxide gases to ethanol and acetate

(Munasinghe et al., 2010).The reaction for the syngas fermentation is given in equations

2.3 & 2.4.

6CO＋3H2O → C2H5OH＋4CO2 ……………………………Equation 2.3

2CO2＋6H2 → C2H5OH＋3H2O…………………………….Equation 2.4

At pH (5.0-7.0), the acetic acid is major product and at pH (4.0-4.5) and without yeast

extract alcohol is a major product. Medium was strictly made under anoxic condition with

continuous passage of nitrogen and carbon dioxide. Batch experiment was done in

continuously stirred tank reactor. Continuous drop in pH level was due to the formation of

acetic acid. But when there is a change in pH to about 4.0 with limited yeast extract ethanol

synthesis is increased. Carbon source from syngas and fructose when transferred to the

reactor ethanol, acetic acid or other organic acid as butyric acid were observed in this

process. Fructose is used to promote cell growth in the medium. It was suggested that pH

is the important factor in ethanol synthesis. When pH was under 5.0, a non-growing stage

results in major synthesis of ethanol. The medium pH can also be reduced by fructose

addition because strong acid such as HCl may prohibit cell growth.

26

2.8 Role of Metabolic Pathways in biochemical synthesis

Methanogenic archaea used to reduce CO2 to CH4 and membrane bound enzymes that

transfer Na+ and H+ which are used to drive the synthesis of ATP by ATPase. Acetogens

use the same path way for CO2 reduction. Energy conversion pathways are different in

sodium and hydrogen ions organisms. Recently it is observed that carbon dioxide is not the

electron sink in acetogens but alternate routes are also present. Organisms that can reduce

CO2 to acetate by suing acetyl coenzyme or Wood-Ljungdahl pathway are called

acetogens. This metabolic capability differentiates acetogen from other organisms that

utilize other path ways to synthesize acetate. Acetogens are anaerobic bacteria which

convert C1 compounds like H2-CO2, CO, or formate to acetate. Acetogenesis produce

billion of tons of acetate each year. Acetogens can grow on different substrates like

hexoses, C1 and C2 compounds. This fermentation is also called as homoacetogenesis. The

Wood-Ljungdahl pathway in anaerobic bacteria is used for both anabolism and catabolism.

CO2 is reduced to formate by formate dehydrogenase, and then formate is bound to

tetrahydrofolate to form H4F. Water is split off and resulting methylene group is reduced

to methyl –H4F. Acetate is produced from acetyl-CoA by phosphotransacetylase and

acetate kinase. One mole of ATP is produced and this ATP is consumed in the formyl-H4F

synthetase reaction. So net gain in ATP is zero. (Gregory et al., 2004) reported that

Acetogens are divided into two groups: the H+-dependent acetogens with Moorella

thermoacetica, the Na+ dependent acetogens with Acetobacterium woodii. The Na+

dependent acetogens does not contain cytochromes but have membrane and follow the

Wood-Ljungdahl pathway. The methanogenic archaea which are used in the path way of

Wood-Ljungdahl they depend on Na+ for CO2 reduction. So it is obvious to look for Na+

dependence of acetogenesis. A. woodii used as a model system to unravel the molecular

basis of the Na+ dependence. Although the mechanisms of energy conservation in

acetogens are still not clear but A. woodii is one in which entire energetic is based on a Na+

across the cytoplasmic membrane and represents first ATPase which combines features of

bacterial and eukaryal enzymes. This enable the organism to regulate its cellular energy

metabolism depends on its growth conditions. Moorella thermoacetica uses nitrate as an

electron acceptor the bioenergetics, and regulatory enzymology, processes involved in the

27

utilization of alternative electron acceptors presents challenge for future. The electron

transport and mechanism is still needs to be solved. Genome analyses are a way toward

understanding of energetic, biochemistry of acetogens. (Byung et al., 1984) studied the

methyl viologen-linked hydrogenase activity of Clostridium acetobutylicum but did not

explain the dehydrogenase activity of carbon monoxide. They found that addition of CO

with increasing pressure inhibited the both growth and hydrogen synthesis by C.

acetobutylicum. Glucose fermentation balance of C. acetobutylicum is dramatically altered

by CO addition. It diverts carbon and electron toward the synthesis of ethanol and butanol

and took them away from H2, CO2, and acetate and butyrate synthesis. The butanol

concentration can be increased from 65 to 106 mM that is increase by 31% when

fermentation was maintained at pH 4.5 and gassed with 85% N2-15% CO, they focused on

three topics, improvement in strain tolerance, understanding of the physiological

relationships between organic acid metabolism and the initiation of solvent synthesis and

continuous fermentation technology. Another important aspect of fermentation is

improvement of solvents produced from a given amount of substrate. The end products of

C. acetobutylicum are formed by pyruvate metabolism. CO addition could inhibit growth

and fermentation of C. acetobutylicum alters by the formation of homo lactic acid. The

purpose of this study was to determine whether exogenous CO addition could inhibit

hydrogenase activity and can enhance solvent synthesis from saccharide fermentations as

a result of decreasing acetate, hydrogen and butyrate synthesis by C.acetobutylicum.so

addition of CO can modulate hydrogenase activity of C. acetobutylicum. The practical

utility of this method improved butanol synthesis from starch. (Wood et al., 1984) studied

about the role of carbon monoxide dehydrogenase in autotrophic path way by acetogenic

bacteria. This pathway involves the reduction of CO2 to formate and its conversion to the

methyltetrahydrofolate. The function of the CO dehydrogenase is to reduce the cobalt of

the corrinoid enzyme to Co+ which is used for methyl acceptor. The dehydrogenase is not

directly involved in the formation of C intermediates. All enzymes required for the

synthesis of acetyl-CoA from CO and methyltetrahydrofolate or from

methyltetrahydrofolate and the carboxyl of pyruvate have now been purified. With these

purified enzymes it is found that CO dehydrogenase is essential for acetyl-CoA synthesis

(Figure 2.2). Furthermore, acetyl-CoA was synthesized using the C1-CO dehydrogenase

28

complex. Thus it is conclusive that CO dehydrogenase has a direct role in the formation of

the carboxyl of acetyl-CoA (Daniell et al., 2012).

Figure 2.2 Role of CO in the formation of acetyl-CoA

Numerous techniques has been utilized to enhance the biofuel productivity but most of

them leading to the consumption of expensive raw materials, catalysts, electrodes, energy

and high temperatures etc. making the processes bit difficult to acquire. Various microbes

cause severe health problems because of their contagious and infectious nature.

2.9 Bio-electrochemical synthesis

Numerous solvents, biofuels and chemicals are synthesized by acetogens by bio-

electrochemical synthesis, fermentation, electrochemical synthesis, genetic mutations and

by metabolic pathways. Bio-electrochemical synthesis consumes electrons from electrode

29

to reduce greenhouse gases like carbon dioxide to extracellular organic valued products.

It’s a potential strategy for capturing the energy in carbon bonds to distributed products

like transportation fuels. Nevin et al., 2011) reported that the acetogens, Sporomusa ovata

has the potential for electrosynthesis. Other acetogenic bacteria like Clostridium

ljungdahlii, Clostridium aceticum, and Moorella thermoacetica consumed current and

produce organic acids. Along with primary product acetate, secondary products like but,

2-oxobutyrate and formate were also formed the identified product of electrosynthesis. It

was reported that C. aceticum. S. sphaeroides, C. ljungdahlii, and M. thermoacetica had

high proficiencies to consume electrons and recovered them into renowned products but

Acetobacterium woodii has not ability for current consumption. Artificial form of

photosynthesis is powered by solar energy where autotrophs renovate water and carbon

dioxide to organic covalent compounds. Bio-electrochemical synthesis over biomass for

the synthesis of fuel by harvesting solar energy is 100 fold higher in efficiency. This avoids

the intensive agriculture and environmental degradation. Microbial electrosynthesis have

the ability to derive electrons from electrodes for the reduction of terminal electron

acceptor. The capacity of electron transfer from electrode to cell is in less consideration.

Geobacter species are capable of reducing the electron acceptors like nitrates, formate and

chlorinated solvents. Anaeromyxobacter dehalogens can reduce fumarate and

dehalogenate 2- chlorophenol. Undefined microbial consortia also contains

microorganisms have the ability to reduce electron acceptors like oxygen with electron

donor electrodes. Methanobacterium palustre reduce carbon dioxide to methane but there

is difficulty in direct electron transfer in methanogens because methanogenesis can produce

hydrogen at low potential. Geobacter and Anaeromyxobacter species can accept electrons

from graphite electrodes for the reduction of fumarate to succinate (Gregory et al., 2004),

U (1V) reduction (Gregory et al., 2005) or reductive dechlorination (Strycharz, et al.,

2010). But it was difficult to verify (Villano et al., 2010) because of the synthesis of

hydrogen required for methanogenesis. Sporomusa ovata has the tendency to derive

electrons from graphite electrodes for the reduction of carbon dioxide to acetate. S.ovata

forms biofilm on the electrode surface and has potential for current consumption to

synthesize acetate and small quantity of oxobutyrate. Acetate formation is through Acetyl

Co-A. (Nevin et al., 2011) studied about the electrodes as electron acceptors to promote

30

the anaerobic degradation of pollutants. Solar energy or other electrical energy source can

provide electrons to these microorganisms for the formation of biofuels and numerous

other valuable chemicals from carbon dioxide. Electrodes can accept electrons to support

anaerobic oxidation of organic compounds or can supply electrons for the respiration of

microorganisms. The most promising application is the formation of bio-cathode and

supply of electrons to microorganisms for useful processes. Indirectly electrons can be

transferred from electrodes to microorganisms by the reduction of electron shuttle

molecules or via the hydrogen gas. Anodes can also provide electrons to cathode at low

potential. Acetogenic microorganisms utilize hydrogen as electron donor to reduce carbon

dioxide for acetate synthesis as end product along with small amount of other organic acids

and alcohols. The cathode biofilms of acetogens prolonged their activity for months

without forming thick biofilms. Acetyl Co-A is the building block for the synthesis of wide

variety of desirable organic products and is the central intermediate in the acetate synthesis.

With genetic engineering carbon and electron flow can be diverted toward butanol

synthesis. Pure culture studies have focused on Geobacter and Shewanella species which

can utilize oxygen as an electron acceptor. Micro- organisms can catalyze proton reduction

to hydrogen gas with hydro-genases by accepting electrons from cathode. In Shewanella

oneidensis extracellular electron transfer is different. Electron transfer is through

conductive pilli. Electron transfer from Geobacter to anode is faster so results in higher

current densities. (Rabaey et al., 2011) reviewed that Microbial electrosynthesis uses

electrical energy for reducing power of microorganisms. In bio-electrochemical

progression microorganisms are used to catalyze oxidation or reduction reactions at

respective electrodes. The best example is MFC in which the electrical current is provided

by microbes to electrode for the reduction of organic substrate. Recently microbial

electrosynthesis has emerged as oxidizing and reducing power for biochemical synthesis

via electricity. In the electrochemical cells microbial cells are used as biocatalysts for

synthesis reactions. So electric current is either supplied or extracted from microorganisms

for sustainable biochemical synthesis. The conversion of carbon dioxide to acetate and

fumarate to succinate are examples of reductive processes while conversion of glycerol to

ethanol is oxidative process. Electrical energy is used to drive carbon dioxide fixation or

formation of acetyl-CoA for the synthesis of target compounds. Instead of Wood Ljungdahl

31

pathway the Calvin-Benson-Bassham cycle is leading to fermentable substrate from

electricity and carbon dioxide. So there is a hybrid metabolism when effective charge

transfers toward the cell. The electron transfer between cell and electrodes are either by

direct contact of biofilm on electrode or by mediators toward the microorganisms. Good

example of autotrophic bio synthesis is butanol synthesis by homoacetogens by using

electric current. The advantages of microbial electrosynthesis are the use of electricity for

bio synthesis and are independent of substrates need arable land and their synthesis

densities are immense.

2.10 Extracellular electron transfer (EET)

In bio-electrochemical synthesis cell the basic challenge is to transfer electrons into

microbes to get industrial level products. Three mechanisms reported are electrolysis of

water, soluble electron mediators and electron transfer directly by C-type cytochromes and

via conductive pili or nanowires

The shuttling of electrons from reductive cathode through electrolysis of water is to

produce hydrogen gas. The cathodic electrons to water form hydrogen by the following

equation 2.5.

2H2O +2e- → H2 +2OH-……………………………….Equation 2.5

The major drawback faced in this system was that large overpotential requiring large

energy input and low solubility of hydrogen reduces the electron availability in solution.

Significant transfer of electron from bacteria to electrode is by four pathways, the reduced

products, the electron shuttles, c-type cytochromes and via conductive pili or nanowires.

2.10.1 Electron transport by fermentation products

Bacterial fermentation products are oxidized an aerobically at the anode to Provide

electrons to anode like in yeast. These products include ammonia, hydrogen, and hydrogen

sulphide and alcohols (Davis et al., 2007). But it was inefficient technique because of slow

reaction of these fermentation products with anode and sometime electrode material were

32

proved to be foul with these products. One reduced product is H2S which can produce

power in MFC by utilizing sulphate reducers. Sulfate reducing bacteria in marine sediments

has ability for the reduction of sulfate compounds to HS-2 or S-2 and their oxidation product

is So. Eight electrons are required for sulphate reduction to sulphide and its oxidation to So

release only two electrons to electrode proves to be an inefficient method. Another example

is of Desulfovibrio species which oxidize their electron donors incompletely to acetate a

limiting efficiency (Lovley et al., 2006).

2.10.2 Electron transport by mediator

Extracellular electron transfer is the ability of bacteria to utilize electron acceptors or donor

external to the cell. The shuttling of electron for industrial application is the use of electro

active molecules that can be reduced or oxidized by bacteria. The oxidized shuttle can be

reduced by electrode and serve as electron donor for various bacteria. These mediators

lessen the necessity of direct contact between electrode and bacteria. Flavins, neutral red,

anthraquinone-2, 6-disulfonate and cobalt sepulchrate are mediators used by bacteria (Von

et al., 2008 and Park et al., 1999). Microbes like Escherichia coli, Pseudomonas,

Bacillus and Proteus are incapable to shuttle electrons from the internal metabolism to

outside the cell. These microbes utilized artificial mediators like thionine, neutral red, Iron

chelates and phenothiazine, thionine, potassium ferricyanide etc. These mediators induce

microbes to produce the electrochemically active reduced products, enter the cell

membrane and capture the electrons from central cell metabolism. In a reduced form they

exit from the cell and transfer these electrons to the electrode and become oxidized to

capture further electrons

Previous researches have shown that along with effectiveness of electron shuttle there are

few disadvantages as well. These mediators are highly toxic to microorganisms like neutral

red and methyl viologen have impact on bacterial viability and diffusion limitation also

affects the rate of synthesis. Finally large quantity of mediator to industrial systems is

costly and impractical. Most feasible way is to engineered the microbes to form their own

mediators with regards to product formation (Thrash et al., 2008). It has been investigated

that few mediators like neutral red capture electrons from NADH and reduced by enzyme

hydrogenase. Proteus vulgaris is another example for transfer of electrons to electrode by

oxidation of sucrose to carbon dioxide where thionine is acting as mediator. Shewanella

33

putrefaciens is efficient in extracellular electron transfer by excreted flavins, and

menaquinone the mediators or by membrane bound cytochromes. Mediators must be of

low cost, efficient reaction rate, able to cross the cell membrane, non-biodegradable and

non-toxic (Huang et al., 2011). Self-produced mediators related to compounds produced

by certain bacteria can shuttle electron naturally to electrodes (Rabaey et al., 2011).

Pseudomonas aeruginosa can produce pyocyanin which can shuttle electron to electrode

along with energy generation in MFC. These electro active compounds also act as

inhibitors and toxins for competitors (Hernandez et al., 2004). Lactobacillus and

Enterococcus species also release mediators the electro active compounds. Electron shuttle

is an adaptive strategy under controlled conditions. Synthesis of mediators in the cell is an

energetically expensive so it must recycled in the system. In microbial fuel cell with fed

batch system these mediators are increased over time without the replacement of medium.

But in continuous flow system these active electron shuttles does not play prominent role

like in waste treatment because replacement of medium continuously flushed out these

mediators from system and reduce the fuel cell efficiency nearly 50% (Gregory et al.,

2004). Another factor limiting the current generation in the MFC is that microbes with

natural mediators incompletely oxidize organic acids like lactate and pyruvate to acetate

even the electrodes are acting as electron acceptors like Shewanella species. Similarly

contaminants can consume the lactate so less electrons are recovered as electricity (Kim et

al., 2002)

34

electron acceptor

oxidized

electron acceptor

reduced

electron acceptor

oxidized

electron acceptor

reduced

Med OX

Med REDe_

e_

cathode

cell

Cytochrome

Figure 2.3 Electron transfer mechanism by Cytochrome and mediator where Cytochrome

directly transfer electron from cathode to cells and mediators in oxidized form capture

electrons from cathode and transfer to cells

2.10.3 Direct transfer of electron

Bacterial species Geobacter and Shewanella possess conductive appendages on the cell

surface labeled as nanowires or pilli. The main route for the reduction of electrodes or

electron acceptors is Mtr pathway. It requires five proteins OmcA, MtrB, MtrC, MtrA and

CymA which reform C-type Cytochrome family. CymA proteins are present on the inner

membrane of the cell while OmcA, MtrC are present on the outer membrane for the

electron transfer from inner side to outer electrodes. MtrA protein links the intra and extra

cellular environment and OmcA proteins are for biofilm and electrode attachment

(Venkata et al., 2014). These pilli or nanowires were first examined under scanning

35

tunneling microscopy (STM). These nanowires were extremely thin with nanometer or less

in diameter. Bacterial nanowires are conductive appendages function as conduits for

electron transport between different members of biofilm layers and are able to carry

electrons from cell to the surface to extracellular electron acceptors. Nanowires produced

by G.sulsurreducens and Shewanella oneidensis are quite different. In Shewanella

oneidensis these nanowires are like thick cables may be the bundle of several conductive

wires whereas in G.sulsurreducens these nanowires are quite thin. Some Photosynthetic

bacteria like oxygenic Cyanobacteria have ability to produce nanowires. They can produce

electricity in MFC in light. It was studied that Shewanella oneidensis are conductive and

can transfer electron at rate 109 per second along with nano fabricated electrodes and 100

mV potential (Naggar et al., 2010). There is a debate on the nanowires of

G.sulsurreducens researchers are considering them as 1V pili made up of C-type

Cytochrome and PilA monomers and their conductivity is because of amino acids. Other

researchers consider nanowires as Cytochrome which connect the biofilm on electrode

(Reguera et al., 2005). Geobacter sulfurreducens with c-type cytochromes can imparts

capacitance to biofilms, which has comparison to synthetic super capacitors. This

investigation leads to effective and sustainable new capacitor designing. A recent

investigation about PilA has shown that they are not only for scaffolding electron but also

for their transport (Vargas et al., 2013). When the amino acids were replaced with aniline

they observe the decrease in conductivity. Another microorganism Pelotomaculum

thermopropionicum produce thick appendages like pilli. These appendages can relocate

electrons among bacteria like fermentative bacteria which regenerate NADH. Similarly

syntrophic methanogenic and sulfate reducing co-cultures have revealed the interspecies

electron transfer. Still detailed investigations are required for proper understanding of

electron transfer mechanism. So pili are capable of electrons transfer across the multilayer

biofilm. It was detected that in G.sulfurreducens can make contact with electrode for

electron conduction by 20um or long pili. G.sulfurreducens can make biofilms of 40 to 50

um thickness at anode so tangled pili transfer electron from cell to cell and produce nano

power network (Reguera et al., 2006).

36

CymA

MtrA

e_

MtrB

OmcA MtrC

electrode

Outer membrane

Inner membrane

CytoplasmNADH

NAD+

Peroplasm

___ _electrons

__

electron acceptor

__

Figure 2.4 4 Mtr respiratory pathway and schematic electron flow from inner membrane

proteins CymA to outer membrane proteins OmcA and MtrC and then to electrode

2.11 Electron transfer from electrode to microorganisms

The reactors studied for straight transfer of electrons from electrode to cells are termed as

electrotrophic reactors where generally the source of electrons is water. The cathode is

poised at negative potential of -400mV which is high enough for hydrogen synthesis and

low to support the anaerobic respiration. Anode and cathode compartments are separated

with selective proton exchange membrane (PEM) to limit the diffusion of oxygen.

Electrons derived from anode chamber with the synthesis of protons and oxygen. Electrons

transferred to cathode by external system and protons by proton exchange membrane.

Geobacter metallireducens can capture electron from cathode for reduction of nitrate to

nitrite. Similarly G.sulfurreducens can capture electron for fumarate reduction to succinate.

The transfer of electron to protons produce hydrogen which is captured by Geobacter

species but it is ruled out because the electrode is supplied with potential which is high for

37

hydrogen synthesis and Geobacter metallireducens is unable to use hydrogen and in

G.sulfurreducens genes for hydrogen uptake have been deleted. Further research

evaluation for transfer of electron from electrode is high research priority (Derek et al.,

2011).

The phenomenon of electron transfer from electrode to cells is still unclear when electron

acceptors are reduced to the inner side of cytoplasm. The electron acceptors consume

protons to reduced end products and proton consumption in the cytoplasm create a proton

gradient across the inner membrane (Nevin et al., 2010a). Geobacter species make quite

thin biofilms at cathode for consumption of electrons whereas biofilms at anode are quite

thick for current generation. Gene expression are quite different in both current producing

and current consuming cells. This suggests that route for transfer of electron from cells to

anode is quite different from transfer of electrons from cathode to cells (Strycharz et al.,

2011).

Figure 2.5 Transfer of electron from cathode to electroactive cells for fumarate, nitrate

and carbon dioxide reduction to succinate, nitrite and acetate respectively

But fortunately there are more way outs of transferring the direct electrons to electroactive

cells and it depends on the direct interaction between bacteria and electrode either by

conductive appendages or membrane bound proteins (Lovley et al., 2012). It was first

demonstrated by pure culture bacteria Geobacter metallireducens in direct association with

cathode along with reduction of nitrate to nitrite (Gregory et al., 2004). There was direct

38

transfer of electrons from electrode to Geobacter metallireducens. Few more examples are

including Anaeromyxobacter dehalogenans, Geobacter lovleyi and Sporomusa ovate,

(Strycharz et al., 2010, Nevin et al., 2010 and Strycharz et al., 2008).

Bio-electrochemical synthesis uses electrical energy as a source of reducing power for

microorganisms which can be easily derived from solar and wind sources. Microbially

derived products are replacing pharmaceuticals, polymers and other commodities. In this

context microbes are provided with energy rich feedstock like sugar, carbohydrates and

other energy rich substances for the synthesis of bio-products making this process costly.

For example butanol synthesis from glucose requires 3 kg glucose per kg butanol. For the

synthesis of fermentable substrate arable land water and nutrients are required and this

poses an economic constraint. The electrochemical synthesis requires basic cell,

Potentiostat and electrodes. Electrolytic conditions like good electrolyte and electrodes

which provide the favorable electron transfer properties and maximize the activation

energy for side reactions. The basic drawback in electrochemical synthesis is the use of

expensive electrodes which corrode quickly in acidic and basic electrolyte and need to be

replaced. The use of inert electrodes like platinum, gold and silver enhance the overall cost

of the reaction. Chemical synthesis requires high temperature which results in the

consumption of fuel or electrical energy thus enhance the cost and temperature of the

surroundings but bio-cathode formation is a major solution for this problem.

Bio-electrochemical treatment is recent and advance technique for the synthesis of biofuels

and biochemicals. Fermentative and respiratory pathways often need additional electrons

for desired products. Bio-electrochemical synthesis use microbes to catalyze the redox

reactions at the electrodes like microbial fuel cell. It has emerged as an alternative option

to provide the redox power via electricity for valuable products. Hence electric current

stimulates the biochemical synthesis. Autotrophic bacterial species has potential to utilize

greenhouse waste gases like carbon dioxide, carbon monoxide, hydrogen and electrons for

biochemical conversions at low temperature. Examples are the synthesis of acetate,

butyrate from CO2. Use of environment friendly microbes, like C. ljungdahlii, S.ovata and

39

C.necator, biocathode development and waste gases as substrates has made this study

attractive for new researchers. Low cost electrodes like carbon cloth and stainless steel are

cost effective. So power synthesis has led to renewed interest in energy storage or

conversion technologies for transport and storage issues with electrical energy. Bio-

electrochemical synthesis is in its infancy but can prove to be a new approach for the

conversion of electrical energy to chemical energy. The basic principal of bio-

electrochemical cell is represented in figure 2.6. The cell with anode and cathode

compartment separated by proton exchange membrane. A simplified bio-reactor with DC

power supply was employed to provide electron instead of hydrogen to electroactive cells

for the reduction of carbon dioxide to volatile fatty acids, alcohols and polyesters. Where

as in previous studies Potentiostat was exploited for the power supply was difficult to

handle on pilot scale. Low cost substrates like molasses and waste greenhouse gas like

carbon dioxide was attractive and effective strategies.

Figure 2.6 Bio-electrochemical cell with bio-cathode and power supply for the synthesis

of bio-fuels

40

In current study, a specific method was designed to initiate anaerobic autotrophic bio-

cathode centered on heterotrophic pre-augmentation. The enrichment of bio-cathode for

autotrophic electrotrophs for biofuel synthesis provides a simplified method to isolate

biochemicals from different inoculum sources. Bacteria were heterotrophically grown first

on glucose, fructose or glycerol and carbon dioxide was delivered as sole electron acceptor

after pre-enrichment and acclimation of culture. Biocathodes catalysis proved to be less

expensive as compared to traditional cathodes which are causing toxicity, corrosion and

denatured material. Selection of microorganisms must be specific which would be able to

shift from heterotrophic to autotrophic metabolism. This mechanism may also deliver

generalized approach along with metabolic activities of microorganisms for various

electron donors or acceptors during the development of anaerobic specialized biocathode.

Commercialization of microbial electrosynthesis is a challenge. In order to harvest valuable

fuels and supplementary organic commodities, pure culture was employed because the

diversity of autotrophic acetogens accepts electrons from negatively poised cathode for

carbon dioxide reduction with high columbic efficiencies. Another trial was the reactor

designing of BES cell. As BES cells relied totally on bio-cathode and its potential was

carefully controlled by Potentiostat as employed in initial studies. Implementation of

Potentiostat was to control the cathode potential and to avoid potential fluctuations that

could damage the cells. It proved to be impractical because of its limited control in large

scale systems and energy intensive for fixed potential. The direct current power source was

utilized to provide potential difference between electrodes. Potentiostat was exploited to

examine the electroactivity of bio-film developed at cathode for both experimental and

control systems. These specific implementations were developed to simplify the reactor

and maintenance of energy efficiencies. All explicit modifications in this technique has

provided general approach and additional potential to electro fuel synthesis.

41

Chapter 3

Materials and methods

42

3 Materials and methods

3.1 Experimental Setup

The present study was conducted for bio-electrochemical harvesting of greenhouse gas

carbon dioxide to organic compounds under specific conditions of substrate, temperature

and pH. Three species of autotrophic bacteria Cupriavidus necator, Sporomusa ovata and

Clostridium ljungdahlii were cultivated to develop multilayer highly structured cathodic

biofilm at specific voltage to convert waste greenhouse gas to spectrum of organics in bio-

electrochemical cell.

3.1.1 Chemicals and Reagents

The following chemicals were utilized for the preparation of medium;

K2HPO4 0.348g, KH2PO4 0.227g, NH4Cl 0.5g, MgSO4.7H2O 0.500g, CaCl2.2H2O 0.250g,

NaCl 2.25g, FeSO4.7H2O 0.002g, yeast extracts 2.0g, Casitone 2.0g, Betaine 6.70g,

NaHCO3 4.0g, Resazurin 1mg, Na2S.9H2O 0.300g (Merk Germany).

3.1.2 Chemicals for vitamin solution

Biotin 2.0 mg, folic acid 2.0 mg, pyridoxine-HCl 10.0 mg, thiamine-HCl.2H2O 5.0mg,

riboflavin 5.0 mg, nicotinic acid 5mg, D-Ca-pantothenate 5.0 mg, vitamin B12 0.10 mg,

p-Aminobenzoic acid 5.0 mg.

3.1.3 Trace element solution

FeCl2.4H2O 1.50 g, ZnCl2 70.0 mg, MnCl2. 4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2.6H2O

190.00 mg, CuCl2.2H2O 2.00 mg, NiCl2.6H2O 24.00 mg, Na2MoO4.2H2O 36.00 mg,

NaHSeO3 10-7 M solution.

43

3.1.4 Standards

Various standards 2-ethylbutyric acid, volatile acid mixture, 3-hydroxybutyric acid,

benzoic acid, methanol, chloroform, acetone, NaOH, HCl, H2SO4 (Merk, Germany) were

utilized for GC-MS analysis.

3.1.5 Gasses

Gas mixture of CO2/H2 (90:10) was used as a source of carbon contents and N2 to provide

anoxic conditions in the anaerobic chamber. Helium was utilized as a carrier gas for GC-

MS analysis.

3.2 Equipment and Instruments

Bioelectrochemical Autoclave used (Shin Saeng Korea) for complete sterilization of

equipment and medium solutions, at 121 0C for 20 minutes.

Vitamin solutions were filter sterilized at 0.45µm pore size filter paper.

pH meter (Rocker EC 400 Microprocessor pH/MV) was used to monitor and maintain the

pH of medium and standards.

44

Samples were centrifuged with centrifuging incubator (Hermle labortechnik GmbH) to

separate cell dry weight, turbidity from medium broth and for further spectroscopic

analysis of supernatant.

Cell concentration in medium was determined by measuring optical density at wavelength

of 600-620nm by UV spectrophotometer (UV-1602 BMS Biotechnology Medical

services). Quartz cuvette with path length of 10 mm was used.

Bio-electrochemical products were analyzed for identification and quantification by GC-

MS analyzer Agilent Technologies (789A US GC system and 5975C inert XLEI CI MSD

detector).

Anaerobic chamber (YQK-11 Jinan Unilab Instrument Co.Ltd) was used to provide anoxic

atmosphere and gases to the exoelectrogens for batch and continuous bio-electrochemical

systems (BES). The chamber was disinfected properly with 70% ethanol and then

completely sterilized by UV irradiation for 30 minutes. Anoxic atmosphere was maintained

by supplying sterilized N2 gas. Moisture was absorbed by palladium beads throughout the

experiment.

45

3.3 Bio-electrochemical reactor configuration

3.3.1 General setup

Bio electrochemical reactor was fabricated with tubular polyacrylate chambers having 2

cm wall thickness. The chambers were partitioned by proton exchange membrane (PEM)

with internal dimensions (12x 8x 12 cm3) and of capacity (100 cm3) each. Non-catalyzed

carbon cloth and round stainless steel mesh plate with 12 cm diameter were used as cathode

for active electron transport and biofilm development whereas chrome plate of diameter 12

cm as an anode. The frame was bolted with bolts between two endplates. Provisions were

made for sampling ports and wire input points along with leak proof sealing for both gasses

and liquids. Bio-electrochemical synthesis of organic compounds in both batch and

continuous system was carried out in explosion proof anaerobic chamber (YQK-11 Jinan

Unilab Instrument Co.Ltd). Both anode and cathode chambers of 100 ml capacity each

were filled with 80 ml of sterile basal medium. Cathode compartment was inoculated by

pre-enriched culture only. The headspace was purged with H2/CO2 to establish a biofilm at

cathode. Voltage of -400mV was supplied by DC power supply (vanal DC power supply

PS-150200) to provide electron at potential for microbial electrosynthesis without the

synthesis of hydrogen gas. Solar energy can be harvested with existing photovoltaic

technology on bio-cathode with DC power supply. This suggests that microbiological

catalysts when coupled with photovoltaic and current-driven microbial carbon dioxide

reduction, represents a new platform of photosynthesis. Bio-electro catalysts were capable

of current consumption and reducing the carbon dioxide, a sole carbon source for the

synthesis of valuable products like acetate, ethanol and small amount of 2- oxobutyrate

(except in Cupriavidus necator for the synthesis of polyhydroxy alkanoates). The medium

was replaced several times to remove planktonic cells. The periodic removal of planktonic

cells enhances the growth of biofilm at cathode surface. A peristaltic pump (Longer pump

BT 100-2J) was attached with BES for converting batch to continuous mode. The

electroactivity of bio-film developed at cathode for both experimental and control systems

was monitored by Potentiostat. The whole reactor performance was conducted in an

anaerobic chamber (YQK-11 Jinan Unilab Instrument Co.Ltd). The bio-reactor was treated

with ethanol, acetone and deionized water before experimental setup. The fabrication of

46

BES was performed in laminar flow cabinet (Stream line Singapore) under UV irradiation

to avoid biological contamination.

3.3.2 Electrodes

Pure carbon cloth (C-Tex20, MAST Carbon International) and stainless steel mesh plate

served as cathode and chromium (Aldrich/GF 99465946) plate as an anode. Both anode

and cathode plates were connected to DC power supply via Platinum wires for electron

conduction. The electrodes were treated with ethanol, acetone and finally with deionized

water before use.

3.3.3 Proton exchange membrane

The proton exchange membrane (PEM, CMI-70005) was utilized for the partition of

cathode and anode and was pretreated to obtain the stable reactor operation. It was placed

in 5 wt. % NaCl solutions for one hour then in 3 wt. % NaOH solution for one hour and

finally rinsed with sterile deionized water for 20 minutes. In case of coagulation the

treatment is done with hydrogen peroxide for 20 minutes.

47

Bio-electro chemical reactor

Figure 3.1 Bio-electrochemical reactor

48

3.4 Coulombic Recovery

Electron recovery in polyhydroxyalkanoates, volatile acid and alcohol synthesis accounted

for high proportion of electrons consumed by culture. The total amount of the current

consumed by system was calculated by integrating the current area (A/m2) against time

(sec).

Electron consumed by electro-autotrophs and recovered in organics were calculated by

equation 3.1

n CE = Idt ……………3.1

x F

x = No of moles of electron per mole of compound

F = Faraday’s constant

n CE = No of electrons recovered in product formation

I = Current

% percent cathodic recovery was calculated by equation 3.2

R Cat = n product / n CE x 100 ………..Equation 3.2

Electrons recovery against the current consumption in continuous system

n CE = Idt ………..Equation 3.3

x Fq S

Where q is the flow rate of substrate in ml / sec and S is the change in the concentration

of substrate in g/L (Giddings et al., 2015 and Ismail et al., 2013).

Where nproduct is the total moles of product recovered in batch and continuous systems

(Giddings et al., 2015, Sleutels et al., 2011 and Logan et al., 2008).

49

3.5 ANOVA Test

Results were tested for significant differences using a one way ANOVA (Analysis Of

Variance) with the help of IBM SPSS statistics version 23 software. Data with statistically

significant findings are stated as being statistically different. The ANOVA test was

performed to compare two variances i.e., the variance within groups and between the

groups. One-Way ANOVA determined the significant difference between the batch and

continuous system for three microbial strains Cupriavidus necator, Sporomusa ovata and

Clostridium ljungdahlii. The significant value was set as 0.05, if the value was less than

this value then the difference between batch and continuous system were considered as

significant while the value greater than this digit was resulted with non-significant effect.

50

3.6 Cupriavidus necator

Cupriavidus necator is facultative gram-negative rod-shaped bacteria. This strain is non-

spore forming and non-pathogenic. Cupriavidus necator was obtained from DSMZ

(Deutsche Sammlung Mikroorganismen and Zellkulturen GmbH) and was cultured in

DSMZ medium 545 at 320C.

3.6.1 Media composition

Cells were grown in DSMZ medium 545 by utilizing chemicals of analytical grade (Sigma

Aldrich chemicals Ltd.) for the composition of medium per liter.

Yeast extract 0.3g, sodium succinate 1.0g, ammonium acetate 0.5g, iron citrate solution

(0.1% in H2O) 5ml, KH2PO4 0.5g, MgSO4. 7H2O 0.40g, NaCl 0.40g, NH4Cl 0.40g,

CaCl2.2H2O 0.05g, vitamin B12 solution (Cyanocobalamin,10mg in 100ml H2O) 0.40ml,

trace element solution 1ml, L-Cystenium chloride 0.30g, Resazurin (0.1%) 0.50ml (Sigma

Aldrich chemicals Ltd.).

Before autoclaving the pH of medium was adjusted to 6.8 with HCl and NaOH. Medium

was boiled for 5 minutes and then cooled in cold water with the purging of N2 gas and then

autoclaved at 1210C for 15 minutes for complete sterilization. Medium was transferred to

anaerobic chamber for the addition of vitamin, trace element, resazurin and L-Cystenium

chloride solutions aseptically to the basal medium after cooling. The final volume was

made up to one liter. The sterilized pH electrode was used again to reconfirm the required

pH of substrate.

3.6.2 Stock Solution for Trace Element

Trace element solution was prepared by utilizing the following components per liter.

ZnSO4. 7H2O 0.10g, MnCl2. 4H2O 0.03g, H3BO3 0.30g, CoCl2. 6H2O 0.20g, CuCl2. 2H2O

0.01g, NiCl2. 6H2O 0.02g, Na2MoO4. 2H2O 0.03g (Sigma Aldrich chemicals Ltd.).

Solution was transferred to medium bottle and stored in the refrigerator at 4 0C.

51

3.6.3 Carbon sources

The main growth substrate for heterotrophic and electro autotrophic growth of Cupriavidus

necator was molasses to supply heterotrophic conditions for cell bio-mass growth and

second source was CO2 gas for autotrophic growth of PHA in both continuous and batch

system (Tatiana et al., 2013). The basic components of molasses are in table 3.1

Table 3.1 Chemical composition of molasses

Components %

Sucrose 46.9

Glucose 5.2

Fructose 6.7

Non-carbohydrate substances 16.4

Water 14.5

protein 0

Fats 0

3.6.4 Culture growth conditions with CO2 and H2

Medium prepared as instructed in DSMZ medium 545 was used as seed medium for cell

growth in serum bottles along with the purging of gas mixture of H2/O2/CO2 (8:2:90) in an

anaerobic chamber. Stock culture of Cupriavidus necator was stored at -20 0C in falcon

tubes with 3:10 ml ratio of liquid broth and glycerol. In order to confirm the purity and cell

growth, culture was observed under microscope by gram staining after 48 hours of

incubation. The stock culture was used to inoculate the medium by transferring 3 to 5 ml

of liquid broth to the cathode compartment of BES.

3.6.5 Medium preparation with Molasses

Molasses were treated with sulfuric acid (0.75wt% pH 1.1) and then heated at 100, 115 and

130 0C for 15 minutes. The pH was adjusted to (6.8) with 5 M NaOH solution. Treated

52

molasses were filtered and sterilized by autoclaving at 121 0C for 15 minutes. Sodium

acetate (10 g/L) was used as supplement [Sharifzadeh et al., 2009).

3.7 Reactor operation

Two substrate phase strategy was employed for bio-electrochemical synthesis of bioplastic.

The first phase was heterotrophic growth of cell bio-mass with molasses followed by

second phase electro autotrophic intracellular growth of PHA content inside the cells by

CO2 source.

The pH of catholyte was controlled to 6.8 by dosing 3M HCl and 1M NaOH solutions by

means of micro pipette.

3.8 Cathode biofilm development

The cathode chamber of microbial fuel cell was loaded with 80 ml of pretreated molasses

inoculated with H2/CO2 grown culture. A carbon cloth, stainless mesh steel plate and

platinum wire were used in the cathode compartment for efficient current conduction.

Cathode was poised at -400 mV without the synthesis of hydrogen. Anode compartment

was loaded with 80 ml medium of molasses without microorganisms. Round chrome plate

as anode with platinum wire was separated from the cathode by proton exchange

membrane. The molasses media was replaced several times to remove planktonic cells.

Optimum conditions of temperature and pH trigger the heterotrophic bio-mass growth to

achieve high cell density. Microbial biofilm was developed on carbon cloth after one week

of inoculation. After the heterotrophic pre-enrichment and acclimation of biofilm at

cathode and dissipation of organic carbon contents of substrate, the system was switched

from heterotrophic to autotrophic growth by purging sterile gas mixture of O2/CO2 (10:90).

The bacterial cell concentration in the cultured media was determined by optical density at

620 nm by using spectrophotometer (UV-1602 BMS Biotechnology Medical services) with

distilled water for appropriate dilution rate. Optical density (OD) is the measure of

53

transmittance of an optical element for given wavelength. OD is the logarithm for the ratio

of transmitted and incident light as in equation 3.4.

OD = -log10 I/I0 …………………….Equation 3.4

I= Intensity of transmitted light and I0 = Intensity of incident light

Absorbance and scattering of light is increased by bacterial concentration. Solution with

higher bacterial concentration exhibits greater absorbance and less concentration reflects

increased transmittance. The enhanced transmittance and reduced absorbance against time

in days indicates the decrease of bacterial concentration in medium and most probably the

deposition of biofilm at cathode.

A curve between the cell dry weight concentrations in ppm against absorbance was made

to measure the optical density of medium to evaluate the biofilm development. The

calibration curve is represented in figure 3.2. The viability of C. necator was further

evident with scanning electron micrographs of carbon cathode surface (Montaser et al.,

2011b).

Figure 3.2 Relationship between cell dry weight concentration and absorbance at 620nm

R² = 0.9557

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12

Ab

sorb

an

ce

Concentration of C.necator

ppm

54

The graph represented in figure 3.3 manifested that there was a decrease in the absorbance

per day because the concentration of cells were reduced in the cultured medium. Decrease

in absorbance was the indication of deposition of biofilm at cathode.

Figure 3.3 The optical density measured at 620nm exhibits the decrease in absorbance

per day

3.9 Experimental conditions of batch system for Cupriavidus necator

The two stage substrate strategy has been employed to keep the substrate and biomass

concentration in BES to a desired level. After the heterotrophic pre-enrichment and

acclimation of biofilm at cathode and dissipation of organic carbon contents of substrate,

the system was switched from heterotrophic to autotrophic growth of cells by blowing the

gas mixture of O2/CO2 (10:90). Sterilized medium was transferred to anode and cathode

compartments of bio-electrochemical reactor. The oxygen and nitrogen in limited

conditions to trigger PHA synthesis at 32 0C. Oxygen supply more than 20% suppress the

PHA synthesis and critical for the safety level in anaerobic chamber. The anaerobic

chamber was continually gassed with gas mixture to maintain anoxic conditions in the

anaerobic chamber too. DC power supply connected to cathode and its potential was

adjusted to -400mV for microbial electrosynthesis without the significant synthesis of

hydrogen. The catholyte was agitated at 250 rpm for 10 minutes with regular interval of

time. pH was controlled by HCl and NaOH solutions and temperature in the incubator of

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Ab

sorb

an

ce

Days

55

anaerobic chamber was measured by thermometer sensor. After one week of cultivation

5ml of the samples were centrifuged for time interval of 24 hours to determine the

concentration of products.

3.10 Control cell for Cupriavidus necator

Experimental performance in the presence of nitrogen was conducted to study the effect of

nitrogen on PHA synthesis. Same set of experimental conditions were supplied to the

control cell along with nitrogen, sample was centrifuged and examined by GC-MS.

3.11 Experimental conditions of continuous system for Cupriavidus

necator

After the completion of batch system the autotrophic growth of PHB was conducted in

continuous chemostat system by connecting it with peristaltic pump (Longer pump BT

100-2J) at the dilution rate of 0.3ml/sec. Dilution of molasses to 50% enables the limitation

of cell growth in chemostat culture under steady state conditions. Steady state is a condition

in which current density, protein and substrate influent concentration is constant. The

concentration of these elements in the culture was determined by medium flow rate with

excessive level of other substrate flowing into the system. The maximum intracellular PHA

contents in autotrophic chemostat culture were obtained with CO2, carbon source at

specific growth rate of cells. The cathode compartment was spurge with gas substrate at

320C and was agitated at 250 rpm for 5 minutes for regular interval of time. DC power

supply was connected to cathode to provide -400mV. Liquid sample was transferred to

falcon tubes nearly 5ml after the time interval of 24 hours for GC-MS analysis. PHA

biosynthesis was based on dry cell concentration, polymer yield and amount of substrate

used.

56

3.12 Cyclic voltammetry for Cupriavidus necator

Cupriavidus enactor’s acceptance of electrons from cathode through direct electron transfer

was evaluated by employing cyclic voltammetry (CV) with carbon cathode the working

electrode against Ag/AgCl the reference electrode. Voltammograms were recorded by

applying a potential ramp at a scan rate of 10mV/s over the range of +0.2 to -0.6 V to the

working electrode. Cupriavidus necator showed the significant electroactivity with definite

redox peaks. CV can also be used to determine the electron shuttles or mediator produced

by bacteria (Venkata et al., 2015).

In the control cell cyclic voltammetry was performed with the fresh medium, the medium

without exoelectrogens under the same set of conditions applied in the experimental cell.

No redox reaction was observed with fresh medium and cell free culture.

3.13 FT-IR analysis

The characterization of functional groups were performed by FT-IR (Nicolet 6700 USA).

Fourier transform infrared spectroscopy is basically an analytical technique which gives

the spectra based on temporal coherence measurements from infrared source. It has ability

to identify unknown substance and chemical bonds present in it by IR-absorption spectrum.

Simple PHA structure were identified by using Nicolet FT-IR spectrometer in photo caustic

mode with spectral range and scan range of 4000-400cm-1 and rate 8m/s respectively

(Dharaiya et al., 2014 and Sergio et al., 2007).

3.14 Analytical Method

3.14.1 The Biopolymer Extraction for GC-MS Analysis

Chloroform extraction method was employed to recover PHA without its degradation and

high degree of purity. Cells with different PHA contents were collected by centrifuging

57

5ml of culture broth at 3500 rpm for 20 minutes at 25 0C. The supernatant was discarded

leaving behind the cell pellets. Solution containing 2ml of chloroform and 2ml of acidified

methanol (3% sulfuric acid) were added to cell pellets in the falcon tube with Teflon screw

caps. Mixture was heated and estrified at 100 0C for 3.5 hours. It was a developed method

for polymer extraction by (Braunegg et al., 1987). The sample was left for one hour at

room temperature for the development of three phases in the solution. The bottom phase

was transferred to the screw caped vial and used for GC-MS analysis while other two

phases of chloroform and methanol were discarded. The endotoxin level was less than

10EU/g of PHA level. The chloroform extraction method was efficient not only to reduce

the endotoxin level of gram negative bacteria but also for PHA recovery with high degree

of purity (Sang et al., 1999).

Addition of benzoic acid (2mg/ml) as an internal standard improved the reproducibility

and accuracy of polymers to be detected at low level of 0.5 ug.

3.15 The Biopolymer Analysis

The biopolymer analysis was based on procedures, SPME sample preparation, GC-MS

analysis and extraction of PHA.

3.15.1 SPME Procedure for Sample Preparation

Solid phase micro extraction (SPME) device and fiber coating polydimethylsiloxane

(100umPDMS) utilized for extraction and concentration of sample from liquid broth. For

extraction solution was placed in 20ml screw-cap vials with silicon/PTFE septa. About 2

ml of sample solution employed for sorption was stirred at 1000 rpm with magnetic stirrer

at 37 0C for 20 minutes. After extraction the fiber was desorbed directly to GC injector for

3 minutes. It was sufficient time for desorption of all analyte studied and reinserting the

fiber after the run without any carry-over (Wejnerowska et al., 2008).

58

3.15.2 GC-MS procedure

The quantification of polyhydroxyalkanoates was performed by GCMS analysis. Gas

chromatogram (Agilent Technologies 789A US) was equipped with MS (5975C inert XLEI

CI MSD detector), data acquisition system with computer software and capillary column

Agilent J&W (HP INNOWAX) with internal diameter 0.32mm and 60 m length. The

column temperature was maintained at 80 0C for 4 minutes followed by ramping at rate of

8 0C to 160 0C maintained for 3 minutes and then at the rate of 30 0C/min after that

temperature was increased to 2000C. The injector and detector temperatures were 250 0C

and 280 0C respectively. Helium was used as carrier gas at flow rate of 1.5ml/min. The

injection volume of the sample for analysis was 1 uL (Montaser et al., 2011b).

3.15.3 Extraction of dry PHA

PHA was extracted from lyophilized cells with chloroform at 80 0C for 6 hours. Solution

was filtered by Whatman filter paper to remove cell debris. After extraction the chloroform

was concentrated. The polymer dissolved in chloroform (2ml) was precipitated with equal

volume of ethanol and cooled at 40C to facilitate the precipitation process. The precipitated

polymer was separated by centrifugation at 5000 rpm at 40C and was placed on pre-

weighed watch glass and supernatant solution was disposed of. Sample was dried in air

for 24 hours at room temperature in fume cupboard for constant weight (Mona et al.,

2001). The w/w % of PHA from bacteria was calculated by equation.

………Equation 3.5

59

Figure 3.4 Wet biomass on pre-weighed watch glass

Figure 3.5 White precipitate of extracted PHA with ethanol

3.15.4 Calibration curve for extracted PHA

The calibration of standard solution of PHA was performed by GC-MS by following the

same set of conditions described. Benzoic acid was used as an internal standard to enhance

the accuracy and reproducibility. Calibration curve was made between standard

concentration of PHA and the peak area against concentration. The concentration of

unknown sample was calculated from calibration curve represented in Figure 3.6.

60

Figure 3.6 Calibration curve between concentration and peak area for polyhydroxy

alkanoates

R² = 0.9698

0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

0 5 10 15 20 25

Standard

peak

area

Concentration

ppm

61

3.16 Sporomusa ovata

Bacterial strain Sporomusa ovata was obtained from DSMZ (Deutsche Sammlung

Mikroorganismen and Zellkulturen GmbH) strictly anaerobic, gram-negative, banana-

shaped and non-pathogenic bacteria cultured in DSMZ medium 311 at 320C.

3.16.1 Media and cultivation method

All chemicals used were of highest purity (Sigma Aldrich chemicals Ltd.) and were used

without any further purification. Cells were grown in the medium with composition

K2HPO4 0.348g, KH2PO4 0.227g, NH4Cl 0.5g, MgSO4.7H2O 0.500g, CaCl2.2H2O 0.250g,

NaCl 2.25g, FeSO4.7H2O 0.002g, NaHSeO3 (10ml of 10-5molar solution), yeast extracts

2.0g, Casitone 2.0g, betaine.H2O 6.70g, NaHCO3 4g, resazurin 1mg, Na2S.9H2O 0.300g,

vitamin solution (10ml) and trace element solution (1ml).

3.16.2 Vitamin solution (medium 141)

Vitamin solution composition per liter was as follows,

Biotin 2.00mg, folic acid 2.00mg, pyridoxine-HCl 10.0mg, thiamine-HCl.2H2O 5.00mg,

riboflavin 5.00mg, nicotinic acid 5.00mg, D-Ca-pantothenate 5.00mg, vitamin B12

0.10mg, p-Aminobenzoic acid 5.00mg and Lipoic acid 5.00mg.

All chemicals were dissolved in deionized water and total volume was made up to 1.0 L.

The solution was mixed thoroughly by passing N2/CO2. After filter sterilization, the stock

solution was stored at 40C.

3.17 Trace element solution SL-10 (medium 320)

HCl (25%, 7.7 M) 10.00ml, FeCl2.4H2O 1.50g, ZnCl2 70.00mg, MnCl2.4H2O 100.00mg,

H3BO3 6.00mg, CoCl2.6H2O 190.00mg, CuCl2.2H2O 2.00mg, NiCl2.6H2O 24.00mg,

Na2MoO4.2H2O 36.00mg.

62

FeCl2 was dissolved first in HCl then in deionized water, other salts were dissolved and

final volume was made up to 1liter.

3.18 Stock solutions preparation

3.18.1 NaHSeO3.10-7 M solution

1M NaHSeO3 = 151g/ 1000ml

10-5M NaHSeO3 = 151x10-5g/1000ml

10-7M solution was prepared from 10-5M solution by dilution method.

10 ml of 10-5 M NaHSeO3 solution was diluted to 1000ml for the preparation of 10-7 M

solution of NaHSeO3.

3.18.2 Resazurin solution 500mg/1000ml

Resazurin solution contained 12.5mg of resazurin in 250ml of deionized water. It was filter

sterilized after thorough mixing and then kept at 40C in the refrigerator.

3.18.3 NaHCO3 solution

Bicarbonate solution was prepared by dissolving 4g of NaHCO3 in 80ml of deionized water

under CO2 + N2 atmosphere.

3.18.4 Lysozyme solution

Lysozyme solution contained 0.1g /100ml of Lysozyme dissolved in 0.01N HCl first and

then volume was made 100ml by deionized water. After thorough mixing it was filter

sterilized.

63

3.18.5 Phosphate buffer solution

K2HPO4 0.348g and KH2PO4 0.227g were dissolved in deionized water and volume was

made up to 200ml. Nitrogen gas was purged for 5 minutes.

3.18.6 Method for preparation of anaerobic medium

According to Sporomusa ovata medium 311 all chemicals were weighed and transferred

to medium bottle except bicarbonates, phosphate buffer, trace elements and vitamin

solution. Chemicals were dissolved in 500ml deionized water and boiled for five minutes.

Medium was cooled in ice water with N2/CO2 purging for 15 minutes. Then solution was

autoclaved for 20 minutes at 121 0C. After autoclaving all solutions were transferred to

anaerobic chamber. The medium was amended with trace element solution 1.0ml,

phosphate buffer solution 200ml, NaHCO3 solution 80ml, NaHSeO3 solution 10ml, vitamin

solution 10ml, resazurin solution 2ml and Lysozyme 10ml. pH of the medium was adjusted

to 7.0 with HCl. 400 uL of 3M HCl of pH 3. Then volume was made one liter with

deionized water.

The 10 ml of the medium was transferred to two serum bottles each sealed with sterile

butyl rubber stopper with 5ml syringe. Only the stopper or septa of butyl rubber are

efficient to prevent the permeation of air into vials. These septa are tightly fixed by sealing

with aluminum crimp. Seed culture about 2.0 ml was used for the inoculation of serum

bottles. These serum bottles were inoculated with Sporomusa ovata and were placed in the

incubator inside the anaerobic chamber at 320C. The 2ml of fresh medium was added to

serum bottles after every 24 hours. After 48 hours bacterial growth was checked under

microscope with proper gram staining. All stock solutions were stored at 40C in

refrigerator. Stock culture was stored at -20 0C in falcon tubes with 3:10 ml ratio of glycerol

and liquid broth for preservation.

64

3.18.7 Medium preparation with CO2 and H2 for Sporomusa ovata

Culture growth for all experiments was exploited in modified growth medium by omitting

betaine, fructose, casitone and resazurin. Culture was grown in serum bottles with medium

and gas mixture of H2/CO2 (10:90) at 320C in an anaerobic chamber. Medium and gas

addition was done during regular interval of time and pure cell growth was observed under

microscope by gram staining (Nevin et al., 2011c).

3.18.8 Cathode biofilm development

Anaerobically prepared 80ml sterile basal medium was added to each chamber of bio-

electrochemical cell. Cathode compartment was inoculated by pre-enriched culture only.

The headspace was purged with H2/CO2 to establish a biofilm at cathode. The medium was

replaced several times to remove planktonic cells. The periodic removal of planktonic cells

enhances the growth of biofilm at cathode surface. Favorable conditions are provided in

heterotrophic bio-mass growth phase to achieve high cell density. Microbial biofilm was

developed on carbon cloth after one week of inoculation. The whole reactor performance

was conducted in an anaerobic chamber (YQK-11 Jinan Unilab Instrument Co.Ltd). In

cathode a round carbon cloth and round stainless mesh plate and in anode a chrome plate

connected with power supply at -400mV (Nevin et al., 2010). The viability of Sporomusa

ovata was further evident with Scanning electron micrographs of carbon cathode surface.

`

3.18.9 Experimental conditions of batch system for Sporomusa ovata

After the heterotrophic pre-enrichment and acclimation of biofilm at cathode, 80ml of the

sterilized H2/CO2 fresh medium without Betaine, fructose, Casitone and resazurin was

transferred to anode and cathode compartment of Bio-electrochemical reactor. The

catholyte consisted of an optimized general growth medium for autotrophic culture of

Sporomusa ovata. The anaerobic chamber was continually gassed with N2/CO2 mixture to

65

maintain anoxic conditions in the chamber. DC power supplier connected to cathode to

provide -400mV electrons at a potential for microbial electrosynthesis without the

significant synthesis of hydrogen. The catholyte was continuously spurge with CO2 at 32

0C with regular interval of time as sole carbon source and electron acceptor to switch

microorganisms from heterotrophic to autotrophic metabolism. Limiting conditions for

oxygen were employed to avoid the gas explosion range and anoxic conditions for the

microbial growth. After one week cultivation 5ml of the samples were collected for time

interval of 24 hours centrifuged and supernatant was transferred to falcon tubes for GC-

MS analysis.

3.18.10 Continuous system for Sporomusa ovata

The batch system was switched to continuous system with peristaltic pump at the dilution

rate of 0.3 ml/sec. Current and total applied voltage was measured by digital voltmeter.

Gas phase was switched to CO2 and consumption of electron was directly from cathode for

the reduction of carbon dioxide the only carbon source to valuable products. The samples

were collected in sterile falcon tubes for time interval of 24 hours for GC-MS analysis.

Samples collected with intervals were centrifuged and supernatant was freezed in the

refrigerator (Nevin et al., 2008).

3.18.11 Control cell without electroactive catalysts

Control cell was employed for fresh medium and cell free culture using the same set of

conditions for experimental cell. The cathode was supplied -400mV by DC supplier.

Samples were analyzed in the absence of electroactive components by GC-MS.

3.18.12 Cyclic Voltammetry (CV) for Sporomusa ovata

Sporomusa ovata, s acceptance of electrons from cathode through direct electron transfer

was evaluated by employing cyclic voltammetry (CV) with carbon cathode the working

66

electrode against Ag/AgCl the reference electrode. Voltammograms were recorded by

applying a potential ramp at a scan rate of 10 mV/s over the range of +0.2 to -0.6 V to the

working electrode. Sporomusa ovata showed the significant electroactivity with definite

redox peaks. Voltammetry studies were employed to determine the redox potential of redox

active components, electrochemical activity of microbial strains and to test the

performance of cathode material with Potentiostat. Cyclic voltammetry was employed to

determine the electron shuttles or mediator produced by bacteria.

3.18.13 Control Cell without electroactive catalysts

Cyclic voltammetry in the control cell was performed in cell free suspension where the

supernatant was obtained by centrifuging cell and in the medium without electroactive cells

under the same set of conditions applied in the experimental cell. No redox reaction was

observed when cyclic voltammetry was analyzed with the fresh medium or cell free culture

(Choi et al., 2014).

3.18.14 Analytical Method for Sporomusa ovata

Organic products were analyzed by head space solid-phase micro extraction (HS-SPME)

followed by gas chromatography mass spectrometry (GC-MS).

3.18.15 Preparation of samples for GC-MS analysis

Liquid broth samples were stored at -160C and 2 ml was pipetted out into 20 ml screw-cap

vials with silicon/PTFE septa with 0.3 uL of 2-ethyl butyric acid as internal standard and

0.75g of NaCl mixed thoroughly. The pH was adjusted to 6.8 to 7 by 3 molar HCl and 0.15

M NaOH.

3.18.16 SPME Procedure

SPME device and fiber coating polydimethylsiloxane (100um PDMS/CAR poly dimethyl

siloxane/carboxene coated fiber) utilized for extraction and concentration of sample from

67

liquid broth. For extraction 2ml of sample solution was placed in 20 ml screw-cap vials

with silicon/PTFE septa. Sample solution employed for sorption was stirred at 1000 rpm

with magnetic stirrer at 37 0C for 20 minutes. After extraction the fiber was desorbed

directly to GC injector for 3 minutes. It was sufficient time for desorption of all analyte

studied and reinserting the fiber after the run without any carry-over (Wejnerowska et al.,

2008).

The quantification of organic products was performed by GCMS analysis. Gas

chromatogram (Agilent Technologies 789A US) was equipped with MS (5975C inert XLEI

CI MSD detector), data acquisition system with computer software and capillary column

Agilent J&W (HP INNOWAX) with internal diameter 0.32mm and 60 m length. The

column temperature was maintained at 70 0C for 2 minutes followed by ramping at rate of

40C/min to 180 0C maintained for 2 minutes and then at the rate of 20 0C/min after that

temperature was increased to 2000C and holding the final temperature for 3 minutes. The

injector and detector temperatures were 250 0C and 280 0C respectively. The GC system

was operated in split less mode with Helium as carrier gas at flow rate of 1.5 ml/min. The

injection volume of the sample for analysis was 1 uL (Montaser et al., 2011 and Lin et

al., 2008).

3.19 Calibration for volatile acids and alcohols

The calibration of standard solution was performed by GC-MS by following the same set

of conditions described above, 2-ethyl butyric acid was used as an internal standard and

volatile acid mixture from (C2 to C7) as an external standard to enhance the accuracy and

reproducibility. Calibration curve was made between known concentration of the standard

volatile acid mixture and 0.3 ml of 2-ethylbutyric acid as an internal standard in each

sample. The concentration of unknown sample was calculated by taking 2 ml of sample

with 0.3ml of internal standard and 0.75g of NaCl from calibration graph as embodied in

figure 3.7 and 3.8.

68

3.19.1 Calibration for volatile fatty acids

Figure 3.7 Calibration curve between concentration and peak area for volatile fatty acid

mixture

0.00E+00

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

1.20E+09

1.40E+09

0.1 0.3 0.5 0.7 1

Pea

k a

rea

Concentration (mM)

ethanoic acid

propanoic acid

butanoic acid

pentanoic acid

hexanoic acid

heptanoic acid

69

3.19.2 Calibration for volatile alcohols

Figure 3.8 Calibration curve between concentration and peak area for alcohols

0.00E+00

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

1.20E+09

0.1 0.3 0.5 0.7 1

Pea

k a

rea

Concentration (mM)

ethanol

butanol

pentanol

hexanol

heptanol

70

3.20 Clostridium ljungdahlii

The third bacterial strain used in this research was Clostridium ljungdahlii, obtained from

DSMZ Deutsche Sammlung Mikroorganismen and Zellkulturen GmbH. Clostridium

ljungdahlii, endospore forming, motile, rod shaped gram positive bacteria cultured in

DSMZ medium 879 at 37 0C (Nevin et al., 2011).

3.21 Medium and cultivation method for Clostridium ljungdahlii

All chemicals used were of highest purity (Sigma Aldrich chemicals Ltd.). Cells were

grown in the medium with composition per liter as follows.

NH4Cl 1.0g, Yeast extract 1.0g, NaCl 0.8g, MgSO4.7H2O 0.2g, KCl 0.1g, KH2PO4 0.1g,

CaCl2.2H2O 0.02g, Na2WO4.2H2O 0.20mg, Fructose solution 50.0ml, Trace element

solution 10.0 ml, Vitamin solution 10.0ml, NaHCO3 solution 10.0ml and Na2S.9H2O

solution 10.0ml.

3.21.1 Vitamin solution (medium 141)

The method followed to prepare Vitamin solution 141 as described in previous section

3.17.2 page 60 for Sporomusa ovata.

3.21.2 NaHCO3 solution per 10.0ml

NaHCO3 was 1.0g dissolved in deionized water and made the volume 10.0ml. Solution

was mixed thoroughly by sparging mixture of N2:CO2 (80:20%) and then filter sterilized.

After filter sterilization, the solution was kept in refrigerator at 40C.

71

3.21.3 Fructose solution per 50ml.

Fructose solution of 5.0g/50ml was prepared in deionized water and was mixed thoroughly

by sparging N2 and then filter sterilized. After filter sterilization the solution was kept in

refrigerator at 40C.

3.21.4 Na2S.9H2O solution per 10.0ml.

Na2S.9H2O was dissolved in deionized water 0.3g and volume was made up to 10.0 ml.

solution was mixed thoroughly and sparged with N2. Solution was autoclaved at 1210C for

20 minutes and then stored anaerobically.

3.21.5 Trace Element Solution per liter

Solution composition per liter was as follows

MgSO4.7H2O 3.0g, Nitrilotriacetic acid 1.5g, NaCl 1.0g, MnSO4.2H2O 0.5g, CoSO4.7H2O

0.18g, ZnSO4.7H2O 0.18g, CaCl2.2H2O 0.1g, FeSO4.7H2O 0.1g, NiCl2.6H2O 0.025g, K.Al

(SO4)2.12H2O 0.02g, H3BO3 0.01g, Na2MoO4.4H2O 0.01g, CuSO4.5H2O 0.01g and

Na2SeO3.5H2O.

Nitrilotriacetic acid was dissolved in 500.0 ml of deionized water by adjusting the pH

6.5with KOH. Rests of the compounds were dissolved and volume was made up to one

liter.

3.22 Method for Preparation of anaerobic medium for C. ljungdahlii

According to Clostridium ljungdahlii medium 879 all chemicals were weighed and

transferred to medium bottle except bicarbonates, fructose, Na2S.9H2O, trace elements and

vitamin solution. All these chemicals were dissolved in 800 ml deionized water and boiled

for five minutes. Medium was cooled in ice water with N2:CO2 (80:20%) purging for 15

minutes then autoclaved for 20 minutes at 121 0C. After autoclaving all solutions were

72

placed in anaerobic chamber. Rests of the solutions were mixed anaerobically and

aseptically.

Trace element solution 10.0ml, NaHCO3 solution 10.0ml, Na2S.9H2O solution 10.0ml,

vitamin solution 10.0 ml, and fructose solution 50 ml. pH of the medium was adjusted to

6.8 with HCl using 400 uL of HCl of pH 3. Then volume was made up to one liter with

deionized water.

The 10.0 ml of the medium was transferred to two serum bottles each sealed with sterile

butyl rubber stopper with 5ml syringe. These septa are tightly fixed by sealing with

aluminum crimp. Seed culture was used for the inoculation of serum bottles. These serum

bottles were inoculated with Clostridium ljungdahlii and were placed in the incubator

inside the anaerobic chamber at 37 0C. Fresh medium was added to serum bottles after

every 24 hours. All stock solutions were kept at 40C in refrigerator. After48 hours the

bacterial growth was checked under microscope with proper gram staining. Stock culture

of Clostridium ljungdahlii was stored at -20 0C in falcon tubes with 3:10 ml ratio of liquid

broth and glycerol for preservation.

3.22.1 Medium preparation with CO2 and H2 for Clostridium ljungdahlii

Clostridium ljungdahlii culture growth for all experiments was exploited in modified

growth medium by omitting fructose. Culture was grown in serum bottles with this medium

and gas mixture of H2/CO2 (10:90) was purged at 37 0C in these culture bottles. Medium

and gas addition was done during regular interval of time and pure cell growth was

observed under microscope by gram staining.

73

3.23 Cathode biofilm development

Anaerobically prepared 80 ml sterile basal medium with fructose was added to each

chamber of bio-electrochemical cell. Cathode compartment was inoculated by pre-enriched

culture only. The headspace was purged with H2/CO2 to establish a biofilm at cathode. The

medium was replaced several times to remove planktonic cells. The periodic removal of

planktonic cells enhances the growth of biofilm at cathode surface. Favorable conditions

are provided in heterotrophic bio-mass growth phase to achieve high cell density. Microbial

biofilm was developed on carbon cloth after one week of inoculation. The whole reactor

performance was conducted in an anaerobic chamber (YQK-11 Jinan Unilab Instrument

Co.Ltd). The cathode was poised at -400mV with DC power supply for potential difference

between cathode and anode. The anaerobic chamber was continually gassed with N2/CO2

mixture to maintain anoxic conditions in the chamber. The microbial cell concentration in

the cultured media was determined by optical density at 620 nm based on calibration curve

by UV spectrophotometer (UV-1602 BMS Biotechnology Medical services). The decrease

in the absorbance per day indicates the deposition of biofilm at cathode (Sharifzadeh et

al., 2009). The viability of C. Ljungdahlii was further evident with Scanning electron

micrographs of carbon cathode surface.

3.24 Experimental conditions for batch system for Clostridium ljungdahlii

After the heterotrophic pre-enrichment and acclimation of biofilm at cathode, 80ml of the

sterilized H2/CO2 fresh medium without fructose was transferred to anode and cathode

compartment of bio-electrochemical reactor. The catholyte consisted of an optimized

general growth medium for autotrophic culture of Clostridium ljungdahlii. The anaerobic

chamber was continually gassed with N2/CO2 mixture to maintain anoxic conditions in the

chamber. DC power supply was connected to cathode at -400mV for microbial

electrosynthesis without the significant synthesis of hydrogen. The catholyte was

continuously spurge with CO2 at 37 0C with regular interval of time as sole carbon source

and electron acceptor to switch microorganisms from heterotrophic to autotrophic

metabolism. Limiting conditions for oxygen were employed to avoid the gas explosion and

74

to provide anoxic conditions for the microbial growth. After one week cultivation 5ml of

the samples were collected for time interval of 24 hours centrifuged and supernatant was

transferred to falcon tubes for GC-MS analysis. The sample were centrifuged and

supernatant was freezed in the refrigerator (Nevin et al., 2010).

3.25 Continuous system for Clostridium ljungdahlii

The batch system was switched to continuous system with peristaltic pump at the dilution

rate of 0.3 ml/sec. Current and total applied voltage was measured by digital voltmeter.

Gas phase was switched to CO2 and consumption of electron was initiated directly from

cathode for the reduction of carbon dioxide the only carbon source. The samples were

collected in sterile falcon tubes for time interval of 24 hours for GC-MS analysis. Samples

collected after the definite time intervals were centrifuged and supernatant was freezed in

the refrigerator.

3.25.1 Control cell without electroactive catalysts

Control cell was employed for fresh medium and cell free culture using the similar

conditions for anode, cathode, temperature and pH. The cathode was poised at -400mV by

DC supplier. Samples were analyzed in the absence of electroactive components by GC-

MS.

3.26 Cyclic Voltammetry (CV) for Clostridium ljungdahlii

Clostridium ljungdahlii acceptance of electrons from cathode through direct electron

transfer was evaluated by employing cyclic voltammetry (CV) with carbon cathode the

working electrode against Ag/AgCl the reference electrode. Voltammograms were

recorded by applying a potential ramp at a scan rate of 10mV/s over the range of +0.2 to -

0.6 V to the working electrode. Clostridium ljungdahlii showed the significant

electroactivity with definite redox peaks. Cyclic voltammetry was employed to determine

the electron shuttles or mediator produced by bacteria.

75

Voltammetry studies were employed to determine the redox potential of redox active

components, electrochemical activity of microbial strains.

When the Clostridium ljungdahlii strains were cultivated with carbon cloth vs. Ag/AgCl

reference electrode, culture showed the current consumption during 48 hours of cultivation

indicating the development of electroactive biofilm at cathode.

3.26.1 Cyclic voltammetry of Control Cell without electroactive catalysts

Cyclic voltammetry in the control cell was performed in cell free suspension where the

supernatant was obtained after centrifuging. In order to rule out the effect of medium on

redox activity of cathode, medium without electroactive catalysts under the same set of

conditions was placed in experimental cell for CV analysis No redox reaction was

observed when cyclic voltammetry was analyzed with the fresh medium or cell free culture

(Choi et al., 2014).

3.27 Analytical Method for volatile fatty acids and alcohols

Organic products were analyzed by head space solid-phase micro extraction (HS-SPME)

followed by gas chromatography mass spectrometry (GC-MS), by following the same

method adopted for Sporomusa ovata. Calibration curves for volatile fatty acids and

alcohols (figure 3.7 and 3.8) were used for the estimation of the products.

76

Chapter 4

Results and discussion

77

4 Results and discussion

Results and discussions for the three microbial strains are discussed below.

4.1 Cupriavidus necator

Current study was conducted for bio-electrochemical synthesis of PHA by exploiting

facultative autotrophic bacteria. The facultative autotrophic bacteria Cupriavidus necator

were selected to capture electron directly from cathode instead of hydrogen to reduce CO2.

Cupriavidus necator contains fln-like adhesion gene cluster which is used for tight, non-

specific adhesion to cathode surfaces for direct transfer of electrons (Pohlmann et al.,

2006). Bio-electrochemical studies have been conducted previously for harvesting waste

greenhouse gas to organic acids and alcohols where electro autotrophs utilize electric

current obtained from renewable energies (Zhang et al., 2015 and Nevin et al., 2011).

However bio-electrochemical synthesis of PHA using electric current is not previously

reported. The previous studies to synthesize PHA were based on fermentation and fed-

batch processes. The drawbacks of processes were long time duration and more chance for

contamination.

In a current study a Specific method was developed for the first time for the synthesis of

PHA based on the development of autotrophic bio-cathode centered on heterotrophic pre-

augmentation. The present research was based on bio-electrochemical synthesis with two

stage-strategy of heterotrophic-autotrophic PHA production from waste low cost

substrates. Heterotrophic approach enhanced biomass production whereas autotrophic

approach was to produce PHA. Cupriavidus necator were heterotrophically grown first on

molasses and after pre-enrichment and acclimation of culture carbon dioxide was delivered

as sole carbon source for autotrophic growth.

In previous bio-electrochemical studies hydrogen was good approach for reduction

reaction during bio production, however hydrogen has shortcomings for microbial

metabolism due to its low solubility the microbial environment has to be pressurized to get

concentrations of the products. So cathodic bio production needs to circumvent hydrogen

(Rabaey et al., 2010). In present study specific implementations were developed for the

78

synthesis of bio-cathode where cathode served as the source of electrons to get desired

product.

The experiment was investigated for both experimental and control reactor in the presence

and absence of nitrogen supply in double chambered reactor partitioned by proton

exchange membrane. The isolated samples were screened for PHA synthesis and were

characterized by GC-MS method of identification and quantification.

Accumulation of PHAs inside the cell is the natural way to store carbon and energy. These

contents were practiced during the nitrogen limitation environment. Presence of nitrogen

leads the metabolic pathway to Krebs cycle for cell growth and energy synthesis. Under

nitrogen and phosphorus limitation and presence of excess carbon, acetyl-CoA pathway is

directed towards PHA accumulation. PHA monomer is represented in the formula with (R)

group. The mechanical properties of PHA depend upon monomer unit. Variation in the

functional group, chain length and unsaturated bonds in PHA structure tailored towards

larger number of application.

Poly-3-hydroxyalkanoates.

The cultivation method for cell mass growth and PHA accumulation by utilizing carbon

dioxide as sole carbon source were according to the equations.

12CO2 + 29H2 2C6H11O3 + 18H2O…....Equation 4.1

2CO2 + 4H2 C2H4O2 + 2H2O………. Equation 4.2

8CO2 + 21H2 C8H16O3 + 13H2O……. Equation 4.3

18CO2 + 49H2 2C9H17O2 + 32H2O….. Equation 4.4

The stoichiometric electro-autotrophic growth of polymers were calculated from the

chemical equations and substrate mole concentration by utilizing ideal gas law.

79

The GC-MS results exhibited the synthesis of organic acids like acetic acid and butyric

acids. These acids were further utilized for the synthesis of PHA in the enriched culture of

Cupriavidus necator. This can be proved from the decrease in the concentration of acetic

acid as represented in the figure 4.3. The optimal experimental conditions employed in

whole experiment, enhanced the dry cell weight and PHA contents to a considerable extent

which was comparable to the PHA contents with pure glucose in fermentation reactors, as

conducted in earlier investigations (Grousseau et al., 2014 and Montaser et al., 2011).

Table 4.1 Synthesis of PHA from dry cell mass after 48 hours

Cell dry weight

mg /50 ml of sample

Extracted Dry

weight of PHA

mg

PHA

synthesis

(%)

342

240

70.17

4.2 Gas Chromatography Mass Spectrometry analysis

The structure and mole fractions of PHA and other compounds were determined by GC-

MS based on peak area of ions. Various identified compounds along with molecular

weight, retention time and displayed formula for both batch and continuous systems were

manifested in the table 4.2.

80

Table 4.2 GC-MS analysis of biodegradable polymer and their chemical composition

(RT; retention time in minutes, CN; compound name, MW; molecular weight, MF;

molecular formula).

RT

CN

MW

MF

8.58

Acetic acid

60

O

OH CH

3

C2H4O2

14.14

3-hydroxy2,2

dimethyl butyrate

131.18 O

O

CH3

CH3

CH3 OH

C6H11O3

14.55

Ethyl 3-hydroxy

hexanoate

160.21 O

O OH

CH3

CH3

C8H16O3

15.19

2, 4 dimethyl

heptanoate

157

C9H17O2

Results exhibited that higher the molecular weight higher the corresponding retention time

like 3-hydroxy 2, 2 dimethyl butyrate and 2, 4 dimethyl heptanoate and lower the molecular

weight and lower the retention time like acetic acid, but in actual the retention time is

independent of molecular mass (Ogunjobi et al., 2013). Peak intensity was related directly

to the concentration of the products.

4.3 Bio-electrochemical synthesis of PHA during batch system

The concentration of polymers during batch system was analyzed by GC-MS analysis.

Concentration of PHA were calculated from the calibration curve after time interval of 24

hours. Concentration was dependent on peak area for every product on gas chromatogram

and maximum concentration was observed at time interval of 120 hours. Figure 4.1

81

represented the maximum concentration of the different polymers estimated against

calibration curve.

Figure 4.1 Bio-electrochemical synthesis of 3-hydroxy dimethyl butyrate, ethyl 3-

hydroxy hexanoate, 2, 4 dimethyl heptanoate concentration in ppm against time duration

for batch system

4.4 Bio-electrochemical synthesis of PHA during continuous system

The bio-electrochemical synthesis of PHA for continuous system was represented in figure

4.2. The concentration of 3-hydroxy dimethyl butyrate was greater than other two

polymers, the ethyl 3-hydroxy hexanoate, 2, 4 dimethyl heptanoate. The comparative study

of batch and continuous systems revealed that bio-electrochemical synthesis of PHA was

greater in continuous system. The concentration obtained for 3-hydroxy dimethyl butyrate,

ethyl 3-hydroxy hexanoate, and 2, 4 dimethyl heptanoate were 79.2, 14.83 and 14.71 ppm

respectively which were greater than batch system, but difference in the concentration was

minute and polymers were in close proximity to both continuous and batch systems. Thus

PHA synthesis by pure culture was more than 70%. In 2013 (Okwuobi and Ogunjobi)

reported the isolated cell dry weights for glucose 0.0203g/L, for fructose 0.25g/L and for

lactose 0.0652g/L and corresponding PHB yields were 0.0143, 0.065 and 0.079 g/L

respectively. The results quoted by (Anjali et al., 2014) from molasses were 2.09g PHA

0

10

20

30

40

50

60

70

80

24 48 72 96 120

Co

nce

ntr

ati

on

(pp

m)

Time ( hours)

3-hydroxy dimethyl

butyrate

ethyl 3-hydroxy

hexanoate

2,4-dimethyl

heptanoate

82

production from dry cell weight of 1.5g/L, similarly the amount of P(3HB) produced was

1.35g/L from cell dry mass 2.0g/L (Sharifzadeh Baei and Najafpour et. al.). The working

methods employed in the above mentioned research was fermentation whereas the present

research was based on bio-electrochemical synthesis of PHA by simplest form of rector.


hexanoate, 2, 4 dimethyl heptanoate concentration in ppm against time for continuous

system

4.4.1 Decrease in Acetic acid concentration during batch and continuous system

It was figured out that decrease in the concentration of the acetic acid reflected its further

consumption during the metabolic reactions of PHA synthesis by Cupriavidus necator.

0

10

20

30

40

50

60

70

80

90

24 48 72 96 120

Con

cen

trati

on

.

(pp

m)

Time (hours)

3-hydroxy dimethyl

butyrate

ethyl 3-hydroxy

hexanoate

2, 4-dimethyl

heptanoate

83

Figure 4.3 Comparison in Acetic acid concentration in both batch and continuous system

Figure 4.3 exhibited that decrease in the concentration of acetic acid increased the density

of PHA granules inside the cells. Acetic acid was the dominant component during the 24

hours round at pH 7, but decrease in its concentration gradually enhance the PHB synthesis

effectively. Moreover butanoic and propanoic acid concentration were observed in minute

quantities at pH level 7which proved to be more beneficial for the synthesis of PHB and

PHH polymers. The volatile fatty acids (VFA) synthesized were consumed further to yield

PHA.

PHA plays a crucial role in priming electro autotrophs for stress survival. Under nutrients-

scarce conditions PHA promotes the long term survival of these PHA producing bacteria

like Cupriavidus necator the metabolic flux from Acetyl-CoA to PHA is nutrient

dependent. The synthesis of high amount of coenzyme A from Krebs cycle under nutrient

rich conditions blocks the PHA synthesis by inhibiting 3-ketothiolase. Actually the Acetyl-

CoA is channeled to the Krebs cycle for cell growth and energy synthesis. Under

unbalanced nutrients conditions like nitrogen, phosphorus limitation and excess of carbon

allows Acetyl-CoA to be directed toward PHA synthetic pathways for PHA accumulation

(Grousseau et al., 2014). When bacterial cells are supplied by inadequate nutrients a

-5

0

5

10

15

20

25

30

24 48 72 96 120

Co

nce

ntr

ati

on

(pp

m)

Time (hours)

Concentrationin

(ppm) batch system

Concentration (ppm)

continuous system

84

build-up of NADH is observed. NADH inhibits the synthesis of citrate resulting in Acetyl-

CoA not being oxidized at enough rate by Krebs cycle and subsequently accumulates. The

supply of -400mV corresponds to the reduction potential of NADH. High concentrations

of NADH and Acetyl-CoA shifted the equilibrium in favour of PHA synthesis. Coenzyme

A is carrier of acyl groups Acetyl-CoA is converted to PHA by three enzymes. Two

molecules of Acetyl-CoA condense to form dimer acetoacetyl-CoA which involves the

action of 3-Ketothiolase. The action of NADPH dependent enzyme acetoacetyl-CoA

reductase reduces the acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (Estelle et al., 2014

and Razaad et al., 2014). By polymerization of (R)-3-hydroxybutyryl-CoA by enzyme

PHA synthase, PHA is formed. This metabolic strategy enables the PHA accumulating

electroactive catalysts in their adaptation to environmental stress conditions.

85

Figure 4.4 Acetyl Co-A pathway for Polyhydroxyalkanoates (Estelle et al., 2014 and

Razaad et al., 2014)

4.5 Coulombic Recovery

Electron distribution value and final products were calculated by converting each

compound to e- equivalent unit by multiplying e- equivalent number per mole by mole of

each compound. For polymers 3-hydroxy dimethyl butyrate, ethyl3-hydroxy hexanoate, 2,

4 dimethyl heptanoate the e- equivalent number per mole were 58, 42, and 98 respectively.

Electron recovered in polymer synthesis were calculated by following Equation 3.1

(Logan et al., 2008).

86

4.6 Electrons recovery against the current consumption

The electron recovery in the products was calculated by comparing the current recovered

in the products against the current consumed by the system. The total amount of the current

consumed by system was calculated by integrating the total area under current (A/m2) vs.

time (sec) over F the Faradays constant (96500 C/mol) and x were the equivalent number

of electrons per mole of the polymer equation 3.3. The equivalent number of electrons per

mole of products were obtained from equations were 58, 42 and 98 for polymers 3-hydroxy

diethyl butyrate, ethyl3-hydroxy hexanoate, 2, 4 dimethyl heptanoate respectively. The

amount of current measured for Cupriavidus necator against -400mV applied potential was

0.0171 A/m2. The electron recovery in the all products during batch system as illustrated

in figure 4.5 were obtained against the total current consumption.

4.6.1 Electron recovery during batch system


polymers during batch system

The electron recovery in polymer formation against the total current consumption during

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

-0.00005

0

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

0 24 48 72 96 120

Cu

rren

t r

ecover

ed in

org

an

ic

com

pou

nd

s

Cou

lom

bs

Co

nce

ntr

ati

on

of

org

an

ic c

om

pou

nd

s

in

(mole

s)

Time (hours)

3-hydroxy diethyl butyrate

ethyl 3-hydroxy hexanoate

2,4-dimethyl heptanoate

total current

87

batch system was maximum for 3-hydroxy diethyl butyrate and minimum for ethyl 3-

hydroxy hexanoate, 2, 4 dimethyl heptanoate respectively.

4.6.2 Percent cathode recovery during batch system

Percent cathode recovery of every PHA molecule was calculated by equation 3.2 and was

maximum for 3-hydroxy diethyl butyrate as compared to ethyl 3-hydroxy hexanoate, 2, 4

dimethyl heptanoate as in figure 4.6.

Figure 4.6 Percent cathode recovery for organic polymers by Cupriavidus necator during

batch system

4.6.3 Electron recovery during continuous system

Coulombic recovery of electrons in continuous system where influent flow rate was 0.3

ml/sec and change in substrate concentration was measured in g/L. The stoichiometric

calculations for the consumption of carbon dioxide were calculated from chemical

equations 4.1-4.4 mentioned above. Data exhibited in figure 4.7 represented the total

current consumed by bio-film and the moles of products recovered from the system.

Maximum recovery was observed at time 120 hours and percent cathode recovery was

obtained maximum for polymer 3-hydroxy dimethyl butyrate compared to other polymers.

0

20

40

60

80

100

120

24 48 72 96 120

Cath

od

e re

cover

y x

100

Time (hours)

3-hydroxy diethyl

butyrate

ethyl 3-hydroxy

hexanoate

2,4-dimethyl

heptanoate

88


polymers in continuous system

4.6.4 Percent cathode recovery during continuous system

Figure 4.8 Percent cathode recovery for organic polymers by Cupriavidus necator in

continuous system

0

0.02

0.04

0.06

0.08

0.1

0.12

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0 24 48 72 96 120

Cu

rren

t re

cov

ered

in

org

an

ic

com

po

un

ds

in

Co

ulo

mb

s

Co

nce

ntr

ati

on

of

org

an

ic

com

po

un

ds

in

(mo

les)

Time (hours)

moles of 3-hydroxy diethyl

butyrate

ethyl 3-hydroxy hexanoate

2,4-dimethyl heptanoate

total current

0

5

10

15

20

25

30

35

0 24 48 72 96 120

Ca

tho

de

reco

ver

y x

100

Time (hours)

% 3-hydroxy diethyl

butyrat

% ethyl 3-hydroxy

hexanoate

%2,4-dimethyl

heptanoate

89

The percent cathode recovery for 3-hydroxy diethyl butyrate was maximum about 30%

and percent cathode recovery of other two polymers were 0.70 and 1.5% respectively in

figure 4.8.

In comparison the results obtained in batch system for 3-hydroxy dimethyl butyrate, ethyl

3-hydroxy hexanoate and 2, 4 dimethyl heptanoate were 97, 19 and 33% respectively at

minimum time duration of 120 hours. Better electron recovery was observed in batch

system due to the inclusive utilization of substrate. The cathode was poised at -0.4V by

DC power supply because the previously used cathode potential -0.6 to 0.7 V were low

enough to produce hydrogen. The hydrogen yield in BES cell triggers the electro

autotrophic biofilms to scavenge hydrogen as electron donor before it was lost through the

gas stream. It was reported that these electro autotrophs were unable to remove all

hydrogen at cathode so this loss of hydrogen instigated low recovery of products which

proved to be expensive. So instead of hydrogen bio-cathode was designed to transfer

electron from cathode to biofilm. In present research DC power supply was used instead

of potentiostat poised cathode as employed in previous BES researches. The fixed potential

with potentiostat was difficult to handle and its constant potential can damage the

biocatalysts. The electron recovery with DC power source was 97% for PHB production

and the result was comparable to previously reported (Nevin et al., 2010) recovery of 87%

for acetate production with potentiostat poised system.

4.7 Gas Chromatograms and Mass Spectrums for Cupriavidus necator

The structure and mole fractions of various organic product of polymers were analyzed by

GC-MS and results are shown in Figures 4.9 - 4.11 based on GC-MS peak area of ions in

the limited supply of nitrogen. By investigating the molecular mass of the fragments the

specific peaks in the spectra were correlated to carbonyl and hydroxyl groups of

polyalkanoates.

Many peaks were observed in gas chromatogram other than desired products.

90

Figure 4.9 Gas Chromatogram of PHA polymer

Figure 4.10 Mass spectrum of poly hydroxyalkanoates

Figure 4.11 Mass spectrum of acetic acid

In the GC-MS graphs the height of peaks represents the concentration of PHA. According

to the literature the molecular weight of polymers should not increase from120. If peaks

exceed from 120 m/z, it indicates the presence of other lipid contaminants. The mass

spectrum of Cupriavidus necator exhibited the peaks less than 120 m/z indicating no

contamination in products. In the gas chromatogram the methyl ester derivative of

91

polyhydroxy alkanoates manifested the peaks with retention time 8.5, 14.14 and 14.55 and

15.19 min. The peaks obtained in mass spectrum at m/z of 87 and 101 were rationalized to

hydroxybutyrate (PHB) and hydroxyvalerate (PHV) species. The peak area of ions at m/z

of 60 corresponding to that of acetic acid species. The m/z of 105 with retention time 13.32

was for benzoic acid, utilized as internal standard in GC-MS analysis (Paramjeet et al.,

2012). The retention time is independent of molecular mass (Ogunjobi et al., 2013).

4.8 Characterization of PHA in the presence of nitrogen supply

The samples from control cell in presence of nitrogen were analyzed by Gas

chromatography. The ambiguous peaks appear in gas chromatogram were not identified as

PHA demonstrated in figures 4.12 and 4.13.

Figure 4.12 Gas chromatogram under nitrogen supply

Figure 4.13 Mass spectrum with nitrogen supply

The mass spectrum of organic compound with m/z 122.1 and retention time 13.3 stand for

benzoic acid with molecular formula C7H6O2. In actual the specific growth rate of the cells

92

decreased when limited by the deficiency of nitrogen. The experiment conducted under

nitrogen supply exhibited the suppression in the PHA synthesis. In the presence of nitrogen

TCA cycle was followed and synthesis of acids and bio-mass was observed. The m/z of

benzoic acid 105 represented the internal standard in the gas chromatogram. No sharp

peaks for PHA were observed, they were formed but not in significant quantity as

compared to the results obtained in limited supply of nitrogen. The highest PHA are

observed under nitrogen, sulfur and phosphorus deficiency while low PHA contents are

obtained under potassium or magnesium deficiency (Tatiana et al., 2013)

4.9 Cyclic voltammetry results for Cupriavidus necator

Cyclic voltammetry of Cupriavidus necator was employed with carbon electrode as

working electrode against Ag/AgCl reference electrode to investigate the electrochemical

activities of these facultative autotrophic catalysts. Voltammograms were recorded by

applying a potential ramp at a scan rate of 10mV/s over the range of +0.2 to -0.6 V to the

working electrode (Marshall et al., 2012). The selection of scan rate was 10 mv/sec

therefore the potential change was very slow, so at each applied potential all proteins

oxidized or reduced multiple time. The culture exhibited the current consumption after the

48 hours of cultivation. After inoculation the peak area was significantly increased revealed

Cupriavidus necator’s significant peak current with definite oxidation and reduction

activity.

93

Figure 4.14 Cyclic Voltammetry of Cupriavidus necator for biotic, abiotic cathode and

fresh medium against Ag/AgCl reference electrode at scan rate of 10mV/s

The shape of reduction peak was different from oxidation peak. The microbial strains

showed the redox peak at (-0.6022644mV~ -1.8921E-04mA/cm2) and oxidation peak at

(0.20397172mV~2.05389E-05mA/cm2) during 48 hours of cultivation.

4.9.1 Cyclic Voltammetry analysis for Control Cell without electroactive

catalysts

No redox reaction was found when cyclic voltammetry was analyzed by fresh medium as

represented in the figure 4.14 implying the absence of electroactive components in the

medium.

For further analysis after 48 hours cultivation and biofilm development the planktonic cells

were drained out and fresh medium was filled in the cathode compartment against Ag/AgCl

reference electrode. The current consumption was recovered again indicating the electron

94

transfer without mediators in the medium. Interruption in the voltage supply affects the

activity of electro-autotrophs and reduce the synthesis.

Scanning Electronic Micrography (SEM) was utilized to visualize the prevalence of

electroactive cells attached to cathode. The biocathode was treated with 2-

bromoethanesulfonic acid, rod shaped bacteria were dominant on electrode. The

cytoplasmic space was packed by inclusion bodies of PHB varying in their diameter. The

PHB inclusion in cytoplasm appeared to be packed in the center suggests that synthesis

and initial storage of PHB is taking place. Even though many inclusions were densely

packed with free space in the cytoplasm reflected that maximum storage of the cells has

not been achieved. Some cells observed were without inclusion bodies in their cytoplasm.

As the carbon source is depleted in the medium these electroactive cells begin to break

down the PHB inclusions via intercellular PHB depolymerase to monomers and dimers.

The consistency of electroactive catalysts at cathode was evidenced from CV, which was

supportive for microbial catalyst at cathode surface. White PHB rich granules were

visualized after 48 hours of cultivation. Small granules represent the low yields but

maximum granules were observed at 120 hours of cultivation

Figure 4.15 Scanning electronic micrographs of rod shaped PHB rich Cupriavidus necator

4.10 FT-IR Analysis

The presence and characterization of functional groups of extracted PHB were confirmed

and identified by Fourier transform infrared spectroscopy FT-IR (Nicolet 6700 USA).

Spectra were recorded between spectral range 4000-400 cm-1 to confirm various functional

groups of extracted polymer in figure 4.16. This spectrum analysis gives further insight

95

into the structure of polymer. Absorption bands occurring at 2935 and 2874 cm-1indicated

the presence of aliphatic –CH3 and –CH2 groups. Absorption bands at 1739 and 1276 cm-1

were corresponding to C=O and C-O stretching groups and were identical to PHB from

Cupriavidus necator. The absorption bands at 3000 and 3400 cm-1 were for C-H and OH

groups. Results presented in figure were identical to the results in literature

Figure 4.16 FT-IR spectroscopy of PHB biopolymer by Cupriavidus necator

The absorption bands are in accordance with those stated in literature (Thawadi et al.,

2012). All absorption bands appeared in spectrum reflected the monomeric units of PHA.

The bio-electrochemical synthesis by Cupriavidus necator with industrial waste molasses

and greenhouse gas CO2 substantially reduced the substrate cost and downsize the cost of

production. The methodology utilized further to simplify the reactor with DC power source

enhanced the coulombic efficiency and electron recovery in products. The 75% PHB

production with recent new technique was equivalent to previous studies.

Statistical analyses were performed using the SPSS statistical package (version 23.0). Data

means were tested at significance levels of P < 0.05 using one way ANOVA.

96

Table 4.3 Comparison (ANOVA) of continuous and batch process for Cupriavidus necator

Source N Mean Std.

Deviation

Std.

Error

95% Confidence Interval

for Mean

Minimu

m

Maximu

m

Lower

Bound

Upper

Bound

Continuous

process (1.00) 15 11.0651 10.13270 2.61625 5.4537 16.6764 -0.05 33.91

Batch Process

(2.00) 15 19.3654 22.47475 5.80295 6.9193 31.8115 2.00 79.20

Total 30 15.2152 17.64176 3.22093 8.6277 21.8028 -0.05 79.20


continuous and batch process for harvesting waste (molasses and carbon dioxide) to

biopolymer (PHB) using Cupriavidus necator

Sum of Squares Df Mean

Square

F value p-value

Between Groups 516.716 1 516.716 1.700 0.203

Within Groups 8509.003 28 303.893

Total 9025.718 29

One-Way ANOVA was performed for Cupriavidus necator manifested in the Table 4.3 &

Table 4.4. It was found that there is no significant difference (p=0.203) between both

processes (p>0.05). Hence, statistically it can be concluded that both batch and continuous

processes do not vary significantly in terms of concentration levels. However, during

performance, standard error for batch process is more than continuous one. It is concluded

that PHB can be synthesized using any process.

97

4.11 Sporomusa ovata

Bio-electrochemical synthesis of plethora of products were compared both in batch and

continuous system by Sporomusa ovata where CO2 was exploited to provide the desired

concentration of carbon contents. An external power supply at cathode as sole electrons

donor for reduction of CO2 to diversity of extracellular multicarbon products. S.ovata can

grow on cathode surface to derive electron for multicarbon compounds providing the proof

for the concept that the carbon dioxide can be converted to valuable products with supply

of electricity. S.ovata the Gram negative bacteria interacts with cathode by C-type

cytochromes and IV pili (Tremblay et al., 2015). It has been mentioned that C-type

cytochromes are responsible for extracellular electron transfer pathway in electrotrophic

bacteria (Nevin et al., 2011). Thus pure culture of acetogen has tendency to accept

electrons directly from cathode to commodities with high coulombic efficiencies. Further

these screened samples were characterized by GC-MS method of identification and

quantification of organic compounds.

Most of the previous research were based on fermentation process for the synthesis of

biofuels like ethanol, butanol and acetone with variety of substrates. Bio-electrochemical

synthesis was explored by (Nevin et al, 2010) who converted CO2 to acetate first time. To

this date BES has been exploited for acetate, butyrate, methane, hydrogen and ethanol

(Batlle et al, 2015) and (Marshall et al, 2012) and other high value added products (Rabaey

et al., 2010). The present research effort was currently focused to synthesize higher value

added compounds ethanol, butanoic acid, pentanol, hexanoic acid and hexanol with pure

culture of acetogens. The implementation of simplified reactor with two stage strategy of

heterotrophic and autotrophic development of biocathode revealed the recent advances

with concomitant production of acetate, ethanol, butanoic acid, pentanol and hexanol. In

recent study pure culture was preferred over mixed communities because according to

previous studies mixed cultures attributed complex metabolic reactions and utilization of

bioproducts by other strains.

98

4.12 Characterization of Bio electrochemically synthesized organic

compounds

The structure and mole fractions of organic acids and other compounds were determined

by GC-MS based on peak area of ions. Various identified compounds along with molecular

weight, retention time is represented in the table 4.5. The major compounds were volatile

fatty acids and alcohols. The highest molecular weight of 116.16 was observed for 2-ethyl

butyrate with corresponding retention time of 21.69. The lowest retention time of 11.2 was

observed for ethanoic acid of molecular weight 60.

Table 4.5 Chemical composition of volatile fatty acids and alcohols revealed by GC-MS

analysis

CN Acetic acid Ethanol Butanoic

acid

Pentanol

Hexanol 2-

ethylbutyr

ate

RT 11.2 16.04 16.9 17.3 20.9 21.69

MW 60 46.06 88.11 88.14 102.17 117.16

DF

MF C2H4O2 C2H6O C4H8O2

C5H12O C6H14O C6H12O2

(RT; retention time in minutes, CN; compound name, MW; molecular weight, MF;

molecular formula; DF displayed formula)

99

4.13 Characterization of organic products synthesized by Sporomusa ovata

The structure and mole fractions of various organic products were based on GC-MS peak

area of ions figures 4.17- 4.20.

Figure 4.17 Gas Chromatogram of various acids and alcohols from Sporomusa ovata

Figure 4.18 Mass spectrum of ethanoic acid

The gas chromatogram of Sporomusa ovata with numerous peaks characterize the presence

of various organic products. Peak with retention time 19.17 exhibited internal standard 2-

ethyl Butanoic acid. The integrated peaks on gas chromatogram exhibiting the various

organic products at m/z 60, 88 and 117 for ethanoic acid, butanoic acid and 2-ethyl butyrate

with retention time 11.2, 16.9 and 21.69 min respectively. Hexanol, ethanol and pentanol

with m/z 102.17, 46.06 and 88.14 with retention time 20.9, 16.04 and 17.3 were clearly

displayed in gas chromatogram figure 4.17. The peaks at retention time 19.01, 21.67, 21.9

indicating the presence of acetic acid diethyl, allyl 2-ethyl butyrate and propyl 2-ethyl

valerate most probably the complex metabolites. The table 4.3 displayed that less retention

time for molecules with low mass and greater the retention time for greater molecular mass,

but in actual the retention time is independent of molecular mass (Ogunjobi et al., 2013).

100

Peak intensity of every product was related with the concentration of the products. Greater

the peak area more the products concentration and vice versa.

Figure 4.19 Mass spectrum of Butanoic acid

Figure 4.20 Mass spectrum of 2-ethylbutyric acid

4.14 Characterization of organic compounds in Control cell

The GC-MS of simple uninoculated medium i.e. without Sporomusa ovata under the same

set of experimental conditions exhibited no sharp peaks for volatile fatty acids and

alcohols. GC-MS results for the control cell showed the absence of desired products

reflected that no electroactive reaction has taken place in the absence of electroactive cells.

Mass spectrum without any peak for volatile fatty acids and alcohols proved that formation

of chemical commodities were not possible without electroactive cells figures 4.21-4.22.

101

Figure 4.21 Gas Chromatogram without Sporomusa ovata

Figure 4.22 Mass spectrum of medium without Sporomusa ovata

The organic product with m/z 143 on mass spectrum represented the compound 5-

Formyltetrahydrofolic acid with molecular formula C20H23N7O7 irrelevant to the volatile

fatty acids and alcohols.

The quantification of each organic products were explored by comparing sample peak area

with the calibration curve after time interval of 24 hours. The peak area was dependent on

concentration from gas chromatogram and maximum products concentration were

observed at 120 hours of inoculation of cells The major products were volatile fatty acids

and alcohols and their concentration in mM are exhibited graphically for both batch and

continuous system.

4.14.1 Bio-electrochemical synthesis of organic acids during batch system

Products obtained after every 24 hours during batch system were analyzed by GC-MS.

The concentration of the VFA and alcohols were estimated by calibration curve.

102


butyrate by Sporomusa ovata in batch system

The biotic synthesis of ethanoic acid, butanoic acid and ethyl butyrate were identified in

figure 4.23. The total ethanoic acid concentration was 0.739 mM produced with average

rate of 0.178mM/day. The butanoic acid concentration was 2.04 mM with average

concentration of 0.408 mM/day and ethyl butyrate 1.23 mM/day with average rate of 0.246

mM/day. Formation of acetate and other volatile fatty acids are followed during

exponential growth phase called Acidogenic phase (Ramio et al., 2015 and Mohammadi

et al., 2014).

0

0.5

1

1.5

2

2.5

24 48 72 96 120

Co

nce

ntr

ati

on

.

(mM

)

Time (hours)

ethanoic acid

butanoic acid

2-ethyl butyrate

103

4.14.2 Bio-electrochemical synthesis of alcohols during batch system

Formation of alcohols were observed in slow growth phase with no ATP formation called

solventogenic phase (LaBelle et al., 2014).

Figure 4.24 Bio-electrochemical synthesis of ethanol, Pentanol and hexanol concentration

in mM by Sporomusa ovata in batch system

The average rate of ethanol, pentanol and hexanol were 0.777, 0.23 and 0.371 mM/day

respectively. Ethanol synthesis was observed 3.526 and maximum 3.886 at 96 and 120

hours respectively. The equivalent number of electrons transfer per mole for ethanol was

12, as the number of electrons are less, it causes the bio-electrochemical conversion of

carbon dioxide to organics at higher rate (Choi et al., 2014).

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Co

nce

ntr

ati

on

.

mM

Time (hours)

ethanol

pentanol

hexanol

104

4.14.3 Bio-electrochemical synthesis of organic acids during continuous system

System was switched to continuous system by peristaltic longer pump at the flow rate of

0.3ml/sec (Nevin et al., 2011). Continuous flow of the substrate used to operate more

amount of medium than batch system in same time duration in less capacity bio-reactor.

Sample collection after equal interval of time was calibrated against calibration curve.

Organic acids concentration in mM were expressed in figure 4.25.


butyrate concentration in mM by Sporomusa ovata in continuous system

The maximum concentrations for ethanoic acid, butanoic acid and 2-ethylbutyrate were

0.723, 1.79 and 1.184 mM respectively with average rate of 0.144, 0.358 and 0.236

mM/day. The butanoic acid production rate was observed maximum close to 0.35 mM/day

at 120 hours. Results were comparable to the (Nevin et al. 2010) where acetate and butyrate

concentration were 0.9 and 0.15 mM per 144 hours. Similarly the results obtained by

(Christopher W. Marshall 2012) were hydrogen, acetate with concentration of 10 mM and

1.0 mM with average rate of 1.3 and 0.1 mM/day respectively. The coulombic efficiency

was 84% after 17 days from start of CO2 flushing. The present research is equivalent to the

previous results with commodities of chemicals in less time duration.

0

0.5

1

1.5

2

2.5

24 48 72 96 120

Con

cen

trati

on

.

mM

Time (hours)

ethanoic acid

butanoic acid

2-ethyl butyrate

105

4.14.4 Bio-electrochemical synthesis of alcohols during continuous system

The bio-electrochemical synthesis of solventogenic phase was perceived under same set of

experimental conditions. The reactor generated the ethanol, pentanol and hexanol after

carbon dioxide reduction figure 4.26.

Figure 4.26 Bio-electrochemical synthesis of ethanol, pentanol and hexanol in

continuous system by Sporomusa ovata

The comparative study of batch and continuous bio-electrochemical synthesis was found

to be best in batch system. The concentration of ethanoic acid, 2-ethyl butyrate, and

butanoic acid were greater i.e. 0.739, 1.231 and 2.04 mM respectively in the 120 hour of

experiment. Similarly ethanol 3.88 and hexanol 1.85 mM concentrations were greater in

batch system in same time duration. Where as in continuous system ethanol, pentanol and

hexanol concentration was 3.144, 1.247 and 1.058 mM in 120 hours. Greater concentration

in batch system reveals the complete consumption of substrate than continuous system.

The simplified reactor designing along with specialized method to start up autotrophic bio-

cathode by heterotrophic pre-enrichment, where pure culture of acetogen S.ovata

synthesized the spectrum of chemicals by replacing hydrogen with bio-cathode. The

synthesis of hexanol was reported first time by BES system during 120 hours of minimum

0

0.5

1

1.5

2

2.5

3

3.5

24 48 72 96 120

Con

cen

tra

tio

n

mM

Time (hours)

ethanol

pentanol

hexanol

106

time duration. The previous research by Logan 2013 had reported ethanol, acetate,

propionate, butyrate and butanol after 54 days of mixed culture cultivation were 0.11 mg/L,

20.3 mg/L, 13.03 mg/L, 10.03mg/L and 0.09mg/L respectively. Keeping all these points in

consideration the present research proved the potential in synthesizing unique products.

During the synthesis of these organics, electrons requirement are fulfilled by extraction

from water at the anode and were delivered to cathode poised at -400mV. Though the

cathode and cell surface are negatively charged and there is electrostatic repulsive forces

but the interaction of cells with cathode for biofilm formation was evidenced by current

consumption which was greater after the biofilm development than the medium without

cells evidenced by cyclic voltammetry. The recovery of electrons in the synthesis of

various organic products were optimum at applied potential of -600 and -700 mV there is

remarkable synthesis of hydrogen (LaBelle et al., 2014). The synthesis of hydrogen

causes hindrance in coulombic recovery of other products. Therefore electroactive cells

can accept electrons directly from cathode in the absence of hydrogen. Higher voltage like

3.0 V are acceptable if there is an excess of electricity to be stored like intermittent

renewable energy source otherwise conservative voltage scheme is desirable. Acetogenic

bacteria utilize the reductive acetyl-CoA pathway to form organics. The supply of -400mV

was more favorable for the reduction potential of NADH. Sporomusa ovata has tendency

to accept electron directly from the cathode due to the presence of genes coding C-type

cytochromes and type 1V pili. Sporomusa ovata with well-characterized components of

extracellular transfer pathways indicates its similarity with electrogenic and electrotrophic

bacteria (Tremblay et al., 2015). During metabolic reactions these electrons are utilized

for the formation of volatile fatty acids and alcohols. These electro-autotrophs are able to

fix CO2 as sole carbon source by accomplishing Wood–Ljungdahl (WL) pathway. Wood-

Ljungdahl pathway is more efficient pathway requires only 8 enzymes, 4 moles of

hydrogen and less than one mole of ATP to convert electrons to electro fuels. In this

pathway two moles of CO2 are reduced to form one mole of acetyl-CoA. Acetyl-CoA goes

through the catabolic pathway to make ATP which provides the energy to bacteria. The

key enzymes in the acetyl-CoA pathway are carbon monoxide dehydrogenase/acetyl-CoA

synthase complex (CODH/ACS) and formate dehydrogenase (FDH), methyltransferase.

107

CODH/ACS complex contains nickel and iron and Formate dehydrogenases contain either

tungsten or molybdenum and has both CO oxidation and acetyl-CoA synthesis activities.

Certain metals play essential role in microbial growth and metabolism but abundance of

these elements are toxic for microbial life. The trace elements are the basic requirement for

their role in enzymes and cofactors in metabolic pathways. Cobalt, nickel, tungsten and

molybdenum are very important metals in homoacetogenic pathways. Most of these

elements are metal center in enzymes or as cofactors. Nickel has been found in many

enzymes like carbon monoxide dehydrogenase. Molybdenum and tungsten are important

trace elements for the growth of microorganisms, gram positive bacteria. Enzymes with

these elements catalyzes the oxidation reactions like formate dehydrogenase (FDH),

formylmethanofuran dehydrogenase (FMDH). FDH catalyzes the first reaction of CO2

reduction to formate in the acetyl-CoA pathway. Formation of acetate and other volatile

fatty acids are followed during exponential growth phase called Acidogenic phase while

alcohols are produced in slow growth phase with no ATP formation called solventogenic

phase.

108

COH2O

CO2

2H+2H+

Formate

Formyl-THF

Methenyl-THF

Methyllene-THF

Methyl-THF Methyl-CFeSP

CFeSP THF

2H+

2H+

H2O

ATP, THF

ADP,Pi

CO2 CO

2H+

Acetyl-CoA

CoA

CFeSP

Acetate

4H+

ADP,Pi

ATP,CoA

Butyryl-CoA

ATP,CoA

ADP,Pi

4H+

4H+

ButanolButyrate

Acetoacetyl-CoA

Acetate

Ethanol

Figure 4.27 Schematic diagram of Acetyl Co-A pathway for Acetoacetyl and Butyryl-Co-A

(Tremblay et al., 2015).

Theoretical yield of VFA and alcohols are calculated from carbon dioxide according to the

chemical equations 4.5- 4.10.

2CO2 + 4H2 C2H4O2 + 2H2O…………Equation 4.5

4 CO2 + 10 H2 C4H8O2 + 6 H2O……….. Equation 4.6

6 CO2 + 16 H2 C6H12O2 +10 H2O……….Equation 4.7

2CO2 + 6H2 C2H5OH + 3H2O………....Equation 4.8

6 CO2 + 18 H2 C6H14O + 11H2O………...Equation 4.9

5 CO2 + 15 H2 C5H12O + 9 H2O................Equation 4.10

Uninoculated reactor (control) did not produce organic acids and alcohols under the same

set of experimental conditions. It was also observed that interruption in the current supply

affects the rate of synthesis of organics. Most of the electrons captured by Sporomusa ovata

by cathode were diverted to reduce carbon dioxide and extracellular products.

109

4.15 Cyclic voltammetry (CV) analysis of biofilm

Cyclic voltammetry was performed to discern possible redox active components associated

with biocathode (carbon cloth) as working electrode against Ag/AgCl reference electrode.

The scan rate was 10mV/sec (Marshall et al., 2012). The culture exhibited the current

consumption during the 48 hours of cultivation. Sporomusa ovata showed the significant

electroactivity Figure 4.28 (black line) with definite reduction peak at (-6.486511E-02mV~

-1.886E-06mA/cm2) and oxidation peak at (2.494812E-01mV~3.20E-06mA/cm2).

Figure 4.28 Cyclic Voltammetry for Sporomusa ovata for biotic, abiotic and fresh medium

against standard Ag/AgCl reference electrode at scan rate of 10mV/s

110

4.15.1 Cyclic voltammetry analysis for Control cell without electroactive catalysts

The cyclic voltammetry of control cell with fresh medium and supernatant with carbon

cloth as a working and Ag/AgCl as reference electrode was without definite redox peaks.

The medium where the bacteria were removed after centrifugation did not show the redox

potential but after two days cultivation the fresh medium was re-filled in the cathode and

it retained the electrochemical activities due to the development of electroactive

components. When the potential was applied the current consumption was recovered

indicated that electron transfer was without mediator between cathode and biofilm. Stirring

the solution did not substantially affect the current consumption and increase in products

provides the evidence for the direct electron transfer from the cathode to biofilm.

Interruption in the voltage supply affects the activity of electro-autotrophs and reduce the

synthesis.

Scanning electron micrography (SEM) was utilized to visualize the prevalence of

electroactive cells attached to cathode figure 4.29. The biocathode was treated with 2-

bromoethanesulfonic acid before scanning, biofilm formation was perceived. The

dominant morphology was rod shaped bacteria varying in size on electrode. The

consistency of electro-autotrophs at cathode was evidenced from CV, which was

supportive for microbial catalyst at cathode surface (Marshall et al., 2012). The

electroactive cells intimately associated with carbon electrode surface would be expected

for direct electron transfer from electrode to cell. No visible turbidity in the cathode

chamber was consistent with previous studies of direct electron driven respiration (Nevin

et al., 2010). The biofilm was responsible further for current consumption and carbon

dioxide reduction.

111

Figure 4.29 Scanning electron micrographs of electrosynthetic cathode biofilms for

Sporomusa ovata

A cathode from abiotic control was examined for the occurrence of electroactive cells using

SEM. The abiotic cathode was without the presence of electroactive catalysts as

characterized in figure 4.30.

Figure 4.30 Scanninng electron micrograph of abiotic cathode without electroactive

catalyst

4.16 Electrons recovery against the current consumption

Electron appearing in the volatile acid and alcohol formation accounted for high proportion

of electrons consumed by culture. The total amount of the current consumed by system was

calculated by integrating the current area (A/m2) against time (sec).

112

Electron consumed by electro-autotrophs and recovered in organics were calculated by

following equation 3.1 (Logan et al., 2008).

Sporomusa ovata capture electrons from bio-cathode with the reduction of carbon dioxide.

Electrons transferred to the cells were diverted to extracellular products rather than biomass

formation. The equivalent number of electrons per mole of the products were 8, 20, 32, 12,

36 and 30 for ethanoic acid, butanoic acid, 2-ethylbutyrate, ethanol, hexanol and Pentanol

respectively. Current measured by Potentiostat for Sporomusa ovata against -400mV was

0.0914 A/m2. Electron recovery was tested for both batch and continuous systems

respectively.



batch system against the total electrons consumed by biofilm

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

0 24 48 72 96 120

Cu

rren

t re

cover

ed in

org

an

ic

com

pou

nd

s in

Cou

lom

bs

Co

nce

ntr

ati

on

of

org

an

ic c

om

pou

nd

s

(mole

s)

Time (hours)

acetic acid

butanoic acid

2ethyl butyrate

ethanol

pentanol

hexanol

total current consumed

113

Electron recovery of every product was obtained by the total current recovered in the

products to the total current consumed by products figure 4.31.


The percent cathode recovery was maximum in batch system like 81% for hexanol, 57%

for ethanol and 50% for butanoic acid as compared to continuous system. The electricity

efficiency to plethora of multicarbon compounds was 80-90% as revealed by (Nevin et al.,

2011 and Nevin et al., 2010) in their previous studies. The present research contributed

the results comparable to the results reported before because of the high electron recovery

during the minimum time duration of 120 hours.

Figure 4.32 Percent Cathode Recovery for organic acids and alcohols by Sporomusa ovata

in batch system

0

10

20

30

40

50

60

70

80

90

0 20 40 60 80 100 120 140

Cath

od

e re

cover

y x

100

Time (hours)

Ethanoic acid

Butanoic acid

2-ethyl butyrate

Ethanol

Pentanol

Hexanol

114


Coulombic recovery of electrons in continuous system where substrate flow rate was

0.3ml/sec and change in substrate concentration was measured in g/L. maximum

recovery was at 120 hours’ time interval figure 4.33.


continuous system against total current consumed by biofilm

The equivalent number of electrons per mole of the products were 8, 20, 32, 12, 36 and 30

for ethanoic acid, butanoic acid, 2-ethylbutyrate, ethanol, hexanol and Pentanol

respectively. As these number of electrons goes up the transfer efficiency falls cause the

lowering of bio-electrochemical conversion of carbon dioxide to organics. The

concentration of ethanoic acid is decreased as two acetyl- CoA converted to Acetoacetyl-

CoA which is leading to butyrate and then to butanoic acid. As more H+ ions increase the

internal pH of electroactive cells, so electroactive catalysts trigger the solventogenesis for

the synthesis of alcohols. As the equivalent number of electrons per mole of ethanol is 12

than pentanol and hexanol so revealing the maximum concentration then pentanol and

hexanol. Pentanol and hexanol are in close proximity because of less difference in their

equivalent number of electrons per mole.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0 24 48 72 96 120

Cu

rren

t re

cover

ed in

org

an

ic

com

pou

nd

s

Cou

lom

bs

Con

cen

trati

on

of

org

an

ic c

om

po

un

ds

mole

s

Time (hours)

acetic acid

butanoic acid

2-ethyl butyrate

ethanol

pentanol

hexanol

total current

115

4.16.4 Percent cathode Recovery during continuous system

Figure 4.34 Percent cathode recovery for organic acids and alcohols by Sporomusa ovata in

continuous system

The Coulombic recovery of volatile fatty acids and alcohols were compared in both batch

and continuous systems. The maximum recovery of electrons in products was observed in

batch system. Continuous addition of fresh medium and removal of planktonic cells

enhanced the electroactivity of cells resulted in more extracellular products in same time

duration. The exceeding recovery range at voltage of -0.4V was attributed by bacterial

reduction of carbon dioxide to organic compounds. The overall recovery of alcohols and

butyrate was more than 80% due to the redox activity of electroactive cells figure 4.34. The

utilization of electrons provided by cathode as a reducing equivalent in biofuel synthesis

resembles to natural photosynthesis but for CO2 reduction there is a need of much higher

electron uptake than microbial bio-electrochemical synthesis from glucose, sugar and

glycerol. This high demand of electron relatively effects the active reduction

pathway and might limit the synthesis of biofuel from CO2. To overcome this challenge

0

5

10

15

20

25

0 24 48 72 96 120

Per

cen

t ca

tho

de

reco

ver

y x

10

0

Time (hours)

ethanoic acid

butanoic acid

2-ethyl butyrate

ethanol

pentanol

hexanol

116

pure cultures were exploited to capture electron directly (without any mediator or electron

shuttle) from negatively poised cathode for carbon dioxide reduction to commodities of

chemicals. Further the recovery of electrons consumed in variety of cathode products with

DC power supply was comparable to the previous studies conducted by Potentiostat. The

cathode potential of -0.65 and -0.73 V were low enough for the production of hydrogen

there for the Sporomusa ovata was utilizing the hydrogen abiotically for the electron

transfer rather than negative cathode as demonstrated in the previous studies (Gregory et

al. 2004). in the present research the cathode was poised at -0.4V potential to avoid

hydrogen production. So direct uptake of electron from cathode was feasible for the

diversity of chemicals.


means were tested at significance levels of P < 0.05 using one way ANOVA.

Table 4.6 Comparison (ANOVA) of continuous and batch process for Sporomusa ovata

Cont. N Mean Std.

Deviation

Std. Error 95% Confidence

Interval for Mean

Minimu

m

Maximu

m

Lower

Bound

Upper

Bound

1 35 1.442823 0.7891982 0.1333988 1.171724 1.713922 0.3503 3.1910

2 35 1.367544 0.7544827 0.1275309 1.108370 1.626718 0.4270 3.1430

Total 70 1.405184 0.7673578 0.0917168 1.222214 1.588154 0.3503 3.1910


continuous and batch process for Sporomusa ovata

Cont. Sum of Squares Df Mean Square F p

Between Groups 2.661 1 2.661 4.189 .045

Within Groups 36.837 58 .635 Total 39.498 59

117

One-Way ANOVA was performed for Sporomusa ovata manifested in the Table 4.6 &

Table 4.7. It was found that there was significant difference (p=0.045) between both batch

and continuous processes (p<0.05). Hence, statistically it can be concluded that batch

process vary significantly in terms of concentration level.

118

4.17 Clostridium ljungdahlii

Clostridium ljungdahlii is anaerobic gram positive bacteria. Gram positive bacteria can

establish the electrical link with negative cathode to capture electrons. Present research was

conducted to evaluate the efficiency of gram positive bacteria for their carbon dioxide

reduction with negatively poised cathode. Previous studies reported that Clostridium

ljungdahlii is devoid of genes responsible for synthesis of cytochromes. In Clostridium

ljungdahlii RnF is multidirectional device for proton translocation, nitrogen fixation and

electron transportation. RnF complexes are ion pumps with membrane bound electron

transfer system. RnF complex develops proton gradient over membrane in both

heterotrophic and autotrophic conditions. Autotrophic growth is completely inhibited

without RnF complex, designates that RnF complex is involved in electron transport as

well as in energy conservation (Pier et al., 2016).

Bio-electrochemical synthesis with mixed communities starting with autotrophic

biocathode was first reported by (LaBelle et al., 2014 and Zaybak et al., 2013), and

Pisciotta et al., 2012) registered six products ethanol, hydrogen, acetate, butyrate, butanol

and propionate in mixed community reactor. The acetate production at rate 1.3

mM/day/cm2 with mixed community was testified by (Jourdin et al., 2014) but

simultaneous production of methane were observed because of the coexistence of

methanogens unless methanogenic inhibitor was introduced to cathode reactor. Pure

culture acetogens were harvested in BES system first time by (Nevin et al., 2011) for the

synthesis of acetate and 2-oxobutyrate. The present research was directed to investigate the

spectrum of chemicals from C!-C7 platform with pure culture of Clostridium Ljungdahlii.

The concomitant production of seven valuable products along with heptanoic acid and

heptanol was a potential achievement in this research.

Bio-electrochemical system was operated by establishing autotrophic biocathode with

Clostridium ljungdahlii where waste greenhouse gas CO2 was sole carbon source

throughout the experiment. Batch and continuous systems were analyzed for coulombic

recovery of biofuels. Identification and quantification of samples were performed by GC-

MS.

119

4.18 Characterization of Bio-electrochemically synthesized organic

compounds

The structure and mole fractions of volatile fatty acids and alcohols determined by GC-MS

were based on peak area of ions. Greater the peak height more the concentration of

products. Various identified compounds along with molecular weight, retention time and

structural formula are listed in table 4.8.

Table 4.8 Chemical composition of volatile fatty acids and alcohols revealed by GCMS

analysis (RT; retention time in minutes, CN; compound name, MW; molecular weight, MF;

molecular formula, DF; displayed formula).

CN Heptanol Acetic

acid

Ethanol Ethyl

butyrate

Hexanol

Hexanoi

c acid

Heptanoi

c acid

RT 4.89 11.32 16.1 20.31 20.9 21.9 23.2

MW 116.20 60 46.06 117.1 102.17 116.16

130.18

MF C7H16O C2H4O2 C2H6O C6H12O2 C6H14O C6H12O2

C7H14O2

DF

Theoretical yields of organics were calculated with aid of following equations.

2CO2 + 4 H2 C2H4O2 + 2H2O.............. Equation 4.11

6 CO2 + 16 H2 C6H12O2 + 10 H2O…… Equation 4.12

7CO2 + 19 H2 C7H14O2 + 12H2O……..Equation 4.13

2CO2 + 6H2 C2H5OH + 3H2O………Equation 4.14

6 CO2 + 18 H2 C6H14O + 11 H2O…….. Equation 4.15

7 CO2 + 21H2 C7H16O + 13 H2O……..Equation 4.16

120

4.19 Gas Chromatogram and Mass Spectrum for Clostridium ljungdahlii

The integrated peaks on gas chromatogram exhibited the various organic products at m/z

ratio of 60, 117, 101 and 83 for ethanoic acid, ethyl butyrate, heptanoic acid and heptanol

with retention time 11.3, 20.31, 23.2 and 4.8 min respectively. The peaks with retention

time 16.1, 21.9 and 20.9 demonstrated ethanol, hexanoic acid and hexanol respectively. As

the m/z ratio was not exceeding from 120 reflected that there was no presence of other

lipids and the resulting products were pure. Peak with retention time 19.1 exhibited internal

standard 2-ethyl butanoic acid (Ogunjobi et al., 2013).

Figure 4.35 Gas chromatogram for Clostridium ljungdahlii

Figure 4.36 Mass spectrum for ethanoic acid

121

Figure 4.37 Mass spectrum for ethyl butyrate

Figure 4.38 Mass spectrum for heptanoic acid

Figure 4.39 Mass spectrum for heptanol

122

4.20 GC-MS Results for control cell without electro-autotrophs

Figure 4.40 Control cell without electro-autotrophs

Figure 4.41 Mass spectrum of Clostridium ljungdahlii without electro-autotrophs

The GC-MS results of uninoculated medium i.e. without cultivation of Clostridium

ljungdahlii with same set of experimental conditions has manifested clearly that no any

metabolic activities have taken place in the medium. Mass spectrum without any sharp

peaks for organic products revealed that without electroactive cells no desirable products

were formed. The peak with retention time 7.79 and m/z 174 was for Benzene sulfonic

acid, 4-hydroxy not of any volatile organic acids figure 4.40 and 4.41.

4.21 Bio-electrochemical synthesis of organic acids during batch system

The quantification of various organic products were analyzed by comparing sample peak

area with the calibration curve. The major products were volatile fatty acids and alcohols

and their concentration in mM were investigated after regular interval of time figure 4.42.

123


Clostridium ljungdahlii for batch system

Based on the information from GC-MS the maximum rate of ethanoic acid, ethyl butyrate,

and hexanoic acid and heptanoic acid synthesis was estimated 0.598, 0.453, 0.437 and

0.403 mM/day. The concentration of ethanoic acid was maximum 2.99 mM at 120 hour.

Concentrations of hexanoic acid and heptanoic acid were 2.187 and 2.0179 mM at 120

hour of medium cultivation. During first 48 hours concentration of each volatile product

enhanced smoothly but at 72 hours there was decline in the concentration of ethyl butyrate

and hexanoic acid, again followed by increase in concentration at 120 hours of incubation.

The sudden decline in concentration was due to flux in metabolic pathway.

The formation of secondary intermediates can also divert the direction toward other

products. At 72 hours of medium cultivation, suppress in ethyl butyrate and hexanoic acid

concentration was observed because of condensation of butyryl-CoA with acetyl-CoA to

hexanoyl-CoA and then to hexanoate and further to heptanoate (Mohammadi et al., 2014).

One product leading to the formation of second product was the basic reason for the sudden

decrease in concentration.

0

0.5

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1.5

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2.5

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3.5

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Co

nce

ntr

ati

on

(mM

)

Time (hours)

Ethanoic acid

Ethyl butyrate

Hexanoic acid

Heptanoic acid

124

4.22 Bio-electrochemical synthesis of alcohols during batch system

Alcohols concentration were observed maximum at 120 hours of experiment. Ethanol

concentration was 3.19 mM, at average rate of 0.638mM/day figure 4.43. Ethanol

concentration was greater than hexanol and heptanol. Hexanol and heptanol concentration

were observed 2.11 and 0.853 mM with average rate of 0.422mM/day and 0.170mM/day

respectively. The equivalent number of electrons transfer per mole for ethanol was 12, as

the number of electrons are greater, it lowers the bio-electrochemical conversion of carbon

dioxide to chemical products. The similar results were observed for hexanol and heptanol

as their equivalent number of electrons transfer per mole were 36 and 42 respectively.

Solventogenic phase was observed at slow growth phase with no ATP formation (LaBelle

et al., 2014).

Figure 4.43 Bio-electrochemical synthesis of alcohols against time duration of Clostridium

ljungdahlii for Batch system

4.23 Bio-electrochemical synthesis of organic acids in continuous system

After batch synthesis, system was switched to continuous system by peristaltic pump with

flow rate of 0.3 ml/sec (Nevin et al., 2011). After regular interval of time screened samples

0

0.5

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1.5

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24 48 72 96 120

Con

cen

trati

on

(mM

)

Time (hours)

Ethanol

Hexanol

Heptanol

125

in falcon tubes were characterized by GC-MS. Concentration of various organic products

were calculated against the standard curve.

There was spontaneous increase in the concentration of ethanoic acid. During the 72 hours

of incubation the concentration was 0.562mM but sudden rise in concentration was

observed at 120 hours i.e. 2.471mM. Other products ethyl butyrate, hexanoic acid and

heptanoic acid concentration were 1.269, 2.66 and 2.23 mM. The production rate was

reached to 0.254, 0.532 and 0.446 mM/day figure 4.44.


Clostridium ljungdahlii for continuous system

Results obtained from Clostridium ljungdahlii were equivalent to the previous research results of

(Nevin et al. 2010 and Marshall 2012) where acetate concentration was 1.0 mM. (Logan 2013)

reported the concentration of ethanoic acid and butyrate 20.3mg/L and 10.03mg/L respectively

after the 54 days of mixed culture cultivation. The present study of BES system synthesized the

ethanoic acid and butyrate 2.47 mM and 1.26 mM after 120 hours cultivation. Keeping all these

points under consideration it is clear that present research contributed potential results in the

research.

0

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2.5

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24 48 72 96 120

Con

cen

trati

on

.

(mM

)

Time (hours)

Ethanoic acid

Ethyl butyrate

Hexanoic acid

Heptanoic acid

126

4.24 Bio-electrochemical synthesis of Alcohols during continuous system

Bio-electrochemical synthesis of ethanol, hexanol and heptanol were observed 3.14, 2.66

and 0.975mM at rate of 0.628, 0.532 and 0.195 mM/day. The smooth increase in the

concentration of volatile acids and alcohols reflects the exponential phase when inoculum

was fully saturated with live and active cells. Under such experimental conditions the

production rate of ethanol, and hexanoic acid were reached up to 3.14 and 2.66 mM at 120

hours figure 4.45.

Figure 4.45 Bio-electrochemical synthesis of ethanol, hexanol and heptanol against time

duration for continuous system by Clostridium ljungdahlii

The comparative study of batch and continuous bio-electrochemical synthesis was found

to be best in batch system. The concentration of ethanoic acid, ethyl butyrate, hexanoic

acid and heptanoic acid were 2.99, 2.66, 2.18.and 2.017mM respectively in the 120 hour

of cultivation. Similarly ethanol 3.19, hexanol 2.11 and heptanol concentration were

0.85mM. The heptanol and hexanol concentration were little greater in continuous system.

As Clostridium ljungdahlii a gram positive bacteria has greater interaction with negative

cathode and this trait aids for the development of bio-film on the cathode surface for direct

capturing of electrons. Metabolic reactions followed both acidogenic and solventogenic

phase for the synthesis of bio commodities. Requirement of electrons are fulfilled by their

0

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1.5

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24 48 72 96 120

Con

cen

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on

(mM

)

Time (hours)

Ethanol

Hexanol

Heptanol

127

extraction from water in the anode compartment and were delivered to cathode by external

circuit evidenced by cyclic voltammetry. These commodities were comparable with

products specified by (Zaybak et al., 2013 and Marshall et al., 2012) in their previous

studies. As Zaybak and LaBelle mentioned the bio-electrochemical synthesis of six

products butanol, ethanol, acetate, butyrate, hydrogen and propionate from mixed

community microbial catalysts in autotrophic BES synthesis. But the present research

provided the better approach for the synthesis of plethora of chemicals by pure culture of

acetogen in minimum time duration of only 120 hours whereas the previous researches

were conducted for more time duration like 256 hours or two months (Zaybak et al., 2013).

Microorganisms are able to fix CO2 as sole carbon source by accomplishing Wood–

Ljungdahl (WL) pathway, in which two moles of CO2 are reduced to form one mole of

acetyl-CoA. The carbonyl or Western branch of WL reduces one mole of CO2 to CO as a

part of bifunctional CO Dehydrogenase/ Acetyl-CoA Synthase complex (CODH/ ACS).

The methyl or Eastern branch utilizes formate dehydrogenase (FDH) for the reduction of

CO2to formate. FDH is subsequently attached to tetrahydrofolate and reduced to methyl-

group. The carbonyl and methyl groups are combined in Acetyl-CoA Synthase along with

a molecule of coenzyme-A to synthesize acetyl-CoA that can either be used for the

formation of acetate thereby regenerate ATP, or for the formation biomass via anabolism.

In substrate level phosphorylation no net ATP is formed in WL pathways figure 4.46. One

ATP is formed during the conversion of acetyl-CoA to acetate and one molecule is

consumed to form 10-formyltetrahydrofolate via the enzyme 10-formyl-H4folate

synthetase. Acetogens must couple reactions within the WL pathway to generate

transmembrane ion gradients, from which gradient-driven phosphorylation produces ATP.

Two mechanisms has been proposed for gradient driven phosphorylation. First, membrane

bound Rnf complexes generate proton-gradient as in the case of C. Ljungdahlii or

cytochromes generate proton-gradient in other acetogens. The reduction of 10-

methylenetetrahydrofolate to 5-methyltetrahydrofolateis coupled to proton gradient

formation to generate ATP. The second mechanism generates ATP through Na+dependent

gradient. However the ATP generated are small and limit the growth rate and these

consideration argue that microorganisms culturing should be heterotrophically on fructose

128

first to achieve high cell densities and then switched to gaseous substrate for autotrophic

growth (Tracy et al., 2012). The adjusted trace metals concentration in medium are helpful

for the accumulation of butanol and hexanol. As metal atoms are active sites for the electron

transfer and substrate binding in WL pathway. Nickel is active site for carbon monoxide

dehydrogenase (CODH) enzyme to receive electrons from CO in WL pathway. Formate

dehydrogenase and aldehyde oxidoreductase (AOR) enzymes are in alcohol synthesis and

critically tungsten dependent. Mo is equivalent to W to bind active sites in enzyme AOR

that are involved in reduction of organic acids to alcohols. Similarly Fe is the basic

requirement for electron transfer centers in enzymes and cofactors.

Acetyl-CoA undergoes sequential metabolic reactions for the synthesis of organic acids

and alcohols that are successively exported from the cell. Volatile fatty acids and alcohol

synthesis occur sequentially in acidogenesis and solventogenesis metabolic phases (Ramio

et al., 2015). Acids are produced during exponential growth phase and when growth rate

slows down and cells enter the stationary phase, alcohol synthesis starts. Clostridium

ljungdahlii produce alcohols at pH 4 to 4.5 under non growth condition and volatile fatty

acids at pH 6 to 7 (Mohammadi et al., 2014). The undissociated acetic acid penetrates

through the cell membrane resulting in a lower internal pH due to excess of H+ ions.

Electro-autotrophs overcome this physiological stress by producing solvents at low pH.

The dense bacterial culture was obtained with fructose due to the availability of intra

cellular enzymes and cofactors. The use of reducing agents trigger the slow growth of

bacteria because of reduced ATP formation. This non growth condition activates the

alcohol synthesis. The figure 4.46 reflects the trends in acidogenesis and solventogenesis

when medium switched from exponential growth phase to non-growth phase under lower

pH values.

In the recent study hexanoic acid, hexanol and heptanol are reported along with other

compatible liquid fuels. Hexanol synthesis is catalyzed by thiolase enzymes which

condense two molecule of acetyl-CoA to acetoacetyl-CoA and eventually forming butyryl-

CoA. For hexanoic acid and hexanol, thiolase condenses butyryl-CoA with acetyl-CoA to

129

form 3-oxo-hexanoyl-CoA that is converted to hexanoyl-CoA. Hexanoyl-CoA conversion

to hexanoate is by same enzyme as for butyrate and to hexanol by enzymes aldehyde and

alcohol dehydrogenases the same enzyme that converts butyryl-CoA to butanol.

Figure 4.46 Overview of Wood–Ljungdahl metabolic pathways involved in the synthesis

of various metabolites phases (Ramio et al., 2015).

4.25 Cyclic voltammetry

Cyclic voltammetry of Clostridium ljungdahlii was performed to evaluate the redox active

molecules associated with biotic cathode. Carbon electrode set as working electrode

against Ag/AgCl reference electrode figure 4.47. The scan rate was 10mV/sec (Marshall

et al., 2012).The culture exhibited the current consumption after the 48 hours of cultivation.

Clostridium ljungdahlii showed the significant electroactivity with definite redox peak

indicates the development of electroactive biofilm at cathode. The reduction and oxidation

peaks were observed at (-6.028 E-01 mV~ -5.1966 E-05mA/cm2) and (2.03E-01 mV ~

3.90 E-05mA/cm2) respectively.

130

In the abiotic control cell of fresh medium without electro-autotrophs did not show the

preferred redox peaks. When CV scan was conducted against the reference electrode. Due

to the absence of electroactive cells i.e. electro-autotrophs, the current transferred through

the circuit was limited because the acceptance of electron at cathode was minimum. The

current density is increased only by increasing the formation of products which was

possible only in the presence of electroactive catalyst. The larger difference in the peak

current in biotic scan and abiotic scan are of note.

Figure 4.47 Cyclic Voltammetry of Clostridium ljungdahlii for biotic, abiotic cathode and

fresh medium against Ag/AgCl reference electrode at scan rate of 10mV/s

When the fresh medium was re-filled in the cathode compartment, it retained the

electrochemical activities due to the development of electroactive catalysts. The current

consumption was recovered again when potential was applied indicated the transfer of

131

current from cathode to electro-autotrophs without mediators. Stirring the solution did not

substantially affect the current consumption and increase in products provides the evidence

for the direct electron transfer from the cathode to biofilm. Interruption in the voltage

supply affects the activity of electro-autotrophs and reduce the synthesis.

Scanning electron microscopy (SEM) was utilized to visualize the prevalence of

electroactive cells attached to cathode figure 4.48. The biocathode was treated with 2-

bromoethanesulfonic acid and then coated with gold 100-Å. Rod shaped electro-autotrophs

were dominant on electrode. The consistency of electro-autotrophs at cathode was

evidenced from CV, which was supportive for microbial catalyst at cathode surface

(Marshall et al., 2012). The electroactive cells intimately associated with carbon electrode

surface would be expected for direct electron transfer from electrode to cell. No visible

turbidity in the cathode chamber was consistent with previous studies of direct electron

driven respiration (Nevin et al., 2010). The biofilm was responsible further for current

consumption and carbon dioxide reduction.

Figure 4.48 Scanning electron micrographs of electrosynthetic cathode biofilms after 24

and 72 hours

The abiotic cathode was examined under scanning electron micrograph for the comparative

studies of electroactive cells. The result signified the absence of electroactive catalyst and

no biofilm was developed as clearly manifested in the figure 4.49.

132

Figure 4.49 Scanning electron micrograph of abiotic cathode without electroactive cells

4.26 Electron recovery against the current consumption

The total amount of the current consumed by system was calculated by integrating the

current (A/m2) against time (sec). The amount of current measured by for Clostridium

ljungdahlii against -400mV applied potential was 0.0848 A/m2. The equivalent number of

electrons per mole of ethanoic acid, ethyl butyrate, hexanoic acid, heptanoic acid, ethanol,

hexanol and heptanol are 8, 32, 32, 38, 12, 36 and 42 respectively.

Clostridium ljungdahlii capture electrons from bio-cathode and reduce carbon dioxide to

extracellular volatile fatty acids and alcohols rather than biomass formation. Electron

recovery was tested for both batch and continuous systems and results were compared by

Coulombic recovery of organic products / total electrons consumption.

Electron recovery for both batch and continuous systems were compared after the biofilm

development at the cathode (Logan et al., 2008).

133


Electron recovery of every product was obtained by the total current recovered in the

products to the total current consumed by products figure 4.50.


batch system against the total electrons consumed by biofilm

The maximum electron recovery was observed in ethanol and ethanoic acid. Minimum

electron recovery was in heptanol and heptanoic acid.

0

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Time (hours)

ethanoic acid

ethyl butyrate

hexanoic acid

heptanoic acid

ethanol

hexanol

heptanol

current consumed

134


Figure 4.51 Percent cathode recovery for organic acids and alcohols by Clostridium

ljungdahlii in batch system

Percent cathode recovery was maximum for heptanoic acid, ethyl butyrate and hexanol and

was minimum for heptanol and ethanoic acid figure 4.51.


Coulombic recovery of electrons in continuous system where influent flow rate was 0.3

ml/sec and change in substrate concentration was measured in g/L.

0

20

40

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Cath

od

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Time (hours)

% Ethanoic acid

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%Hexanoic acid

%Heptanoic acid

%Ethanol

%Hexanol

%Heptanol

135


continuous system against the total electrons consumed by biofilm

Numerous products formation causes the complexity and non-linearity in the end results.

In batch system ethanoic, butanoic, hexanoic and heptanoic acids were produced with

slight increase in concentrations till 48 hours, but at 72 hours there was slight decrease in

ethyl butyrate concentration, according to Wood Ljungdahl pathways two acetyl-CoA

converted to Acetoacetyl-CoA which further generating butyryl-CoA leading to butyrate.

But the concentration of butyrate was decreased as butyryl CoA with one more acetyl CoA

was converted to hexanoyl-CoA for the synthesis of hexanoate and then hexanol which in

turn reflected a slight decrease in hexanoic acid graphically. As more and more H+ ions

penetrated through cell membrane and increased the internal pH. To overcome this

physiological stress electroactive catalysts trigger the solventogenesis for the synthesis of

alcohols. This trend reflected in acidogenesis and solventogenesis when medium was

switched from exponential growth phase to non-growth phase under lower pH values.

During batch system ethanoic acid and ethanol were with maximum concentration while

heptanoic acid and heptanol were at low concentration than other products as the transfer

of electrons increased gradually, the reduction of carbon dioxide to organic value added

0

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Moles Ethanoic acid

Moles Ethyl butyrate

Moles Hexanoic acid

Moles Heptanoic acid

Moles Ethanol

Moles Hexanol

Moles Heptanol

Total current consumed

136

products decreased slightly. During the continuous system as the medium was added

uninterruptedly with constant removal of products from the system resulted in smooth

increase in the concentrations of organic products. Continuous system revealed the same

trend of decrease in products concentration with increase in electron transfer as illustrated

in figure 4.53.

4.26.4 Percent cathode recovery in continuous system

Figure 4.53 Percent Cathode Recovery for organic acids and alcohols by Clostridium

ljungdahlii in continuous system

The coulombic recovery of volatile fatty acids and alcohols at applied voltage were

observed different for batch and continuous system. The maximum recovery was 93%,

89%, 98% and 97% for ethyl butyrate, hexanoic acid, heptanoic acid and hexanol for batch

system. The other products were ethanoic acid, ethanol and heptanol but their recovery was

comparatively less 30%, 49% and 45% respectively. The maximum recovery was at 120

hours of the cultivation at temperature 37 0C and pH 7 when CO2 was merely the carbon

source.

0

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Cath

od

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Time (hours)

%Ethanoic acid

%Ethyl butyrate

%Hexanoic acid

%Heptanoic acid

%Ethanol

%Hexanol

%Heptanol

137

Where as in continuous system the coulombic recovery of heptanoic acid, hexanoic acid

and hexanol was 94%, 72% and 80%. The utilization of electrons provided by cathode as

a reducing equivalent in biofuel synthesis resembles to natural photosynthesis.

Bio-electrochemical synthesis of commodity of chemicals by electroactive acetogen like

Clostridium ljungdahlii with simplified reactor was competitive with previous research.

The pre-enrichment technique to start-up biocathode with pure culture was unique

methodology resulted in an electrotrophic community for the plethora of metabolites and

biofuels like, acetate, hexanoic acid, heptanoic acid, ethanol, and heptanol in limited time

duration than previous research. Pre-enrichment with different substrates used for the

autotrophic communities could be helpful for unique metabolic capabilities.


means were tested at significance levels of P < 0.05 using one way ANOVA for Clostridium

ljungdahlii exhibited in the tables below

Table 4.9 Comparison (ANOVA) of continuous and batch process for Clostridium

ljungdahlii

Cont. N Mean Std.

Deviation

Std. Error 95% Confidence

Interval for Mean

Minimum Maximu

m

Lower

Bound

Upper

Bound

1 35 1.442823 0.7891982 0.1333988 1.171724 1.713922 0.3503 3.1910

2 35 1.367544 0.7544827 0.1275309 1.108370 1.626718 0.4270 3.1430

Total 70 1.405184 0.7673578 0.0917168 1.222214 1.588154 0.3503 3.1910

138


continuous and batch process for Clostridium ljungdahlii

Sum of Squares df Mean Square F P

Between Groups 0.099 1 0.099 0.166 0.685

Within Groups 40.531 68 0.596

Total 40.630 69

One-Way ANOVA was performed for Clostridium ljungdahlii manifested in the Table 4.9

& Table 4.10. It was found that there was no significant difference (p=0.685) between both

processes (p>0.05). Hence, statistically it can be concluded that both batch and continuous

processes don not vary significantly in terms of concentration level.

139

4.27 Conclusions

The bio-electrochemical synthesis by autotrophic acetogens having tendency to accept

electrons from cathode has extended the horizon of research. The main aim of current

research was to setup simplified bioelectrochemical reactor to synthesize commodities of

chemicals from waste greenhouse gas carbon dioxide by electro autotrophic acetogens in

less time span. The most investigated configuration in BES reactor was the development

of renewable bio-cathode, more practicable than the precious electrodes of gold, platinum

and copper. Bio cathodes catalysis proved to be less expensive as compared to traditional

cathodes which were causing toxicity, corrosion and denaturation of material. Two stage

strategy employed for heterotrophic pre-enrichment to enhance the anaerobic bio-cathode

development followed by supply of CO2 the sole carbon source to switch microorganisms

from heterotrophic to autotrophic growth. The electroactive catalysts selected for the

current research were Sporomusa ovata, Clostridium ljungdahlii and Cupriavidus necator,

able to capture electrons directly without any mediator making it more operational

technique, was further proved by cyclic voltammetry of the medium inoculated in BES

reactor. The key advantages of direct electron capture from cathode is the direct catalysis

and retention of electroactive catalyst in the cathode chamber. Further the bio-cathode

development was evidenced by scanning electron micrography. The pre-enrichment

technique of bio-cathode development by pure culture rather than mixed community

proved better strategy to reduce waste greenhouse gas CO2 to extracellular carbon

compounds by following the Acetyl-CoA pathway. The acetogens selected for the recent

research were following the Wood-Ljungdahl pathway rather than other carbon fixation

pathways. Wood-Ljungdahl pathway is more efficient pathway requires only 8 enzymes, 4

moles of hydrogen and less than one mole of ATP to convert electrons to electro fuels.

Bio-cathode the electron donor in this research was poised at -0.4V to avoid the hydrogen

production which can causes the loss in coulombic recovery of biofuels. In previous studies

the cathode was poised at fixed potential by potentiostat which was difficult to handle and

fixed potential can damage the cells. Therefore another implementation to simplify the

reactor was DC power supply system which was comparable to the electron recovery of

84% as reported previously for potentiostat poised system. The solar powered UPS can also

140

be attached with BES for the conversion of carbon dioxide to multicarbon compounds. The

electricity driven microbial electrocatalysis of CO2 and water to extracellular multi-carbon

compounds offers the possibility to overcome the problems concerning to the difference

between energy demands and supply and storage difficulties of renewable electricity by

converting it to covalent carbon compounds.

The electroactive catalysts are capable of redox reactions at the electrode and synthesis

of short chain volatile fatty acids, alcohols and polyhydroxyalkanoates etc. The

uninoculated control cell proved that without electroactive cells the redox activity of the

BES reactor was not possible. No redox active cell no valuable products. Carbon dioxide

as substrate in BES reactor offers some advantages like unlimited availability, reduction of

global warming, limited toxicity to the environment and land independence. One

disadvantage associated with the use CO2 as final electron acceptor is the requirement of

large number of electrons. The formation of poly hydroxybutyrate (PHB) and

Polyhydroxyheptanoates (PHH) require 58 and 98 electrons, acetate and ethanol requires

8 and 12 electrons but hexanoic acid, heptanoic acid require 32 and 38 electrons. This large

requirement of electrons implies large current and equivalently high power demand. The

electron recovery in the product formation was compared in both batch and continuous

systems. The study of Clostridium ljungdahlii exhibited the ethanoic acid, hexanoic acid

and heptanoic acid concentrations 2.99 mM, 2.18 mM and 2.017 mM while ethanol yield

was 3.19 mM, hexanol and heptanol was 2.11, 0.853 mM respectively. These results were

observed at 120 hours of batch system at optimum conditions of pH 6.8 and temperature

37 0C. The results for continuous system under the same set of experimental conditions

showed greater concentration in case of heptanoic acid 2.23 mM, hexanoic acid 2.66 mM

hexanol 2.66 mM. The percent cathode recovery was maximum in heptanoic acid 98.39%,

hexanol 97% and in ethanol was 50%. The ANOVA test, a statistical analysis for

comparison between batch and continuous system was performed, from ANOVA table it

was found that F=1.70, with p-value= 0.203, as p-value >0.05. Hence, statistically it can

be concluded that both processes don not different significantly in terms of concentration

level.

Sporomusa ovata the second microbe revealed the products butanoic acid, 2-ethylbutyrate,

ethanol, Pentanol and hexanol with concentration of 2.04, 1.23, 3.88, 1.15 and 1.85 mM

141

were greater in the batch system at 120 hours of batch system. percent cathode recovery

was maximum in hexanol 81.6 %, in butanoic acid 50%, in ethanol 56% and in 2-

ethylbutyrate was 48.14% at optimal conditions of pH 7.0 and temperature 32 0C. The

ANOVA test revealed that value of F= 4.189 and p= 0.045. The p value was lesser than

0.05 which proved the significant difference between the concentration level of batch and

continuous system.

The third facultative bacteria Cupriavidus necator accumulated the 3-hydroxydimethyl

butyrate 33.90 ppm, ethyl-3-hydroxy hexanoate 11.6 ppm and 2, 4-dimethyl heptanoate

8.27 ppm in batch system with percent cathode recovery of 97%, 19.9% and 33.71% and

in continuous system the concentrations were 79.2, 14.8 and 14.7 ppm respectively. The

comparative studies of batch and continuous system exhibited the best results in batch

system with maximum electron recovery i.e., more than 90%. For comparison between the

concentrations level of both batch and continuous system ANOVA test was performed.

The result from ANOVA table represented the F= 0.166 and significant figure was 0.685.

The p value was greater than the set value of 0.05. Hence, statistically it can be concluded

that both processes don not different significantly in terms of concentration level.

The maximum yield in batch studies was due to the complete reduction of substrate to

organic products than in continuous system. Whereas continuous system has some

advantages over batch system for industrial point of view. Continuous system deals with

greater amount of substrate concentration than batch system, all stages of reactions are

carried out simultaneously so overall time required for the process is reduced. Secondly

the continuous system operated in small bio-reactor than batch reactor. On industrial scale

the continuous system is better due to the continuous synthesis in small volume reactor.

The bioplastic yield with electron recovery more than 90% has potential for industrial

implementation. Meanwhile the ethanol synthesis in both strains of Sporomusa ovata and

Clostridium ljungdahlii was 3.88 mM and 3.19 mM respectively with electron recovery of

57% and 49% could be recognized for industrial application. The best achievements were

observed with the microbial strains Clostridium ljungdahlii, the synthesis of heptanoic acid

and heptanol not reported before in the bio-electrochemical synthesis. The concentration

obtained was greater in continuous system 2.23 mM and 0.975 mM at 120 hours of its

cultivation time. Though the hexanol was observed in bio-electrochemical synthesis with

142

microbial strain Sporomusa ovata too but its concentration was 1.855 mM. The

concentration of hexanoic acid and hexanol observed with Clostridium ljungdahlii was

greater than Sporomusa ovata.

The use of fixed current indicates the high synthesis rates in short startup time. Interruption

in the current supply causes disruption in synthesis. Uninoculated cultures did not show

any consumption of the current due to lack of electroactive cells.

BES technology proved its worth not only for the synthesis of high grade multiple

compounds but also for its promising environmental and industrial concerns due to its

sustainability, renewability and environmental friendly traits. The synthesis of VFA, PHB

and alcohols with low cost substrates like molasses and waste gases has made it a novel

bio-synthetic strategy.

143

4.28 Highlights

The bio-electrochemical synthesis of renewable chemicals were investigated from

waste greenhouse gas carbon dioxide.

Nontoxic acetogens Sporomusa ovata, Cupriavidus necator and Clostridium

ljungdahlii were studied for cathode biofilm development and direct capturing of

electrons from cathode.

Specific selection of microorganisms which were able to switch from heterotrophic

to autotrophic metabolism.

Recent study investigated the bio-electrochemical synthesis of PHA by

Cupriavidus necator not reported before.

Cupriavidus necator were electrotrophs proved by 75% yield of PHB in bio-

electrochemical reactor.

The synthesis of hexanoic acid, heptanoic acid, hexanol and heptanol from

Clostridium ljungdahlii were not reported in previous studies.

The synthesis of 2-ethylbutyrate, Butanoic acid, Acetic acid, ethanol, pentanol and

hexanol were remarkable achievements from Sporomusa ovata, in bio-

electrochemical cell.

144

4.29 Future perspective and challenges

Bio-electrochemical synthesis (BES) utilizing CO2 as feedstock an electricity driven

product formation must be demonstrated at pilot scale. The lack of fundamental knowledge

about the electroactivity of bio-catalysts, the cathode materials, the reactor designing and

product recovery require multidisciplinary approaches for sustainable biochemical

synthesis. Optimization and scaling of process are strong requirement for

commercialization. For strong BES technology both operational and technical

considerations are key requirement. A key feature of BES system is the electron exchange

at cathode. The mechanisms of electron shift from cathode to cells are still poorly

understood. The lack of understanding of electron transfer (ET) process make it difficult

to optimize the cathode surface. Comprehension knowledge about microorganisms-

electrode interaction will open new horizons for BES process for effective bio-synthesis.

A major challenge is the need of material to interface with microbial community because

electrodes are not extracellular interfaces for microorganisms. Adaptive evolution can

prove an effective strategy for electron exchange between electro-autotrophs and external

electron acceptors. Material science can play a great role in providing cheap, durable and

highly conductive electrodes with appropriate surface properties. Good electron transport

properties, better attachment of electro-autotrophs, high surface area must be the qualities

of electrode material. The treatment of carbon cloth with gold, palladium or nickel

nanoparticles can promotes the bio-fuel electrosynthesis. These modification of cathode

surface could prove to be a promising approach for CO2 reduction acting as terminal

electron acceptors. Achieving high PHB density by autotrophic cultivation is not simple

process due to low solubility of gases to liquid phase which is a limiting factor for both

bio-mass as well as PHB synthesis. So to enhance the synthesis of biofuels and bio

commodities efficient electrode material, mechanism of electron transport needed because

BES is not only an environmental sustainable approach but also have great tendency to

reduce the fossil fuel dependence and diversity for biofuel synthesis.

145

Chapter 5

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146

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6 List of Publications

1. Robina Farooq, Gugan Jabeen, Maria Siddique, Saleem Farooq Shaukat, Saif Ur

Rehman. ‘Bioelectrochemical Reduction of Carbon Dioxide to Organic

Compounds’, International Journal of Scientific & Engineering Research, Vol. 7,

Issue 10, (2016), ISSN 2229-5518 (IF = 3.8).

2. Jabeen. G, R. Farooq, 2016. Bio-electrochemical synthesis of commodity

chemicals by autotrophic acetogens utilizing CO2 for environmental remediation,

Journal of Biosciences. 41(3), 367-380. (IF 2.06)

3. Jabeen. G, R. Farooq, et al., 2015. Acetyl-CoA Pathway for Biosynthesis of

Organics, Asian Journal of Chemistry. 27 (1), p 1-8. (IF 0.45)

4. Jabeen. G, R. Farooq, 2015. Microbial Fuel Cells and Their applications for Cost

Effective Water Pollution Remediation, Proceedings of National Academy of

Sciences, India, Section. B Biological Sciences. DOI 10.1007/s40011-015-0683-x.

(IF 0.4).

5. Jabeen. G, R. Farooq, 2016. Synthesis of bio-plastic an innovative technique for

biodegradable and biocompatible polymers. Paper Accepted by Asian Journal of

Chemistry. (IF 0.45).

6. Robina Farooq, Gugan Jabeen, ‘Bioelectro trophic synthesis of

polyhydroxyalkanoates from waste molasses and carbon dioxide’, Patent

application NO. 307/2016, dated 30/05/2016, Government of Pakistan, Intellectual

Property Organization, The Patent Office, Karachi.

163

7 Appendix I

164

Cell dry weight concentration and absorbance

Dry weight

concentration

ppm

Absorbance

0 0

2 1.139

4 1.185

6 2.51

8 3.02

10 3.9

Optical density between days and absorbance

Days Absorbance

1 0.4485

2 0.813

3 0.416

4 0.386

5 0.347

6 0.0994

7 0.0909

165

Calibration between concentration and standard peak area for polymers

Sample

ID

Concentration

ppm

Peak

area

1 0 0

2 5 34063368

3 10 76286069

4 15 95019054

5 20 113757850

Concentration of standard volatile acid mixtures in mM and peak area from GC-MS

analysis

S

N

Standard

concentratio

n

mM

Response

Ethanoic

acid

R Response

Propanoic

acid

Response

Butanoic

acid

Response

Pentatonic

acid

Response

Hexanoic

acid

Response

Heptanoic

acid

1 0.1 15098654 54324176 96324176 564324176 328296634 412057673

2 0.3 45139719 214405316 378265625 748906006 506389282 544189090

3 0.5 75405263 370324213 711841799 818620251 592013781 660452397

4 0.7 89956481 444924191 1106032971 936908978 641811241 690802415

5 1.0 124310996 558485271 1299740406 1026032971 746433069 805078125

166

Standard concentration of alcohols in mM and peak area from GC-MS analysis

SN

Standard

concentration

mM

Response

Ethanol

Response

Butanol

Response

Pentanol

Response

Hexanol

Response

Heptanol

1 0.1 290486201 201486299 334405316 343744450 203745450

2 0.3 334405316 318461004 412057673 466389282 337185855

3 0.5 390324213 378261625 486892803 612013781 486892803

4 0.7 444924191 544924191 546902654 671811241 736908978

5 1 538485271 746433069 645930916 805078125 1026032971

3-hydroxy dimethyl butyrate concentration of Cupriavidus necator for batch system

Time

hours

Concentration

ppm

Peak

area

24 11.043 72262184

48 16.555 105335076

72 24.304 151826724

96 27.029 168176487

120 33.905 209435739

167

Ethyl 3-hydroxy hexanoate concentration of Cupriavidus necator for batch system

Time

hours

Concentration

ppm

Peak

area

24 3.29 25749567

48 4.23 31432830

72 3.29 25748897

96 10.84 71094079

120 11.65 75945173

Table 7.1 2, 4 dimethyl heptanoate concentration of Cupriavidus necator for batch system

Time

hours

Concentration

ppm

Peak

area

24 0.0472 5716340

48 3.66 28005709

72 3.307 25847587

96 4.65 33943107

120 8.27 55657301

3-hydroxy dimethyl butyrate concentration of Cupriavidus necator for continuous

system

Time

hours

Concentration

ppm

Peak

area

24 11.714 76286069

48 25.51 159062396

72 46.78 286681909

96 50.98 311890976

120 79.2 481235571

168

Ethyl 3-hydroxy hexanoate concentration of Cupriavidus necator for continuous system

Time

hours

Concentration

ppm

Peak

area

24 2.003 18022733

48 2.96 23790365

72 11.18 73119674

96 11.26 73560231

120 14.83 95031184

2, 4 dimethyl heptanoate concentration of Cupriavidus necator for continuous system

Time

hours

Concentration

ppm

Peak

area

24 2.0037 18022733

48 3.004 24025339

72 3.166 24998769

96 11.18 73119674

120 14.71 94311089

Acetic acid concentration of Cupriavidus necator for batch system

Time

hours

Concentration

ppm

Peak

area

24 2.86 23178749

48 0.0167 6100704

72 0.262 4424078

96 0.597 2417749

120 0.1125 5324891

169

Acetic acid concentration of Cupriavidus necator for continuous system

Time

hours

Concentration

ppm

Peak

area

24 25.83 160990349

48 6.21 43297154

72 1.807 16842264

96 3.11 24664501

120 2.606 21640611

Total current consumed by Cupriavidus necator and current recovery in polymers in

batch system

Time

hours

Total

Current

Coulombs

3-hydroxy diethyl

butyrate

moles

ethyl 3-hydroxy

hexanoate

moles

2,4-dimethyl

heptanoate

moles

0 0 0 0 0

24 0.000784727 8.3786E-05 2.05355E-05 -3.00637E-07

48 0.001569454 0.000125607 2.64028E-05 2.33121E-05

72 0.002354181 0.000184401 2.05355E-05 2.10637E-05

96 0.003138908 0.000205076 6.76612E-05 2.96178E-05

120 0.003923635 0.000257246 7.27171E-05 5.26752E-05

170

Percent Cathode Recovery for organic polymers by Cupriavidus necator in batch system

Time

hours

Percent

3-hydroxy diethyl

butyrate

Percent

Ethyl 3-hydroxy

hexanoate

Percent

2,4-dimethyl

heptanoate

0 0 0 0

24 31.74 5.63 -0.0192

48 47.58 7.24 14.92

72 69.85 5.633 13.48

96 77.68 18.56 18.95

120 97.45 19.94 33.71

Total current consumed by Cupriavidus necator and current recovery in polymers in

continuous system

Time

hour

3-hydroxy

diethyl butyrate

moles

ethyl 3-hydroxy

hexanoate

moles

2,4-dimethyl

heptanoate

moles

Total

Current

Coulombs

0 0 0 0 0

24 8.92971E-05 1.25023E-05 1.27624E-05 0.021204703

48 0.000194466 1.84758E-05 1.91338E-05 0.042409408

72 0.000356609 6.97834E-05 2.01656E-05 0.063614113

96 0.000388626 7.02828E-05 7.12102E-05 0.084818817

120 0.000603751 9.2566E-05 9.36943E-05 0.106023521

171

Percent Cathode Recovery for organic polymers by Cupriavidus necator in continuous

system

Time hours Percent

3-hydroxy

diethyl butyrate

Percent

ethyl 3-hydroxy

hexanoate

Percent

2,4-dimethyl

heptanoate

0 0 0 0

24 4.373425264 0.095433882 0.210518856

48 9.524165826 0.1410306 0.315615433

72 17.46532643 0.532676387 0.332635972

96 19.03339764 0.536488025 1.174627342

120 29.56934274 0.706582363 1.545506995

Ethanoic acid concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 0.668 11063031

48 0.634 10046234

72 0.741 13253831

96 0.742 13289882

120 0.739 13188983

Butanoic acid concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 1.502 250802549

48 1.686 305902629

72 1.727 318111124

96 1.765 329513300

120 2.04 412057673

172

2-ethyl butyrate concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 1.086 125980229

48 1.176 152846493

72 1.195 158523097

96 1.209 162919910

120 1.231 169401115

Ethanol concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 1.858 311486201

48 2.478 348694790

72 3.022 381369701

96 3.526 411600426

120 3.886 433197118

Pentanol concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 0.227 318221124

48 0.367 329407531

72 0.545 343667282

96 0.521 341721515

120 1.15 392057673

173

Hexanol concentration of Sporomusa ovata for batch system

Time

hours

Concentration

mM

Peak

area

24 0.014 201462343

48 1.436 343667282

72 1.437 343744450

96 1.704 370467988

120 1.855 385512300

Ethanoic acid concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 0.627 9823031

48 0.639 10185289

72 0.672 11187987

96 0.711 12332876

120 0.723 12700923

Butanoic acid concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 1.169 150903539

48 1.165 149788668

72 1.352 205802639

96 1.431 229407531

120 1.79 337180788

174

2-ethyl butyrate concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 1.068 120480229

48 1.018 105480229

72 1.164 149320870

96 1.176 152813390

120 1.184 155302256

Ethanol concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 0.394 223645960

48 0.556 233386630

72 0.898 253899780

96 2.534 352099788

120 3.144 388640251

Pentanol concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 0.025 302057973

48 0.108 308668722

72 0.522 341822675

96 0.574 345940315

120 1.247 399832134

175

Hexanol concentration of Sporomusa ovata for continuous system

Time

hours

Concentration

mM

Peak

area

24 0.12 212057973

48 0.418 241822675

72 0.986 298668722

96 1.059 305940315

120 1.058 305832134

Total current consumed by Sporomusa ovata and current recovery in organic acids and

alcohols in batch system

Time

hours

Total

current

consumed

Coulombs

Ethanoic

acid

moles

Butanoic

acid

moles

2ethyl

butyrate

moles

Ethanol

moles

Pentanol

moles

Hexanol

moles

0 0 0 0 0 0 0 0

24 0.028698653

0.000668 0.001502 0.001086 0.001858 0.000227 0.000014

48 0.057397306 0.000634 0.001686 0.001176 0.002478 0.000367 0.001436

72 0.086095959 0.000741 0.001727 0.001195 0.003022 0.000545 0.001437

96 0.114794611 0.000742 0.001765 0.001209 0.003526 0.000521 0.001704

120 0.143493264 0.000739 0.00204 0.001231 0.003886 0.00115 0.001855

176

Percent cathode recovery of organic products of Sporomusa ovata vs. time duration in

batch system

Time

hours

Percent

recovery

Ethanoic

acid

Percent

recovery

Butanoic

acid

Percent

recovery

2ethyl

butyrate

Percent

recovery

Ethanol

Percent

recovery

Pentanol

Percent

recovery

Hexanol

0 0 0 0 0 0 0

24 6.53 36.71 42.47 27.24 8.3 0.615

48 6.9 41.21 45.99 36.33 13.45 63.17

72 7.2 42.21 46.73 44.31 19.98 63.22

96 7.25 43.14 47.28 51.7 19.1 74.96

120 7.22 49.86 48.14 56.98 42.16 81.61

Total current consumed by biofilm and electrons recovered in organic compounds in

continuous system by Sporomusa ovata

Time

hours

Total

current

consumed

Coulombs

Ethanoic

acid

moles

Butanoic

Acid

moles

2-ethyl

butyrate

moles

Ethanol

moles

Pentanol

moles

Hexanol

moles

0

0

0

0

0

0

0

0

24

0.014349326

0.000627

0.001169

0.001068

0.000394

0.000025

0.00012

48

0.028698653

0.000639

0.001165

0.001018

0.000556

0.000108

0.000418

72

0.043047979

0.000672

0.001352

0.001164

0.000898

0.000522

0.000986

96

0.057397306

0.000711

0.001431

0.001176

0.002534

0.000574

0.001059

120

0.071746632

0.000723

0.00179

0.001184

0.003144

0.001247

0.001058

177

Percent cathode recovery of organic products of Sporomusa ovata vs. time duration in

continuous system

Time

hours

Percent

Recovery

Ethanoic

acid

Percent

Recovery

Butanoic

acid

Percent

Recovery

2-ethyl

butyrate

Percent

Recovery

Ethanol

Percent

Recovery

Pentanol

Percent

Recovery

Hexanol

0 0 0 0 0 0 0

24 0.57 10.43 17.8 1.97 0.25 1.71

48 0.58 10.4 16.96 2.78 1.08 5.97

72 0.61 12.71 19.4 4.49 5.22 14.08

96 0.64 13.7 19.67 9.25 5.74 15.12

120 0.66 16.27 19.73 15.72 12.47 15.3

Ethanoic acid concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.35029 1508921

48 0.48941 5682310

72 0.55031 7509508

96 2.168 56045050

120 2.993 80816740

178

Ethyl butyrate concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 1.875 362501313

48 1.939 381862810

72 1.536 260897141

96 1.171 151571986

120 2.266 480032894

Hexanoic acid concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 1.3231 432314798

48 1.5772 457727616

72 1.0775 407751354

96 2.1833 518335535

120 2.187 518708486

Heptanoic acid concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 1.0123 391109100

48 1.2885 415968147

72 1.998 479858943

96 1.8429 465865059

120 2.0179 481549506

179

Ethanol concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.4475 173148317

48 0.7316 243899242

72 1.014 260897141

96 2.861 371717202

120 3.191 391500347

Hexanol concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.416 241628806

48 0.445 244597812

72 1.717 371717202

96 1.911 391109100

120 2.11 411865059

Heptanol concentration for batch system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.671 74373646

48 0.711 82216463

72 0.753 90608530

96 0.821 104373646

120 0.853 110609530

180

Ethanoic acid concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.45297 4589202

48 0.50238 6071502

72 0.56323 7897181

96 0.56406 7921811

120 2.471 65139719

Ethyl butyrate concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 1.01 103098791

48 1.074 122396863

72 1.2139 164172874

96 1.2377 171334995

120 1.2692 180785376

Hexanoic acid concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.9069 390696796

48 1.6255 462550801

72 1.618 461807102

96 1.6273 462730436

120 2.6638 566389282

181

Heptanoic acid concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.7658 368922538

48 1.6473 448260917

72 1.832 464883681

96 1.6336 447031124

120 2.2314 500802415

Ethanol concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.427 225664596

48 0.74 244443269

72 0.728 243733980

96 1.201 272098768

120 3.143 388620251

Hexanol concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 1.096 309694289

48 1.784 378489968

72 2.482 448260917

96 2.648 464883681

120 2.663 466389282

182

Heptanol concentration for continuous system by Clostridium ljungdahlii

Time

hours

Concentration

mM

Peak

area

24 0.432 26575335

48 0.793 98664631

72 0.929 125868050

96 0.913 122680108

120 0.975 135018689

Total current consumed by Clostridium ljungdahlii and recovered in volatile fatty acids

and alcohols in batch system

Time

hours

Total

current

consumed

Coulombs

Ethanoic

acid

Moles

Ethyl

butyrate

Moles

Hexanoic

acid

Moles

Heptanoic

acid

Moles

Ethanol

Moles

Hexanol

Moles

Heptanol

Moles

0 0 0 0 0 0 0 0 0

24 0.027164 0.00035029 0.001875 0.0013231 0.0010123 0.0004475 0.000416 0.000671

48 0.054329 0.00048941 0.001939 0.0015772 0.0012885 0.0007316 0.000445 0.000711

72 0.081493 0.00055031 0.001536 0.0010775 0.001998 0.001014 0.001717 0.000753

96 0.108658 0.00218 0.001171 0.0021833 0.0018429 0.002861 0.001911 0.000821

120 0.1358228 0.002993 0.002266 0.002187 0.002017 0.003191 0.002115 0.000853

183

Percent cathode recovery of organic products of Clostridium ljungdahlii vs. time

duration in batch system

Time

hours

Percent

Ethanoic

acid

Percent

Ethyl

butyrate

Percent

Hexanoic

acid

Percent

Heptanoic

acid

Percent

Ethanol

Percent

Hexanol

Percent

Heptanol

0 0 0 0 0 0 0 0

24 3.59 77.027 54.354 49.384 6.893 19.226 36.179

48 5.02 79.656 64.793 62.858 11.270 20.566 38.336

72 5.65 63.100 44.265 97.470 15.621 79.353 40.601

96 22.38 48.106 89.692 89.904 44.075 88.319 44.267

120 30.73 93.090 89.844 98.397 49.158 97.747 45.993

Total current consumed by biofilm and electrons recovered in organic compounds in

continuous system by Clostridium ljungdahlii

Time

hours

Total current

consumed

Coulomb

Ethanoic

acid

Moles

Ethyl

butyrate

Moles

Hexanoic

acid

Moles

Heptanoic

acid

Moles

Ethanol

Moles

Hexanol

Moles

Heptanol

Moles

0 0 0 0 0 0 0 0 0

24 0.141624982 0.00039025 0.00101 0.0009069 0.0007658 0.000427 0.001096 0.000432

48 0.282476948 0.00050238 0.001074 0.0016255 0.0016473 0.00074 0.001784 0.000793

72 0.423715421 0.00056323 0.0012139 0.001618 0.001832 0.000728 0.002482 0.000929

96 0.564953895 0.00056406 0.0012377 0.0016273 0.0016336 0.001201 0.002048 0.000913

120 0.706192369 0.002471 0.0012692 0.0026638 0.00223 0.003143 0.002063 0.000975

184

Percent cathode recovery of organic products of Clostridium ljungdahlii vs. time duration in

continuous system

Time

hours

Percent

Ethanoic

acid

Percent

Ethyl

butyrate

Percent

Hexanoic

acid

Percent

Heptanoic

acid

Percent

Ethanol

Percent

Hexanol

Percent

Heptanol

0 0 0 0 0 0 0 0

24 0.475 18.671 24.589 32.595 1.065 42.817 8.385

48 0.611 19.854 44.073 70.116 1.846 69.695 15.392

72 0.685 22.440 43.869 77.977 1.8168 96.963 18.032

96 0.686 22.880 44.122 69.533 2.997 80.0089 17.722

120 3.008 23.463 72.225 94.918 7.843 80.594 18.925

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