Exploring the Impact of Electron Shuttles on Methane

Production

by

Nik Mohd Syazwan Nik Mohd Zamri

A thesis submitted as partial fulfillment

of the requirement for the degree of

Bachelor of Science – Biotechnology (Honours)

School of Biotechnology and Biomolecular Sciences

University of New South Wales

November, 2010
ABSTRACT

Electron shuttles are compounds that can accept and donate electrons reversibly.

They have received a lot of attention due to their capacity and capability in altering routes

of the electron flow through microbes. The transfer of electron through electron shuttles

enhanced bioremediation rate and energy productivity in microbial fuel cells. This study

tried to test the effect of electron shuttles on methane production rate as a final step of

coal conversion to methane. Neutral red, anthraquinone-2,6- disulfonate (AQDS) and

cyanocobalamin were tested on a pure culture of Methanococcoides burtonii as well as on

complex methanogenic community using zero-valent iron as the reducing agent. In the

absence of carbon source, reduced neutral red has been shown to modestly increased

methane production by the methanogenic community while reduced electron shuttles did

not have any impact on methane production by M. burtonii. Inhibition also was observed

in methane production by methanogenic community in treatments with AQDS and more

intense in treatments with cyanocobalamin.

i
ORIGINALITY STATEMENT

I hereby declare that this submission is my own work and to the best of my knowledge

contains no materials previously published or written by any other person, or substantial

proportion of material which have been accepted for the award of any other degree or

diploma at UNSW or any other education institution, except where due acknowledgement

is made in the thesis. Any contribution made to the research by others with whom I have

worked with at UNSW or elsewhere, is explicitly acknowledged in this thesis. I also declare

that the intellectual content of this thesis is the product of my own work, except to the

extent that assistance from others in the project’s design and conception or in style,

presentation and linguistic expression is acknowledged.

Signed...............................................

Date..................................................

ii
ACKNOWLEDGEMENT

• Dr Mike Manefield for your continuous support since the beginning I joined your

group, your patience of teaching me and guiding me from scratch to what I am

now. It was an invaluable experience being under your supervision.

• Dr Matt Lee for all the basics about gas chromatography, experimental setup and

the electrochemistry. I started to appreciate more about organic chemistry and

electrochemistry after I know you.

• Adrian Low for helping me a lot with experimental methods and materials since I

enjoy wasting syringe and needles, and for always be there when I need help.

• Nur Hazlin Hazrin Chong for being a very sweet lab manager.

• Maria-Luisa for DNA extraction advice.

• Joanna Koenig for helping me with GC and ferrous ion determination.

• Whole lab 141 team: Iman, Sania, Olivier, Onder, Sofea for helping me in every way

and any way and continuous moral support.

• My partner, Nadhiah for going through with me this honours year through thick

and thin.

• The Centre for Marine Bioinnovation.

• And you who read my thesis, hope you find it very useful.

iii
TABLE OF CONTENTS

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

1.1 COAL AS AN ENERGY SOURCE ................................................................................................. 1

1.2 METHANE GAS ........................................................................................................................ 2

1.3 METHANOGENESIS .................................................................................................................. 3

1.4 METHANOGENS ...................................................................................................................... 4

1.5 METHANOCOCCOIDES BURTONII ............................................................................................. 4

1.6 THERMODYNAMICS AND REDOX POTENTIAL ........................................................................... 5

1.7 BIOCHEMISTRY OF METHANOGENESIS .................................................................................... 7

1.8 ELECTRON SHUTTLES ............................................................................................................. 9

1.9 IRON AS ELECTRON DONOR FOR METHANOGENESIS .............................................................. 13

1.10 HYPOTHESIS ..................................................................................................................... 14

1.11 AIM OF THE STUDY ........................................................................................................... 14

2 MATERIALS AND METHODS ........................................................................................... 16

2.1 MEDIA FOR BIOASSAYS ......................................................................................................... 16

2.1.1 Carbon-free minimal media ....................................................................................... 16

2.1.2 Carbon-free complex media ....................................................................................... 16

2.1.3 Carbon-free complex media with HEPES buffer ........................................................ 16

2.1.4 Modified methanogen growth medium (MFM)........................................................... 17

2.2 ELECTRON SHUTTLES ........................................................................................................... 18

2.3 MICROORGANISMS AND CULTIVATION.................................................................................. 18

2.3.1 Methanococcoides burtonii ........................................................................................ 18

2.3.2 Methanogenic sludge .................................................................................................. 19

2.4 METHANE ANALYSIS ............................................................................................................. 19

3 RESULTS ................................................................................................................................ 21

3.1 ASSESSING HYDROGEN PRODUCTION FROM IRON CORROSION .............................................. 21

iv
 3.2 IMPACT OF ELECTRON SHUTTLES ON M. BURTONII ................................................................ 22

3.3 IMPACT OF ELECTRON SHUTTLES ON METHANOGENIC SLUDGE ............................................. 24

4 DISCUSSION .......................................................................................................................... 33

4.1 ELECTRON SHUTTLES AS ELECTRONS CARRIER ..................................................................... 33

4.2 IRON AS ELECTRON DONOR AND HYDROGEN SOURCE ........................................................... 34

4.3 IMPACT OF ELECTRON SHUTTLE ON METHANE PRODUCTION BY M. BURTONII ....................... 37

4.4 IMPACT OF ELECTRON SHUTTLES ON METHANE PRODUCTION BY METHANOGENIC SLUDGE .. 39

5 CONCLUSION ....................................................................................................................... 43

v
LIST OF FIGURES

Figure 1: The three steps of coal degradation to methane.. ..................................... 2

Figure 2: The biochemical pathway of reduction of CO2 to CH4 ................................ 8

Figure 3: Structure of certain electron shuttles. ........................................................ 9

Figure 4: Quinone model of reduction and oxidation. ............................................. 10

Figure 5: The action of electron shuttle as an electron carrier ................................ 11

Figure 6: (A). Schematic diagram of microbial fuel cells. ......................................... 12

Figure 7: Electron transfer through the production of hydrogen ............................ 13

Figure 8: Electron from biofilm can come from fermentation................................. 13

Figure 9: Proposed mechanism of electron transfer through electron shuttles.. ... 15

Figure 10: Hydrogen evolution rate over in the absence of biomass. ..................... 21

Figure 11: Hydrogen evolution rate over in the presence of biomass..................... 22

Figure 12: Methane production with different shuttles using iron. ........................ 23

Figure 13: Methane production with different shuttles using trimethylamine....... 24

Figure 14: Methane production with different shuttles using glucose ................... 25

Figure 15: Methane production with different shuttles using iron and 40 mM of

carbonate as a buffer ........................................................................................................... 26

Figure 16: Methane production using 80 mM of carbonate as a buffer. ................ 27

Figure 17: Methane production with different shuttles using iron. ........................ 29

Figure 18: Methane production with different shuttles in the absence of iron. ..... 29

Figure 19: Experimental setup using agar as a physical barrier ............................... 30

Figure 20: Methane production using different shuttles. ........................................ 31

Figure 21: Methane production with different shuttles. ......................................... 32

Figure 22: Schematic illustrations of cathodic depolarisation ................................. 36

vi
 Figure 23: Methylotrophic pathway and biomass production in M. burtonii.......... 38

Figure 24: Central part of methanogenesis - formation of coenzyme M (CoM). .... 41

LIST OF TABLES

Table 1: pH change for treatment in 80 mM carbonate buffer……………………………. 28

Table 2: pH change within cultures in contact with the iron…………………………………32

Table 3: Hydrogen and hydroxide ion evolution from iron corrosion……………………42

vii
ABBREVIATIONS

ES Electron shuttle
AQDS anthraquinone-2,6-disulfonate
mV milli-Volt
∆G°’ Gibbs free energy
E’o Redox potential
CoM Coenzyme-M
CoB Coenzyme-B
CoM-CoB Complex of Coenzyme-M and Coenzyme-B
MFM Methanogenic growth medium

viii
 1 INTRODUCTION

1.1 Coal as an energy source

Coal has been defined by “a compact, stratified mass of mummified plants which
have been modified chemically in varying degrees, interspread with smaller amounts of
inorganic matter” (Ehrlich 1925). It is a form of ancient plant deposit exposed to chemical
and physical conversion to form highly reduced carbonaceous material over very long
geological time scales. The carbon content in coal is generally between 75-95% dry weight
which is higher than peat (51-59% dry weight) and typical wood (49.2% dry weight)
making it a high value fuel.
The Australian Coal Association (ACA) recognises four major classes of coal. Lignitic
coal is the least developed form generated during the Tertiary period (2-60 mya) and
anthracite coal is the most reduced and formed during the Paleozoic period (250-550
mya). In between are the sub-bituminous and bituminous coals that developed during the
Paleozoic and Mesozoic period (60-250 mya). Of all the classes, anthracite and bituminous
coals are considered high rank coals as they have high carbon content and heat value
compared to others. A study made by Ma et al (1991) on five different Australian coals
showed that on average, the burning of coal has a specific heat of combustion of 29.27 MJ
kg-1. Such enormous potential energy within coal explains the high value of the fossil fuel.
In regards to value, the coal mining and combustion process leads to several
environmental impacts, primarily the emission of CO2 to the environment. In 1980, 100.11
million metric tons of carbon dioxide were released to the environment just from the
consumption of coal (EIA 2006). This figure increased up to 231.84 million metric tons in
2006. Such CO2 gas release creates a greenhouse effect and global warming, hence there
is widespread interest in reducing emissions.

1
 Introduction

The conversion of coal to methane gas utilising microbial metabolism for electricity
generation is a promising approach to reducing CO2 emissions. There are three major
steps involved in this process (Figure 1). First, coal is biofragmented to a complex mixture
of aromatic, carboxylic and aliphatic hydrocarbons followed by fermentation to even
simpler compounds such as acetate, CO2 and molecular hydrogen. Finally the action of
methanogens will ultimately turn all the by-products to methane gas.

Figure 1: The three steps of coal degradation to methane. First

biofragmentation by coal degrading bacteria occurs to produce simpler
compounds. Then fermentation occurs converting them to even simpler
molecules before being reduced to methane by methanogens.

1.2 Methane gas

Methane is a colourless and odourless gas and the most abundant organic

chemical in the Earth’s atmosphere (Cicerone and Oremland 1988). It comes from the

anaerobic breakdown of organic carbon from wetlands, landfills, forests and oceans

(Denman et al 2007) and accounts for 70% of total global emissions. It is a potent

greenhouse gas (Heimann 2010) and interacts directly with the Earth’s infrared radiation

2
 Introduction

to ensure the warmth of Earth’s surface and near-surface atmosphere (Cicerone and

Oremland 1988).

The heat of combustion of methane gas is 890 kJ/mol, which is considered low

compared to other common gaseous fuels like ethane (1,560 kJ/mol) and propane (2,220

kJ/mol) (NIST Chemistry WebBook 2008). However, being the simplest hydrocarbon,

methane has the highest heat produced per mass unit (55.6 kJ/g) compared for example

to ethane (52.0 kJ/g) and propane (50.3 kJ/g). Hence, methane gas is favourable to be

used as an alternative fuel for daily uses.

1.3 Methanogenesis

The process involved in methane production by microbes is called

methanogenesis. It is a form of anaerobic metabolism unique to certain members of the

Archaea (Thauer 1998). Organisms with this type of metabolism have received a lot of

attention due to their ecological importance and biochemical features. Methanogenesis is

a very important process for global carbon cycling. For example, organic matter in

wetlands are mainly degraded through fermentation processes to smaller low molecular

weight organic acids and alcohols like lactic acid and ethanol (Torres et al 2005). Bacteria

syntrophically convert these products to acetate, formate and CO2. Ultimately,

methanogens will remove all these products in the form of methane gas through

methanogenesis. Without this final process, large amounts of carbon waste from

fermentation would be accumulating in the environment and become toxic.

3
 Introduction

1.4 Methanogens

While methanogens are all Archaea, the morphology of this group is diverse

including short and long bacilli, cocci, basic forms of large chains and aggregated clumps

(Torres et al 2005). They are now classified into six separate orders: Methanobacteriales,

Methanococcales, Methanomicrobiales, Methanopyrales, Methanocellales and

Methanosarcinales. Different orders have different substrate preferences and different

pathways involved in methanogenesis. The first five groups listed above use H2 and CO2 as

substrates for methanogenesis. The Methanosarcinales lineage has a more versatile

electron donor range including methanol, methyl-amines, methylthiols or acetate

(Deppenmeier et al. 1999).

1.5 Methanococcoides burtonii

M. burtonii is a psychrophilic microorganism adapted to cold temperatures (1-2

°C), isolated from Ace Lake, Antartica and capable of growing up to a maximum

temperature of 28 °C (Williams et al. 2009). This organism is an obligate methylotrophic

methanogen that can utilise C-1 compounds like methylamine and methanol, but not

formate, acetate or carbon dioxide as a carbon source (Goodchild et al. 2004). Many

researches have been done to study the mechanisms and proteins involved in its

adaptation to cold temperature. Recently the genome sequence (Allen et al. 2009) and

proteomic studies (Williams et al. 2009; Williams et al. 2009) of this organism has been

completed allowing the study of genomic and proteomic basis and survival of this

organism.

4
 Introduction

1.6 Thermodynamics and redox potential

Gibbs free energy (∆G°’) indicates the spontaneity and direction of a process at a

certain constant temperature and pressure (Voet and Voet 2004). Exergonic processes

(with –∆G°’ sign) release energy for work spontaneously while endergonic processes (with

+∆G°’ sign) requires the addition of external energy for a reaction to proceed.

Redox potential (E’o) is the tendency for a chemical species to acquire or lose

electrons thereby becoming reduced or oxidised. For example, the ½ O2/H2O redox couple

has a potential of +816 mV (Nelson and Cox 2008) indicating O2 has a high affinity for

electrons. On the other hand, the NADH/NAD+ redox couple has a very low potential of –

320 mV so NADH has a strong tendency to donate electrons to oxygen, as occurs through

protein complexes in electron transport chains during aerobic metabolism. The amount of

energy liberated in this process calculated based on Equation 1 is –220 kJ mol-1 (Box 1). As

∆G’° is directly proportional to redox potential difference (∆E’°), then the potential of the

electron acceptor indicates the amount of energy as ATP obtained in the metabolic

process (Thrash and Coates 2008).

5
 Introduction

Box 1 – Relationship between Gibbs free energy and redox potential difference

∆G’° = –nF ∆E’° [1]

Equation 1: The standard free energy equation for thermodynamic potential of

reactions; n = Number of electrons transferred; F = Faraday proportionality constant

(96.48 kJ mol-1 V-1); ∆E’° = Difference in redox potential between electron donor and

electron acceptor.

Calculation of free energy based on the redox potential difference between

NADH/NAD+ and ½ O2/H2O.

(1) NAD+ + 2H+ + 2e- → NADH + H+ E’° = -0.32 V

(2) ½ O2 + 2H+ + 2e- → H2O E’° = 0.816 V

∆E’° = E’° (electron acceptor) - E’° (electron donor)

∆E’° = +0.816 – (-0.32)

∆E’° = +1.14 V

∆G’° = –2 x (96.48 kJ mol-1 V-1) x 1.14 V

= –220 kJ mol-1

6
 Introduction

1.7 Biochemistry of methanogenesis

There are four overlapping pathways for methanogenesis; hydrogenotrophic,

acetoclastic, methylotrophic and methyl reduction (Welander and Metcalf 2005). The

hydrogenotrophic pathway is the basic reduction of CO2 to CH4 by obtaining electrons

from the oxidation of H2 (Figure 2) (Equation 2). The enzymes involved in the oxidation of

H2 are formylmethanofuran dehydrogenase, which takes part in the first step of CO2

reduction, and coenzyme F420, which is mainly involved in reducing CO2 to methenyl

radical (CH-) and then to methyl radical (CH3-) (Figure 2) (Thauer 1998). The methyl radical

is then transferred to coenzyme M (CoM) and reduced to CH4 during the formation of a

coenzyme M and coenzyme B (CoM-CoB) complex. The reduction of CoM-CoB to its

original substrates is coupled with a phosphorylation process to produce ATP. Note that

the hydrogenotrophic pathway has the highest Gibbs free energy released compared to

other pathways (Equation 3-5) (Thauer 1998).

The acetoclastic pathway utilises acetate to form CO2 as a source of electrons to

reduce methyl radicals to methane (Equation 3) while methylotrophic and methyl

reduction pathways use C-1 compounds e.g. methanol (CH3OH). The methyl reduction and

methylotrophic pathways are essentially the same except the latter occurs when there is

no H2 present to produce the reducing power for reduction of three methanol molecules

to methane (Equation 4 and 5). Similar to hydrogenotrophic, these other pathways also

converge at the formation of methyl-CoM (refer to Welander and Metcalf (2005) for

complete biochemical pathway for all methanogenesis pathways).

7
 Introduction

Hydrogenotrophic: 4H2 + CO2 → CH4 + 2H2O ∆G°’= –131 kJ mol-1 [2]

Acetoclastic: CH3COO- + H+ → CO2 + CH4 ∆G°’= –36 kJ mol-1 [3]

Methylotrophic: 4CH3OH + 2H2O → CO2 + 3CH4 + 4H2O ∆G°’= –106.5 kJ mol-1 [4]
Methyl reduction: CH3OH + H2 → CH4 + H2O ∆G°’= –112.5 kJ mol-1 [5]

∆G°’ = +16 kJ mol-1

∆G°’ = -4.4 kJ mol-1

∆G°’ = -4.6 kJ mol-1

∆G°’ = -5.5 kJ mol-1

∆G°’ = -17.2 kJ mol-1

∆G°’ = -30 kJ mol-1

∆G°’ = -40 kJ mol-1 ∆G°’ = -45 kJ mol-1

Figure 2: The biochemical pathway of reduction of CO2 to CH4 adapted from

Welander and Metcalf (2005). Initially energy was spent to reduce CO2 by
Formylmethanofuran dehydrogenase (Fd). Then, subsequent reduction was done
mainly by coenzyme F420 leading to formation of methyl-coenzyme M. Another
reduction then leads to the formation of methane gas. ∆G°’ values obtained from
Thauer (1993). They add up to –130.7 kJ mol-1 which is almost identical to
calculated value of –131 kJ mol-1 from free energy of formation data (Equation 1)
(Thauer et al 1977).

8
 Introduction

1.8 Electron Shuttles

Electron shuttles are redox active compounds that can accept and donate electron

reversibly without being degraded

degraded. Such compounds can be ubiquitous in the

environment like humic substance

substances that comes from degradation of dead organic matter

(Watanabe et al. 2009) and can al

also
so be biologically made by microbes like cyanocobalamin

(Vitamin B12). Electron

lectron shuttles like neutral red and anthraquinone-2,6--disulfonate (AQDS)

can also be synthetic (Figure 3)

3).

The property of being able to be reduced and oxidised reversibly enables electron

shuttles to be electron carrier

carriers in redox reactions and biochemical processes. For example,

this type of compound has an important role in microbial metabolism, specifically

intracellular electron carriers. Compound like nicotinamide adenine dinucleotide (NAD) is

regarded as an electron shuttle since it involves transferring electrons from the citrate

cycle to the respiratory

atory electron transport chain
chain. In methanogenesis, the coenzyme F420

C.
A.

B.

Figure 3: Structure of certain electron shuttles, AQDS (A); Neutral red

(B); and Cyanocobalamin (C).
9
 Introduction

is also an electron shuttle.

Organic electron shuttles are mostly aromatic compounds connected to redox

active functional groups like amine, di-ketone and hydroxyl (Watanabe et al. 2009). In

humic substance, the quinone groups (aromatic compounds attached to di-ketone

moieties e.g. AQDS) are the electron-accepting moiety. This has been proved by Scott et al

(1998) when they made a study on AQDS using electron spin resonance (ESR)

spectroscopy. They speculated that any electron transfer to quinone leads to the

formation of semiquinone and hydroquinone radicals (Figure 4). Based on ESR

spectroscopy, the reduction process of AQDS detects an increase in radical’s formation

from below detection levels to 7 x 1018 spins/g. Then the signals disappeared after the

introduction of oxygen due to its very high affinity for electrons.

Figure 4: Quinone model of reduction and oxidation proposed

by Scott et al (1998). Electron transfer to the quinone group
leads to the formation of semiquinone (contains an unpaired
electron) and hydroquinone.

10
 Introduction

The activity of electron shuttles in shuttling electrons for microbial metabolism is

influenced by the redox potential of the shuttles (Wolf et al. 2009). It can serve as an

electron carrier from external electron donors to the cells (Figure 5) or from cells to an

external electron acceptor. Being so, it reduces the activation energy of the total reaction

allowing it to occur faster (Van der Zee and Cervantes 2009).

Figure 5: The action of electron shuttle as an electron carrier. The shuttles are
reduced by electron donors (e.g. zero-valent iron) and donate electrons for
microbial metabolism. Figure modified from Watanabe et al (2009).

Electron shuttles’ researches currently focus on utilising bacterial metabolism for

energy production and bioremediation. In microbial fuel cells (MFC), electricity was

generated from decomposition of organic matter and waste by microbes (Watanabe et al.

2009). In this device, electron shuttles carry electrons from bacterial respiration systems

(NADH) to an external electrode allowing electricity generation (Figure 6) (Park and Zeikus

2000). Similar mechanisms also occurr in bioremediation of chlorinated waste as the

transfer of electrons through shuttles allows reduction of the chlorinated waste to non-
11
 Introduction

chlorinated gas (James et al. 2008) (Figure 6). Electron shuttles have also been shown to

enhance degradation of azo-dyes (Watanabe et al. 2009) and nitroaromatic pollutants

(Van der Zee and Cervantes 2009)

A.

B.

Figure 6: (A). Schematic diagram of microbial fuel cells in which neutral

red is used as electron carrier between cells and electrode (Park and
Zeikus 2000). (B) Schematic representation of shuttle-mediated
dechlorination process in the presence of an electron donor (acetate) and
cyanocobalamin (CC) (James et al. 2008).

12
 Introduction

1.9 Iron as electron donor for methanogenesis

Following the promising application of electron shuttles in MFC and

bioremediation, a project has been designed to apply electron shuttles to improving rates
of methanogenesis using iron as electron donor to reduce the shuttles.
Methanogenesis requires CO2 and H2 as the main substrates. In the presence of
iron, anaerobic microbial activities lead to biocorrosion of iron to produce H2 from H2O
(see section 4.2). These H2 molecules can then act as an electron donor for
methanogenesis (Figure 7) (Dinh et al. 2004). In addition, electrons can be obtained from
the complex organic carbon or biomass. The degradation of dead cells releases electrons
in the form of hydrogen by fermentative bacteria (Figure 8). These H2 will also be
consumed by methanogens keeping the concentration level of H2 sufficiently low in the
complex environment.

Figure 7: Electron transfer through the production of hydrogen as a

result of iron corrosion. Open oval represents biomass; Feo represents
zero-valent iron.

Figure 8: Electron from biofilm can come from fermentation of dead cells
and be used for methanogenesis. Open oval represents biomass.

13
 Introduction

1.10 Hypothesis

Given that 1) the final step in production of methane from coal is methanogenesis

and that 2) methanogens can harness energy from a variety of reducing equivalents, it

was hypothesised that electron shuttles in the reduced form can act as electron donors for

methane production and that this can increase methane production rates.

To address this hypothesis an experimental system was devised to supply pure and

mixed species methanogenic cultures with reduced electron shuttles. This involved

incubating biomass in the presence of electron shuttles and zero valent iron (Figure 9).

Zero valent iron was included to maintain the electron shuttles in the reduced state.

Unavoidably, the corrosion of iron in water generates hydrogen, thus the electron shuttles

must compete with hydrogen as an electron donor for methanogenesis in order for their

impact to be observed. Additionally, the fermentation of microbial biomass to hydrogen,

the production of other extracellular reducing equivalents by biomass and the direct

transfer of electrons from iron to methanogens, represents other sources of reducing

power with which the electron shuttles must compete for a role in methanogenesis.

To address the project hypothesis the following aims were tested experimentally.

1.11 Aim of the study

i) Test the impact of neutral red, AQDS and cyanocobalamin on methane

production rates and growth rates of a pure culture of M. burtonii with zero

valent iron or trimethylamine as a source of reducing equivalents.

14
 Introduction

ii) Test the impact of neutral red, AQDS and cyanocobalamin on methane

production by anaerobic sludge in the presence and absence of zero valent iron

as a source of reducing equivalents.

Figure 9: Proposed mechanism of electron transfer through electron

shuttles. The shuttles will be reduced by iron and carry electron to the
biochemical pathway of methanogens. Open oval represents biomass;
Feo represents zero-valent iron.

15
 2 MATERIALS AND METHODS

2.1 Media for bioassays

2.1.1 Carbon-free minimal media

Media was prepared as a 10x concentrated carbon-free minimal media (1 L) that

contained 1 g of KCl, 15 g of NH4Cl, 6 g of NaH2PO4. The working media was made by

diluting the 10x media (100 ml) in 900 ml dH2O containing 2.5 g NaHCO3 as a buffer for pH

7.2.

2.1.2 Carbon-free complex media

Media was prepared based on Karri et al (2005) without the addition of any carbon

source. The media (1 L) contained 280 mg of NH4Cl, 5 g of NaHCO3, 250 mg of K2HPO4, 10

mg of CaCl2.2H2O, 183 mg of MgCl2.6H2O and 1 ml of trace element solution. The trace

element solution (1 L) contained 100 mg of MnCl2.4H2O, 170 mg of CoCl2.6H2O, 100 mg of

ZnCl2, 251 mg of CaCl2.2H2O, 19 mg of H3BO3, 50 mg of NiCl2.6H2O, 20 mg of

Na2MoO4.2H2O and was adjusted to pH 7 with NaOH.

2.1.3 Carbon-free complex media with HEPES buffer

The working media (1 L) used was modified from Shin and Cha (2008) and

contained 1 ml of trace element solution, 100 mg of MgCl2.6H2O, 25 mg of CaCl2.2H2O,

300 mg of KH2PO4 and 300 mg of NaHCO3 buffered with 60 mM of HEPES acid

(C8H18N2O4S) and 30 mM of HEPES salt (C8H17N2NaO4S). The trace element solution (1 L)

contained 100 mg of MnCl2.4H2O, 170 mg of CoCl2.6H2O, 100 mg of ZnCl2, 251 mg of

CaCl2.2H2O, 19 mg of H3BO3, 50 mg of NiCl2.6H2O, 20 mg of Na2MoO4.2H2O and adjusted

to pH 7 with NaOH.

16
 Materials and Methods

2.1.4 Modified methanogen growth medium (MFM)

A modification of methanogenic growth media from Franzmann et al (1992) known

as MFM was prepared as described by Thomas et al (2000). The medium (1 L) contained

335 mg of KCl, 600 mg of MgCl2.6H2O, 100 mg of MgSO4.7H2O, 250 mg of NH4Cl, 140 mg of

CaCl2.2H2O, 23.37 g of NaCl, 2 mg of ferrous ammonium sulfate, 1 mg of resazurin, 5 mg

trimethylamine HCl, 2 g of yeast extract, 10 ml of vitamin solution, 10 ml of mineral

solution, 100 mg of CH3COONa and 140 mg of K2HPO4. The pre-dissolved media was then

flushed with N2 gas (15 minutes) followed by N2:CO2 (80:20; 15 minutes). Then, 500 mg of

pre-dissolved reducing agent cysteine HCl in 1 ml dH2O was added to the media followed

by 2.52 g Na2CO3 as a buffer. Then the media was degassed until the colour change to pale

yellow (indicating reduced condition). The pH was then adjusted to 6.8 using 32% HCl and

autoclaved in 100 ml aliquots at 121°C for 15 minutes in separate serum bottles. The

autoclaved media was shaken overnight to dissolve any precipitate formed and then 1 ml

of 2.5% Na2S solution was added into each serum bottle. The media was allowed to

equilibrate for 2 hours before inoculation.

The vitamin solution (1 L) contained 2 mg of biotin, 2 mg of folic acid, 10 mg of

thiamine-HCl, 5 mg of riboflavin, 5 mg of nicotinic acid, 5 mg of DL-Ca pantothenate, 0.1

mg of vitamin B12, 5 mg of para-aminobenzoic acid and 5 mg of lipoic acid. The vitamin

solution was filter sterilized using 0.22 μm syringe filter (Millipore) and frozen as 10 or 15

ml aliquots.

The mineral solution (1 L) contained 1.5 g of nitrilotriacetic acid (pH then adjusted

to 6.75 with KOH), 3 g of MgSO4.7H2O, 0.5 g of MnSO4, 0.1 g of NaCl, 0.1 g of FeSO4.7H2O,

0.1 g of CoSO4, 0.116 g of CaCl2.2H2O, 1 g of ZnSO4, 250 μl of 4% w/v solution of CuSO4,

0.01 g of Alk(SO4)2, 0.01 g of H3BO3, 0.01 g of Na2MoO4·2H2O. The mineral solution was
17
 Materials and Methods

filter sterilized using a 0.22 μm syringe filter (Millipore) and stored at -20 °C as 10 or 15 ml

aliquots.

Another carbon-free media was also prepared using the same recipe and method

without the addition of trimethylamine, CH3COONa (sodium acetate) and yeast extract.

2.2 Electron Shuttles

Known electron shuttles; Neutral red (-325 mV), anthraquinone-2,6- disulfonate

(AQDS) (-200 mV) and cyanocobalamin (Co(III)/Co(II): +200 mV) were prepared as 50x

stock solutions by dissolving in dH2O in 50 ml tubes and stored at 4 °C.

2.3 Microorganisms and cultivation

2.3.1 Methanococcoides burtonii

A pure culture of Methanococcoides burtonii provided by Dr Tim Williams was

incubated in a modified methanogen growth medium (MFM) under anaerobic condition at

room temperature. The stock culture was stored in a fume hood to prevent any release of

unpleasant odour to the environment.

Batch experiments were performed in triplicate in 30 ml Hungate tubes filled with

12 ml MFM media and 100 μM electron shuttles (Neutral red, AQDS or cyanocobalamin).

A control culture was also prepared without the addition of any electron shuttle. M.

burtonii (3 ml) culture was added and vessels were sealed with a Teflon coated butyl

rubber stopper and purged with N2:CO2 (80:20) for 15 minutes. All cultures were

incubated at room temperature.

18
 Materials and Methods

Another batch experiment using iron as electron donor was also performed by

modifying the setup using carbon-free MFM media and addition of 0.3 g iron into each

vessel.

2.3.2 Methanogenic sludge

Methanogenic sludge was obtained from Malabar Wastewater treatment plant

(Sydney, Australia) and stored in a 200 ml serum bottle under anaerobic condition sealed

with rubber stopper. The sludge was stored at 37 °C shaking at 40 rpm.

An initial batch experiment was performed in triplicate in 60 ml serum bottles

filled with 50 ml carbon-free media, 100 μM electron shuttles (Neutral red, AQDS and

cyanocobalamin) and 1 g iron dust (Fluka, diameter 6-9 μm). Control cultures without iron

and without shuttles were also prepared and then all cultures were sealed and purged

with N2:CO2 (80:20) for 15 minutes. The experiment was initiated by injecting the

methanogenic sludge (0.5 ml) into each culture. All cultures were incubated in 37°C room

temperature.

Subsequent experiments using media with HEPES buffer were done using the same

setup but with the following modifications: less starting media volume (27 ml), more

biomass volume (3 ml) and incubation at 37°C shaking at 40 rpm. The iron concentration

was 20 mg/ml for all subsequent experiments. Sterilised samples were prepared by

autoclaving at 121 °C for 15 minutes three times.

2.4 Methane analysis

Gas chromatography with a Flame Ionisation Detector (FID) was used to analyse

the methane concentration in each treatment. The injection was 100 μl of headspace gas

with 1:10 split ratio in the Agilent Technologies 7890A GC System with J&W Scientific GS-

19
 Materials and Methods

GASPRO column installed. Helium was used as the carrier gas at flowrate 3 ml/min. The

oven temperature was kept at 100°C and the FID was kept at 250°C throughout the

analysis. Each analysis ran for 3 minutes with the methane peak appearing at

approximately 2.8 minutes. A standard curve was prepared to relate the peak area to

methane concentration.

20
 3 RESULTS

3.1 Assessing hydrogen production from iron corrosion

To study the rate of iron corrosion in producing hydrogen from water, 0.6 g iron

dust was incubated in 30 ml of HEPES buffered media at 37°C under anaerobic conditions.

The rate of hydrogen production was constant over 7 days with an observed increase in

hydrogen concentration of 7.5 mM/day/g iron (Figure 10).

35
y = 4.4904x + 3.1032
Hydrogen Concentration (mM)

30
R² = 0.9818
25

20

15

10

0
0 1 2 3 4 5 6 7
TIme (Days)
Figure 10: Hydrogen concentration and evolution rate over 6-days of incubation
in the absence of biomass.

To assess the impact of microbial biomass on the production of hydrogen by iron in

water, a second experiment was conducted whereby 3 mL of methanogenic sludge was

included in the 30 mL reaction. Whilst the rate of hydrogen production was again constant

over 7 days (Figure 11), it was 3.4 fold slower than in the absence of biomass (2.2

mM/day/g iron).

21
 Results

12

Hydrogen Concentration (mM)

y = 1.314x + 2.2554
10
R² = 0.9853

0
0 1 2 3 4 5 6 7
TIme (Days)
Figure 11: Hydrogen concentration and evolution rate over 6-days of incubation
in the presence of biomass.

3.2 Impact of electron shuttles on M. burtonii

To test if electron shuttles serve as electron donors for methane production by M.

burtonii, the organism was incubated anaerobically in the presence of 100 μM neutral red,

AQDS or cyanocobalamin. Zero valent iron was used to ensure the shuttles were

maintained in a reduced state and CO2 is the only carbon source available for the

organism.

Very little methane was produced in all treatments throughout the experiment

(Figure 12). There was variability within the triplicates of each culture. Statistical analysis

(two-way ANOVA) shows that there is no significant difference between all four

treatments tested (P = 0.2462).

22
 Results

0.4
No Shuttle AQDS

Methane Concentration (mM)

0.35
Neutral Red Cyanocobalamin
0.3
0.25
0.2
0.15
0.1
0.05
0
0 5 10 15 20 25 30 35
Time (Days)

Figure 12: Methane concentration over 29-days of incubation with different shuttles
using iron as the electron source. Treatments were established and monitored in
triplicate. Error bars represent standard deviation.

Second experiment on M. burtonii was performed in the presence of its favoured

carbon source i.e. trimethylamine using the same setup as in the previous experiment, in

the absence of iron (Figure 13). After a week, methane production occurred in the control

treatment lacking a shuttle, and in the AQDS and cyanocobalamin treatments. Methane

production was inhibited by neutral red (P = 0.0197).

High variability was observed within the treatments throughout the experiment

and the gas pressure for the no shuttle controls increased steadily in the 15 ml headspace

to a level that some leakage occurred during sampling.

23
 Results

140
No Shuttle AQDS
120
Methane Concentration (mM) Neutral Red Cyanocobalamin
100

80

60

40

20

0
0 5 10 15 20 25 30 35
Time (Days)

Figure 13: Methane concentration over 29-days of incubation with different

shuttles using trimethylamine as the electron source. Treatments were
established and monitored in triplicate. Error bars represent standard deviation.

3.3 Impact of electron shuttles on methanogenic sludge

To test the impact of electron shuttles on methanogenesis by Archaea using a

variety of metabolic pathways, anaerobic sludge was incubated with and without shuttles

in the presence of zero-valent iron or glucose as a source of electrons. The first

experiment was performed in a simple carbon free minimal media to support microbial

growth buffered with 40 mM carbonate buffer in the presence of 100 μM shuttles and 5

mM glucose (Figure 14).

All treatments had a lag phase of 35 days, after which the AQDS treatment had an

increase in methane production while the others remain unchanged. After 50 days, the

control treatment without shuttles began to produce methane. All other treatments
24
 Results

generated very little methane. The pH of all cultures was between 6.5-7 during the 70-

days experiment.

50
45 Glucose only AQDS
Methane Concentration (mM)

Neutral Red Cyanocobalamin

40
No Glucose
35
30
25
20
15
10
5
0
0 10 20 30 40 50 60 70 80
Time (Days)

Figure 14: Methane concentration over 69-days of incubation with different shuttles
using glucose as an electron source. The pH only dropped from 7 to 6.5 throughout
the experiment. Treatments were established and monitored in triplicate. Error bars
represent standard deviation.

At the same time, a similar experiment was also performed using 20 mg/ml zero-

valent iron as the electron source for the biomass (Figure 15). Very little methane was

produced during 17 days of incubation. The treatment with AQDS (P = 0.00003) shows the

best improvement in methane production followed by the treatment with

cyanocobalamin (P = 0.000007). However, the presence of neutral red did not significantly

affect methane production. A small but significant difference (P = 0.0065) in methane

production was observed in the presence and absence of iron suggesting that had little

impact on the experiment.

25
 Results

After 17 days, the pH of all cultures with iron was 8.5-9 and 6.5-7 for the cultures

lacking iron. The pH change in the presence of iron suggested that the buffer used was not

adequate to control pH.

1.6
Methane Concentration (mM)

1.4
Iron only AQDS
1.2
Neutral Red Cyanocobalamin
1
No Iron No Shuttle
0.8
0.6
0.4
0.2
0
0 5 10 15 20
Time (Days)

Figure 15: Methane concentration over 17-days of incubation with different shuttles
using iron as sole electron source using 40 mM of carbonate as the buffer. At day 17,
the pH of the culture has already reached in between 8.5-9. Treatments were
established and monitored in triplicate. Error bars represent standard deviation.

26
 Results

Following the failure of 40 mM carbonate buffer to control the pH in cultures

containing iron (Figure 15), the experiment was repeated using a more complex growth

media based on Karri et al (2005) which contained a more concentrated carbonate buffer

(80 mM). Three treatments were preformed to test the capacity of the buffer in the new

media; two with 20 mg/ml iron, and one without iron. One of the treatments containing

iron was sterilised to assess the impact of abiotic processes on pH and to check for the

abiotic formation of methane gas.

Again, very little methane production was observed (Figure 16). Small quantities

were produced by both viable cultures whilst no methane was observed in the sterile

culture. Methane was produced at a faster rate in the presence of iron but this ceased

after day 10. The pH of iron culture also increased significantly reaching 8.5 by day 6 and

increasing with time (Table 1). In contrast, the pH of the culture lacking iron remained

relatively constant. The pH of the sterile culture also increased over time suggesting iron

corrosion was responsible for hydroxide release.

0.30
Methane Concentration (mM)

Viable+Iron Sterile+Iron
0.25
Viable+No Iron
0.20

0.15

0.10

0.05

0.00
0 2 4 6 8 10 12 14
Time (Days)

Figure 16: Methane concentration over 12-days of incubation to test the ability
of 80 mM of carbonate acting as a buffer. Treatments were established and
monitored in triplicate. Error bars represent standard deviation.
27
 Results

Table 1: pH change for treatment in 80 mM carbonate buffer.

Day 20mg/ml Sterile No Iron
0 7 7 7
6 8.5 9.5 7
10 8.5 10 7.5
12 9 10 7.5

A third experiment was performed in another carbon-free media based on Shin &

Cha (2008) using HEPES as the active buffer. In this experiment, the total working culture

volume was reduced to 30 ml with 3 ml of methanogenic sludge, 100 μM of electron

shuttles and 20 mg/ml iron dust.

The presence of iron greatly accelerated methane production in all treatments

including the treatment without shuttle (Figure 17). For the first 13 days, the methane

production rates were similar for all treatments. Between day 13 and day 43, the role of

the shuttles started to emerge but no statistically significant different between them and

the control treatment. However, on average neutral red treatment was always higher

than the other treatments, while cyanocobalamin was always the lowest.

Methane production in the treatments lacking iron was much lower than in the

iron amended treatments (Figure 18). However, these cultures produced more methane

than in previous experiments without iron as the electron source (Figure 15 and Figure

16). From figure 18, the methane concentration started to increase after a short lag phase

(5 days). Initially, the control treatment without shuttles has the highest methane

production rate followed closely by the neutral red culture. After day 50, the methane

concentration in the neutral red culture suddenly increased threefold after another 10

days of incubation (P= 0.0045). Both AQDS and cyanocobalamin inhibited methane

production as they did in the presence of iron (Figure 17). Methane production was
28
 Results

observed in the AQDS culture throughout the experiment but less than the control. Very

little methane was produced in cultures containing cyanocobalamin. Methane production

stagnated after day 41 except for treatment with neutral red.

70
Methane Concentration (mM)

60
No Shuttle AQDS
50
NR CC
40

30

20

10

0
0 10 20 30 40 50 60
Figure 17: Methane concentration over 62-days of incubation with different
Time (Days)
shuttles using iron as sole electron source. Treatments were established and
monitored in triplicate. Error bars represent standard deviation.

16
Methane Concentration (mM)

14
No Shuttle AQDS
12
NR CC
10
8
6
4
2
0
0 10 20 30 40 50 60 70
Time (Days)
Figure 18: Methane concentration over 62-days of incubation with different
shuttles in the absence of iron. Treatments were established and monitored in
triplicate. Error bars represent standard deviation.

29
 Results

Following the success of HEPES to control the pH, another experiment was

designed to test the effect of limiting contact between biomass and iron using agar. Agar

(2%) was used as a physical barrier between the iron and the biomass (Figure 19).

Media

Biomass + Iron Biomass only

Agar
Agar Agar + Iron

Figure 19: Experimental setup using agar as a physical barrier between iron and
methanogenic sludge to minimise direct access of biomass to electron source.

When agar served as a barrier, the presence of shuttles in the treatments

increased methane production higher than the no shuttle treatments (Figure 20). There

was a small increase during the first 7 days before methane started to build up with the

highest one in the neutral red treatment. In contrast, both AQDS and cyanocobalamin

treatments also had high methane amount but not significantly different to the iron only

treatment (P = 0.3831 for AQDS and P = 0.3540 for cyanocobalamin). The pH maintained

at 7 and rise up to 7.5 during day 19 for both controls and AQDS treatments.

30
 Results

25

Methane Concentration (mM)

20 Iron only AQDS
Neutral Rd Cyanocobalamin
No Iron
15

10

0
0 5 10 15 20 25
Time (Days)

Figure 20: Methane concentration over 23-days of incubation with different

shuttles. Iron was covered in agar and covered again with another layer of agar.
Treatments were established and monitored in triplicate. Error bars represent
standard deviation.

On the other hand, different trend was observed in the treatment where biomass

was in contact with iron (Figure 21). Inhibition was observed in the treatments with

shuttles as well as high variability of methane produced within each treatment throughout

the experiment. Only the AQDS treatment was significantly different to the no iron

treatment (P = 0.0322) while others were not (P = 0.1240 for neutral red and P = 0.3411

for cyanocobalamin). In contrast to previous experiment, there was a major change in the

pH (Table 2). Experiments were terminated after day-23 because the experimental setup

was flawed. Hydrogen gas evolved below the agar that contained iron pushing the agar

upward reducing the volume of the headspace.

31
 Results

25

Methane Concentration (mM)

20 Iron Only AQDS
Neutral Rd Cyanocobalamin
No Iron
15

10

0
0 5 10 15 20 25
Time (Days)

Figure 21: Methane concentration over 23-days of incubation with different

shuttles. Iron was not covered in agar. High variability of the readings was
observed. Treatments were established and monitored in triplicate. Error bars
represent standard deviation.

Table 2: pH change within cultures in contact with the iron.

Time (days) Iron Only AQDS Neutral Rd Cyanocobalamin No Iron
0 6.5 6.5 6.5 6.5 6.5
6 7.5 7.5 7.5 7 7.5
14 8 8 8 7 8
19 8 8 8 7 8

32
 4 DISCUSSION

4.1 Electron shuttles as electrons carrier

Methanogenesis is a form of anaerobic respiration that utilises hydrogen and

carbon dioxide producing methane gas as the main end-product. The gas emitted can be

harnessed and used for energy production (e.g. electricity). Thus, many studies have

characterised the pathways and mechanisms of the reaction including the enzymes and

co-enzymes involved (Thauer 1998; Welander and Metcalf 2005) as well as the

transmembrane proteins involved in energy production (Deppenmeier et al. 1999;

Kulkarni et al. 2009).

This study is designed to assess the impact of certain electron shuttles (neutral red,

AQDS and cyanocobalamin) on methane production. Previous studies have proved the

capability of the selected shuttles in facilitating microbial extracellular respiration. Neutral

red has been shown to interact with microbial metabolism in Clostridium acetobutylicum

(Kim and Zeikus 1992) and Butyribacterium methylotrophicum (Shen et al 1996) by

altering electron and carbon flow. This shuttle is a water and lipid soluble molecule with a

midpoint potential (-325 mV) similar to NADH (-320 mV). Neutral red can also mediate

electricity production in microbial fuel cells by Escherichia coli (Park and Zeikus 2000) and

mixed methanogenic culture (Park et al. 1999).

AQDS has been shown to improve the azo-dyes decolourisation process by

microbes (dos Santos et al. 2003; Costa et al. 2010). Reduction of AQDS produces

anthrahydroquinone-2,6,-disulfonate (AHQDS) and its oxidation to AQDS removes two

electrons with a redox potential of -184 mV (Wolf et al. 2009). Cyanocobalamin (Vitamin

B12) also has been shown to enhance the rate of carbon tetrachloride degradation by both
33
 Discussion

pure culture and anaerobic microbial enrichment (James et al. 2008). Cyanocobalamin is a

shuttle that has cobalt as the central metal with three different oxidation states, Co(III),

Co(II) and Co(I). The reduction of Co(III)/Co(II) and Co(II)/Co(I) has redox potentials of +200

mV (Kim and Carraway 2002) and -590 mV (Kliegman and McNeill 2008) respectively . As

many previous studies have proved that the shuttles are able to be utilised for microbial

extracellular respiration, it is highly likely that they will have certain impact on

methanogenesis.

4.2 Iron as electron donor and hydrogen source

Zero-valent iron (ZVI) was used as the artificial electron source to reduce the

electron shuttles. Iron has been a great interest in improving microbial metabolism. It has

been used as an electron donor to support reductive conversion of contaminants,

bioremediation of halogenated and explosive compounds as well as in permeable reactive

barriers (Karri et al. 2005). Iron also is a highly reactive metal that can corrode with oxygen

and dissolve in oxygen-free water due to the oxidative action of the water itself (Reardon

1995).

In anaerobic conditions, the dissolution of ZVI to release e– (Equation 6) was driven

by the presence of protons from water. This process ends up in the production of H2 as

well as OH– into the solution (Equation 7). The corrosion rate of various iron preparations

was summarised by Reardon (2005) but the corrosion rates obtained in the literatures

were hard to be compared to this experiment due to the nature of their complex

experimental setup and the way of representing the iron’s size (literature used mesh size,

not in μm). The iron dust that was used in this experiment has a diameter between 6-9 μm

which is very small. Due to its small size, more total surface area of iron exposed to water

34
 Discussion

leading to high rate of corrosion (Figure 10). Even so, lower amounts of hydrogen were

detected in the presence of microbial biomass (Figure 11). This may simply be due to the

biomass covering the iron dust, reducing surface area in contact with water, but the

consumption of the hydrogen by the biomass cannot be ruled out.

The dissociation of Fe to Fe2+ has a redox potential of -440 mV (Dinh et al. 2004).

Nernst equation can be used to calculate the amount of reduced shuttle produced at this

potential. Calculation based on Nernst equation (Appendix 1) shows that this potential is

low enough to reduce 100% of 100 μM AQDS and Co(III) to AHQDS and Co(II) respectively

while only 99% of neutral red were reduced. However, iron is not strong enough to further

the reduction of Co(II) to Co(I) (E’°= -590 mV) as only 0.3 μM of Co(I) was produced. Since

Co(II) dominates, cyanocobalamin in this study will have a midpoint potential of +200 mV.

Primary dissolution: Fe ↔ Fe2+ + 2e (E0 = – 0.44 V) [6]

Overall process: Fe(s) + 2H2O(l) → Fe2+ + 2OH- + H2(g) [7]

Additionally electrons from iron in the culture can be utilised by methanogens

directly through a cathodic polarisation process (Daniels et al. 1987) (Figure 22). In the

absence of other electron acceptors (i.e. no shuttle treatment), the electrons released will

dissociate the water molecule to form H2 molecules. The accumulation of H2 in the

treatment serves as electron source as well as freely available substrates for

methanogenesis (Equation 8) leading to higher rates of methane production. This can be

seen in Figure 9 and Figure 11 by comparing the treatment with iron and without iron,

where the presence of iron is not toxic and has a positive impact on methane production.

Methanogenesis: 4H2 + CO2 → CH4 + 2H2O [8]

35
 Discussion

Figure 22: Schematic illustrations of cathodic depolarisation by methane-

producing bacteria. Cathode-derived electrons and water-derived protons form
hydrogen (the form of “hydrogen” is not specified) and used for
methanogenesis.

The impact of the iron corrosion on methanogenesis can be seen in Figure 17.

During the first 13 days, methane formation rate was similar in all treatments indicating

no impact of the electron shuttles. Methane production were very fast at this stage even

faster than reported by Daniels et al (1987) in their experiment of analyzing

methanogenesis with iron as the sole electron source. In their study, Methanosarcina

barkeri produced lower amount of methane than the sludge used in this study as

interaction between complex communities within the sludge might have

released/contained chemicals or carbon source that can accelerate methane production

rate.

36
 Discussion

4.3 Impact of electron shuttle on methane production by M. Burtonii

The pathway used by M. burtonii is methylotrophic pathway (Figure 23) where one

molecule of C-1 compound is oxidised to carbon dioxide to provide electrons for reduction

of another three C-1 molecules to methane (Welander and Metcalf 2005). In this study,

the carbon source for M. Burtonii is trimethylamine and the metabolism using this

compound releases methane, carbon dioxide and ammonia (Hippe et al. 1979).

The presence of shuttles did not promote methane production by M. Burtonii

when grown in trimethylamine, in contrast, a total inhibition was observed for treatment

with neutral red (Figure 13). The presence of neutral red disrupts the methanogenesis by

taking up electron from the biological cofactor involved in the process. In methanogens,

cofactor F420 is the universal electron carrier that involves in transferring electrons in

methanogenesis. In hydrogenotrophic pathway (Figure 2), cofactor F420 (E’° = –360 mV )

accept hybrid electrons from H2 (E’° = –414 mV) (Deppenmeier et al. 1996) and donates

them to molecule involved in the formation of methyl-CoM. However, in methylotrophic

pathway, this cofactor gets reduced by electrons from the biochemical pathways due to

the absence of hydrogen in the M. Burtonii experiment (Figure 23). As there is a difference

in midpoint potential between cofactor F420 and neutral red, calculation based on Nernst

equation estimates that 87.5% of the neutral red get reduced at -360 mV, suggesting that

most electrons were used to reduce the shuttle rather than involved in methane

production. However, reduction of 87.5% neutral red should not completely inhibit

methane formation. It can be hypothesised that the shuttle might get involved in breaking

down the trimethylamine to a form that cannot be utilised by M. burtonii. In a study by

Colby and Zatman (1974), they have shown that phenazine methosulphate, a derivative of

37
 Discussion

phenazine, can acts as electron acceptor in trimethylamine degradation. As neutral red is

also a phenazine derivative, it might as well have the capacity to chemically involve in

oxidation of trimethylamine through another mechanism.

Figure 23: Methylotrophic pathway and biomass production in M. burtonii.

The substrates enter the pathway through methyl-corrinoid protein (CH3-
CP). Refer to Allen et al (2009) for complete details of each pathway and
each compound.

38
 Discussion

The presence of iron is toxic to M. burtonii (Figure 12). Methane production was

greatly inhibited compared to Figure 13. However, there was some methane produced

during the first 7 days of incubation reaching a level almost similar to experiments of

sludge with in the presence of iron (Figure 15 and Figure 16). Hence, it can be concluded

that it was not the zero-valent iron being the toxic agent; it was the accumulation of

product from iron corrosion inhibiting this organism. M. burtonii originates from cold

climate. However, this organism is able to grow at temperatures as high as 23 °C; the

temperature at which the pure culture was grown in this study. Based on a study by

Williams et al (2009), the shifting of growth temperature leads to several changes

including less surface layer protein and less integral membrane proteins which are

believed to be for higher temperature adaptations. However, more ferrous ion transport

proteins and ferritin (iron storage protein) were produced by the methanogens at 23 °C.

Therefore it would seem unlikely that an increase in Fe2+ would have a deleterious effect

on the growth of M. burtonii.

The presence of neutral red completely inhibited methane production when M.

burtonii was incubated with trimethylamine, but not as severe as when incubated with

iron. Neutral red did not have a negative impact because it was reduced by iron and not

by the cofactor F420. Hence, the biochemical pathway was not disrupted by neutral red

and methane can be produced.

4.4 Impact of electron shuttles on methane production by methanogenic sludge

The presence of the different electron shuttles had different impacts on methane

production for both pure culture and methanogenic sludge. The shuttles will only be

effective if it lowers activation energy of reactions involved in methanogenesis. To achieve

39
 Discussion

that, midpoint potential of the shuttles should be ideally in between the two eventual half

reactions involved in energy generation (Van der Zee and Cervantes 2009). As for azo dye

reduction, electron transfer is from biological carrier to the extrinsic waste. Thus, the

shuttles should have midpoint potential higher than NADH (E’° = –320 mV) and lower than

of the azo dye for improvement in decolourisation rate to occur. However, the context of

this project is looking for shuttles that donate electrons into the biological system to

enhance methanogenesis rate. Hence, the shuttles will be effective if it has a midpoint

potential lower than the universal electron carrier involves in methanogenesis i.e. cofactor

F420. Since this cofactor has a midpoint potential of –360 mV, which is lower than all the

three shuttles testes, the cofactor F420 is unlikely to be reduced by any of the shuttles.

Thus, if any acceleration in methane production rate observed in this study, then it was

not influenced through this compound i.e. direct reduction of the cofactor by the shuttles.

From all the shuttles tested, neutral red seemed to have a modest positive impact

on methanogenic sludge while others did not (Figure 18). In fact, cyanocobalamin seemed

to have an inhibitory effect. A proposed hypothesis for this observation is due to the

disruption of methanogenic biochemical pathway by the shuttles. Figure 23 shows the

central part of methanogenesis which is the formation of coenzyme-M. The coenzyme-M

(CoM) has an attached methyl group that when oxidised with coenzyme-B (CoB) forming a

heterodisulphide complex of CoM-CoB, the methyl group will be reduced to methane

molecules. The CoM-CoB complex will then be reduced back to its original substrate and

this process has a potential of –200 mV (Equation 9). As neutral red has a potential of –

325 mV, it will be able to donate electrons for the reduction of CoM-CoB process, causing

acceleration and more methane were produced. In contrast, AQDS (–187 mV) and

cyanocobalamin (+200 mV) have potentials higher than the reduction step. Instead of
40
 Discussion

donating electrons, these shuttles may extract electrons from the reduction process,

causing deceleration and less methane produced.

Figure 24: The central part of methanogenesis i.e. the formation of coenzyme M
(CoM) and its reaction with coenzyme B (CoB) to produce CoM-CoB complex and
CH4. The oxidation of the complex is coupled with ADP phosphorylation to ATP.

CoM-S-S-CoB + 2[H] → H-S-CoM + H-S-CoB E’o = -200 mV [Equation 9]

Experiments with iron required HEPES as the working buffer. HEPES is a strong

buffer and has been described as one of the best buffers available for biological research

(Good et al. 1966). It has a pKa value of 7.48 with a buffer range 6.8-8.2. The iron

corrosion produces H2 and OH- at a molar ratio of 1:2. Table 3 summarise the calculated

amount of hydrogen and hydroxide iron present from iron corrosion experiment (Figure

17 & 18). The presence of 60 mM of HEPES was strong enough to buffer the reaction

solution maintaining a pH in between 7-8 for 40 days.

41
 Discussion

Table 3: Hydrogen and hydroxide ion evolution from iron corrosion.

With biomass Without biomass
-
Day H2 (mM) OH (mM) H2 (mM) OH- (mM)
0 0 0 0 0
1 3.7 7.3 7.1 14.2
2 5.0 9.9 12.3 24.6
3 5.7 11.5 15.7 31.4
4 7.9 15.8 23.0 46.1
5 8.6 17.2 25.9 51.9
6 10.2 20.5 28.9 57.7

42
 Discussion

5 CONCLUSION

Different electron shuttle have different impact on methane production depending on

their midpoint potential. Reduced neutral red, AQDS and cyanocobalamin have no

significant impact on methane production by M. burtonii. In fact, neutral red can become

an inhibitor for methane production when the preferred substrate for M. burtonii

(trimethylamine) is present in the treatment.

Neutral red is able to increase methane production by methanogenic community to a

modest degree relative to AQDS and cyanocobalamin. In contrast, the presence of AQDS

and cyanocobalamin become an inhibitor for methanogenesis. The level of inhibition

depends on the midpoint potential of each shuttle to the potential of molecules involved

in biochemical pathway.

43
 Discussion

(Thomas et al. 2000) (Karri et al. 2005) (Shin and Cha 2008) (Stookey 1970) (Ma et

al. 1991) (Thomas et al. 2000) (Reardon 2005) (Williams et al. 2009) (Scott et al. 1998)

(Park and Zeikus 1999) (Allen et al. 2009)

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7 APPENDIX

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50
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51
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