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by
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
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
i
ORIGINALITY STATEMENT
I hereby declare that this submission is my own work and to the best of my knowledge
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,
Signed...............................................
Date..................................................
ii
ACKNOWLEDGEMENT
• Dr Mike Manefield for your continuous support since the beginning I joined your
• Dr Matt Lee for all the basics about gas chromatography, experimental setup and
• 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.
• Whole lab 141 team: Iman, Sania, Olivier, Onder, Sofea for helping me in every way
• My partner, Nadhiah for going through with me this honours year through thick
and thin.
• And you who read my thesis, hope you find it very useful.
iii
TABLE OF CONTENTS
1 INTRODUCTION .................................................................................................................... 1
3 RESULTS ................................................................................................................................ 21
iv
3.2 IMPACT OF ELECTRON SHUTTLES ON M. BURTONII ................................................................ 22
4 DISCUSSION .......................................................................................................................... 33
5 CONCLUSION ....................................................................................................................... 43
v
LIST OF FIGURES
Figure 10: Hydrogen evolution rate over in the absence of biomass. ..................... 21
Figure 12: Methane production with different shuttles using iron. ........................ 23
Figure 14: Methane production with different shuttles using glucose ................... 25
Figure 15: Methane production with different shuttles using iron and 40 mM of
Figure 17: Methane production with different shuttles using iron. ........................ 29
Figure 18: Methane production with different shuttles in the absence of iron. ..... 29
LIST OF TABLES
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
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.
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
1.3 Methanogenesis
Archaea (Thauer 1998). Organisms with this type of metabolism have received a lot of
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
methanogens will remove all these products in the form of methane gas through
methanogenesis. Without this final process, large amounts of carbon waste from
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,
pathways involved in methanogenesis. The first five groups listed above use H2 and CO2 as
°C), isolated from Ace Lake, Antartica and capable of growing up to a maximum
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
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
5
Introduction
Box 1 – Relationship between Gibbs free energy and redox potential difference
(96.48 kJ mol-1 V-1); ∆E’° = Difference in redox potential between electron donor and
electron acceptor.
∆E’° = +1.14 V
= –220 kJ mol-1
6
Introduction
acetoclastic, methylotrophic and methyl reduction (Welander and Metcalf 2005). The
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
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
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
7
Introduction
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]
8
Introduction
Electron shuttles are redox active compounds that can accept and donate electron
The property of being able to be reduced and oxidised reversibly enables electron
regarded as an electron shuttle since it involves transferring electrons from the citrate
C.
A.
B.
active functional groups like amine, di-ketone and hydroxyl (Watanabe et al. 2009). In
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
from below detection levels to 7 x 1018 spins/g. Then the signals disappeared after the
10
Introduction
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
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).
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
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
A.
B.
12
Introduction
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
the production of other extracellular reducing equivalents by biomass and the direct
power with which the electron shuttles must compete for a role in methanogenesis.
To address the project hypothesis the following aims were tested experimentally.
production rates and growth rates of a pure culture of M. burtonii with zero
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
15
2 MATERIALS AND METHODS
diluting the 10x media (100 ml) in 900 ml dH2O containing 2.5 g NaHCO3 as a buffer for pH
7.2.
Media was prepared based on Karri et al (2005) without the addition of any carbon
The working media (1 L) used was modified from Shin and Cha (2008) and
to pH 7 with NaOH.
16
Materials and Methods
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
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,
0.01 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.
(AQDS) (-200 mV) and cyanocobalamin (Co(III)/Co(II): +200 mV) were prepared as 50x
room temperature. The stock culture was stored in a fume hood to prevent any release of
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
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.
(Sydney, Australia) and stored in a 200 ml serum bottle under anaerobic condition sealed
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
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
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
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.
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
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.
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
22
Results
0.4
No Shuttle AQDS
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.
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
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
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)
variety of metabolic pathways, anaerobic sludge was incubated with and without shuttles
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
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)
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
cyanocobalamin (P = 0.000007). However, the presence of neutral red did not significantly
production was observed in the presence and absence of iron suggesting that had little
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
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
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
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
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
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
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
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
Figure 19: Experimental setup using agar as a physical barrier between iron and
methanogenic sludge to minimise direct access of biomass to electron source.
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
10
0
0 5 10 15 20 25
Time (Days)
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
31
Results
25
10
0
0 5 10 15 20 25
Time (Days)
32
4 DISCUSSION
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
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
red has been shown to interact with microbial metabolism in Clostridium acetobutylicum
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
microbes (dos Santos et al. 2003; Costa et al. 2010). Reduction of AQDS produces
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.
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
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).
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
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.
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
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.
35
Discussion
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
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
rate.
36
Discussion
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).
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
accept hybrid electrons from H2 (E’° = –414 mV) (Deppenmeier et al. 1996) and donates
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
Colby and Zatman (1974), they have shown that phenazine methosulphate, a derivative of
37
Discussion
also a phenazine derivative, it might as well have the capacity to chemically involve in
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
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
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
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
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
(CoM) has an attached methyl group that when oxidised with coenzyme-B (CoB) forming a
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,
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.
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
41
Discussion
42
Discussion
5 CONCLUSION
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
modest degree relative to AQDS and cyanocobalamin. In contrast, the presence of AQDS
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)
44
6 REFERENCES
45
References
Wolf, M., A. Kappler, J. Jiang and R. U. Meckenstock (2009). "Effects of Humic Substances
and Quinones at Low Concentrations on Ferrihydrite Reduction by Geobacter
metallireducens." Environmental Science & Technology 43(15): 5679-5685.
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7 APPENDIX
49
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50
References
51
References
52