The Effects of Temperature and Feed Concentration on
Methane Production in Thermophilic Bacteria.
(Denise-30507106, Mark 30441382, Caleb-30833366, Mandeep, Michael, Matt Tye30385839 and James- 30037902)
The Effects of Temperature and Feed Concentration on
Methane Production in Thermophilic Bacteria.
1. Abstract
A chemostat bioreactor was established using a mixed culture of thermophilic
methanogenic bacteria sourced from AnaeCo. A specific methane production rate of 1.5
mmole methane/mmole glucose/h was observed at glucose feed concentration at 1M with
D = 0.001 h-1. The conversion efficiency of glucose to methane at varying glucose
substrate concentrations was determined to be on average 54%. Methane production was
determined at 55, 62, 70 and 80 ° C over a 24 hour period. Most significantly this
chemostat project determined that optimal methane production occurred at 62 ° C with a
conversion efficiency of 86%.
2. Introduction
2.1 The recent oil crisis and the consequent price rises have generated considerable
interest in the exploration of renewable energy sources. However one alternative,
methane, was discovered centuries ago and still may be the one that civilization turns to
once the oil dries up. (V. Nallathambi Gunaseelan 1997) Methane is an attractive
alternative energy source. Compared to other fossil fuels, methane produces few
atmospheric pollutants and generates less carbon dioxide per unit energy (Chynoweth D
P., J M. Owens and R. Legrand. 2000). Methane currently represents about 20% of the
US energy supply (Chynoweth D P., J M. Owens and R. Legrand, 2000). One of the most
attractive qualities of methane as a renewable energy source however is that by-products
of food processes and organic waste can be utilized to produce energy rather than
discarded. The most common process of methane production is via anaerobic digestion.
2.2 Anaerobic digestion is a process in which fermenting and methanogenic bacteria
convert organic matter into methane (CH4) and carbon dioxide (CO2). This process,
which is actually carried out by a consortium of several different microorganisms, can be
found in numerous environments, including sediments, flooded soils, animal intestines,
and landfills.(Chynoweth D P., J M. Owens and R. Legrand. 2000) The process can be
divided into three sections; fermentation, acid production and finally methane production.
2.3 In the first stage biomass is digested by anaerobic digestion into alcohols and fatty
acids, which are soluble (Macleod et al. 1990). Acetogenic bacteria are then able to
utilize these compounds to produce acetic acid. (Macleod et al, 1990). The final stage
involves the actual production of methane. The bulk of the methane is produced by
utilization of the acetate, CO2 and hydrogen produced by the stage 2 bacteria. (Rohlich et
al, 1977) The biogas composition is typically 60% methane and 40% carbon dioxide with
traces of hydrogen sulfide and water vapor. (Chynoweth D P., J M. Owens and R.
Legrand. 2000) The product gas can be used directly or processed to remove carbon
dioxide and hydrogen sulfide.
2.4 Thermophilic bacteria have optimum growth above 50˚C and are enriched in
bioreactors operating between 50 and 60˚C (Isaacson, 1991). With this experiment
requiring thermophilic methanogens a mixed culture was sourced from AneaCo
(anaeco.com) through Murdoch University. AnaeCo have developed a patented
bioconversion process called DiCom that incorporates anaerobic digestion of municipal
waste for production of biogas into its recycling business in Perth. This system operates
at an optimum temperature of 55˚C which was adopted as the baseline temperature in this
experiment.
2.5 Aims
The aims of this experiment were to establish a functioning chemostat of thermophilic
methanogenic bacteria to initially establish methane production for a given substrate
loading and to then determine optimal temperature for methane production.
3 Materials and Methods
3.1. Medium and Inoculum
A 500ml sample of activated thermophilic methanogenic bacteria was acquired from
Aneaco through Murdoch University. This was placed into a 1 L glass Braun bioreactor
which was sealed with a rubber bungs in outlet holes and a rubber seal between the top
and bottom sections of the reactor (Figure 1). The bottom of the bioreactor was filled
with crushed pumice to a depth of 25 mm to provide surface area for biofilm formation.
A hole was drilled in one rubber bung to allow tight insertion of a 1 mL syringe tube
(with ends removed). Silicon tubing was then attached to these ends and secured with
cable ties. This method was used for all fittings and it is important to perfect this
technique. Access to the reactor for pH testing was through a bung with a tube hanging in
the liquid and an outside tube sealed with a clamp.
A stock glucose solution was prepared and stored in refridgeration.
Stock Glucose Solution (g/L) (Jarell and Kalmokoff, 1988)
D-glucose
180
NH4Cl
1.07
Yeast extract 1.0
MgCl2.6H2O
1.02
Tryptone
1.0
CaCl2.2H2O
0.01
KCl
0.52
Na2SO4
0.14
KH2PO4
0.30
NaHCO3
2.10
The Braun reactor sat in an insulated water bath incubator initially set to a temperature of
55oC. The water bath incubator was covered with a 25 mm thick polystyrene sheet, a hole
was cut in the sheet into which the Braun reactor sat. The incubator was filled with water
and kept topped up during the course of the experiments.
Initial research was carried out under batch conditions with the layout of the equipment
illustrated in Fig.1. Emitted gas was passed through to an inverted water-filled container
where gas entering displaced an equal volume of water into a collection vessel, enabling
gas production (mL) to be recorded by measuring water displaced.
The main aim for this initial experimentation was to check for gas production and find
any leaks in the reactor.
3.2 Carbon Dioxide Capture
Carbon dioxide was removed from the total gas output from the system by passing it
through a 1M solution of NaOH according to the simple acid-base reaction:
CO2 + NaOH → NaHCO3 or Na2CO3 where ratio of acid:base >= 1:2 + H2O.
This solution was tested regularly to ensure that the pH remained in excess of 10.
3.3 Chemostat Reactor
A chemostat reactor was then set up with the full layout of the equipment illustrated in
Figure 2. The glucose solution was placed in a 1 litre Schott bottle, this sat in a container
filled with ice (to minimize fermentation) on top of a magnetic stirrer (to prevent
precipitation of glucose solution). The pump was connected to a timer and set for
inflow/outflow of 2 mL every 2 hours. The outflow was also collected in a Schott bottle.
The glucose solution was fed into the reactor through a 5 mL syringe that had been
pushed through a rubber bung preventing contamination of the feed line. The outlet tube
was placed to hang in the middle of the reactor liquid and the end was covered with
perforated dishcloth to prevent blockage and methane bubbles entering. Two safety
bottles were included to prevent mishaps with the NaOH.
3.4 Gas Recording
The gas produced passed through the safety bottles and NaOH solution to an oil-filled Utube delivering gas under uniform pressure to an electro-mechanical gas flow recorder,
calibrated to issue a pulse at 9mL gas volume increments. This sensor was linked to a
computer with a LabView program data-logger and analyser. The gas flow recorder also
had a manual counter which proved invaluable as problems were encountered with the
program.
3.5 Volatile Fatty Analysis
In order to evaluate, to some degree, the efficiency and possibly stability of the process
with regard to production of volatile fatty acids (VFAs), samples of the culture media
were taken at intervals over the course of the procedure.
Volatile fatty acids were analysed by first taking 1.5 mL of digester liquor in 2 mL
Eppendorf tubes and centrifuging (@12000rpm-2min). Next 0.5% v/v phosphoric acid
was added to free all VFAs as well as to control bacterial numbers. The samples were
then analysed on Varian’s 3300/3400 gas chromatograph, the following parameters were
used.
Column:
Packing:
Column Temp:
Carrier Gas:
N2 flow rate
Injection Mode:
Injection Temp:
Sample Volume:
Detector:
Detector Temp:
Glass, 1.8m x 2 mm ID
Poropack QS, 80/100 mesh
190º C
Nitrogen
12 mL/min
On Column
250º C
1 μL
Flame Ionisation (FID)
250º C
3.6 Methane Production at varying glucose feed concentrations
Fresh batches of the glucose feed were made up at 0.5, 0.75 and 1 M. The reactor was fed
for 24 hours with each feed concentration and gas produced recorded.
3.7 Methane Production at varying Temperature
The temperature of the water bath incubator surrounding the bioreactor was increased to
62, 70 and 80oC, and emitted gas volume recorded over a 24 hour period. After the water
bath reached the new temperature 1 hour was allowed to pass before gas recording began
to allow for gas expansion at the higher temperature.
Batch Culture
Gas
Collection
vessel
Water bath
55ºC
NaOH
Culture
Safety vessel
vessel
Direction of inflow (feed and biogas)
Tube to extract culture to test for pH and
to add feed
Figure 1: Batch culture
The Chemostat Set-up
CPU
Peristaltic
Pump
Counter
valve
Magnetic
Water bath
stirrer
55ºC
NaOH
Culture
vessel
Collection
vessel
Safety vessel
You tube
filled
with oil
Direction of inflow (feed and biogas)
Direction of outflow (biomass with waste)
Tube to extract culture to test for pH
Figure 2: The Chemostat Apparatus
4. Results
In order to address the aims of the experiment the results are presented in three sections.
4.1 Establishment of a functional chemostat
To get a functional chemostat working effectively is the initial aim of the project
 The batch culture after inoculation onto the pumice was fed 12 mmole of glucose
in one dose principally to confirm gas was being produced and leaks were sealed.
This resulted in methane production of approximately 400 mL in 24 hours.
Temperature was maintained at 55˚C
 The batch was converted to a chemostat but failed due to pump or timer
malfunction causing 300 mL of substrate to enter the reactor. 15 mL of 3M NaOH
stabilized pH. 200 mL of batch culture was removed and 200 mL H2O added. The
pump was replaced (see recommendations)
 This culture was put into a batch reactor and left until the volatile fatty acid
concentration (VFA) reduced (Table 1).
Table 1: [VFA] after substrate over-addition
Acetate (mM) Propionate (mM) Butyrate (mM)
1 Day after 300 mL substrate added 179.46
28.07
91.17
8 Days after 300 mL substrate 51.55
18.71
89.61
added


After reinoculation with a new culture the chemostat again failed due to
pump/timer malfunction however the gas outlet blocked causing the reactor to
explode (see recommendations)
The chemostat was successfully established using the original culture after [VFA]
was reduced (Table 1) and the reactor was connected to the U-tube counter.
4.2 Methane production at varying substrate concentrations
The chemostat was run at three glucose substrate concentrations (0.5 M, 0.75 M and 1 M)
for 24 hours at D = 0.001 h-1 (1 ml/1 L/h) in order to determine methane production by
the mixed culture (Figure 3)
1000
Methane Production (mL) in 24 hours
900
800
700
600
500
400
300
200
100
0
0.5
0.6
0.7
0.8
0.9
1
1.1
Glucose Feed concentration (M)
Figure 3: Methane production at [glucose] of 0.5, 0.75 and 1.0 M at 55˚C over a 24 hour
period at D = 0.001 h-1 (2 mL every 2 hrs into 1L)
Table 2: Methane production at [glucose] of 0.5, 0.75 and 1.0 M at 55˚C
[Glucose]
M
Methane
produced
mL (24
h)
Methane
mL
prod/h
Total
Substrate
Loading
mmoles
Methane
Production
mmoles
Expected
Methane
Production
mmoles
Specific
Methane Prod
Rate
(mmol/mmol
glucose/h)
Conversion
Efficiency %
0.5
0.75
1.0
500
700
900
20.8
29.2
37.5
12
18
24
20.4
28.5
36.7
36
54
72
1.7
1.6
1.5
56.6
52.9
51.0
Conversion efficiency is calculated by converting methane produced/hour (mL) to mmole
produced/hour and comparing to that expected.
Example calculation: 0.5 M (Table 2)
500 mL methane/24.5 (L gas per mole) = 20.4 mmole methane
C6H12O6 → 3CH4 + 3CO2
Expected 1 mole glucose = 3 moles methane so 36 mmoles expected
Conversion efficiency = (20.4/36)*100 = 56.6%
4.3 Methane production at varying temperatures
The chemostat was run with glucose substrate concentration of 1 M for 24 hours at four
temperatures (55, 62, 70 and 80˚C) in order to determine methane production by the
mixed culture at varying temperatures (Figure 4) and the conversion efficiencies (Table
4). The dilution rate D = 0.001 (1 mL/1L/h) however an extra 4 ml of glucose substrate
was added at the start of each run hence the altered substrate loading rate of 1.16 mmol/h.
Table 3: Methane Production rate over a range of Temperatures in a 24 hour period
Temperature Methane
Methane
Substrate
Specific Methane Conversion
˚C
Prod ml/h Prod
loading
Production Rate
Efficiency %
mmol/h
mmol/h
mmol
methane/mmol
glucose/h
55
48.9
1.996
1.16
1.72
57.3
62
71.6
2.992
1.16
2.52
86.0
70
26.6
1.086
1.16
0.94
31.2
80
0
0
1.16
0
0
80
Methane Production (mL/h)
70
60
50
40
Series1
30
20
10
0
55
60
65
70
75
Temperature C
Figure 4: Methane production at varying temperatures
80
85
The highest specific methane production of 2.52 mmoles methane/mmole glucose was
achieved at 62˚C with an efficiency of 86%. No methane production was recorded at
80˚C.
4.4 VFA analysis
The VFA concentrations inside the reactor were analysed at the varying temperatures
(Table 3)
Table 3: VFA concentrations inside reactor at 55, 62 and 70 ˚C
Temperature C Acetate (mM) Propionate (mM) Butyrate (mM)
55
64.50
17.31
78.91
62
126.40
32.33
52.51
70
175.79
31.22
40.13
Total VFA (mM)
160.72
211.24
247.14
Overall VFA concentration increased with rising temperature, acetate concentrations in
particular have increased markedly while butyrate concentrations have decreased.
The chemostat was successfully run at varying substrate concentrations and temperatures
in accordance with the aims of the project.
5. Discussion
This project successfully established a working chemostat of thermophilic methanogenic
bacteria and tested methane production rates over a range of substrate concentrations and
temperatures.
5.1 To establish a functional chemostat
The first challenge for this project was to get a functioning air tight chemostat. This was
the most difficult and important part of the experiment. A batch culture was set up first to
test the system of any leakage. There were many leakage problems whilst setting up the
batch culture. In order to detect where exactly the leakages were, the system was put
under water and air pressure applied to detect leakage. After sealing the leaks, 400ml of
methane was obtained in 24hrs after being fed a total of 12 mmol of glucose.
The batch culture was then converted to a chemostat but failed to obtain results after the
first run due to faulty pumps or timer. Over the weekend, there was a malfunction with
either the timer or inflow pump which caused an excess of 300mL of substrate to enter
the bioreactor causing a blockage in the pipe of the first safety bottle. Due to a buildup of
pressure this led to the chemostat exploding. Perseverance eventually paid off and a
functioning chemostat was established, enabling the second and third aims of the
experiment to be met.
5.2 Effects of Concentration of Glucose
Methane is produced from glucose in a ratio according to the equation
C6H12O6 → 3 CH4 + 3 CO2
At 0.5 mM glucose concentration 0.84 mM methane was produced. Using the
stoichiometric equation for this reaction 1.5 mol of methane is produced per 0.5 mol of
glucose. So at 0.5 mM glucose concentration the chemostat ran at 56% efficiency. The
remainder has been used for biosynthesis of VFAs or converted to energy for growth and
maintenance.
Production of methane increased as glucose concentration increased. At 1M glucose
concentration 900 mL of methane was produced. `As substrate feed concentration
increased, production of methane increased while production efficiency decreased
slightly.
5.3 Effects of Temperature
Methane production increased when temperature was increased to 62°C However, the
total VFA (mM) concentration, especially acetate increased substantially (doubled)
between 55° to 62° C. Between 62° C to 70°C there was a less significant increase of
acetate. Acetate oxidation is a rate limiting step and higher temperatures favour acetate
formation (Yue et al., 2008). Therefore the higher the temperature, the higher the acetate
formation hence up to an optimum of 62°C in this case. If the temperature rises further,
more acetate is formed which possibly limits the methanogenesis pathway halting
methane production to almost zero at 80° C. Another explanation is that the thermophillic
archaea were inhibited or killed by raising temperature above 62 ° C.
Butyrate concentration decreased with increasing acetate concentration and increasing
temperature (Martina et al, 2005). Some Acetate was converted into butyrate by the
following equation:
2CH3COO− + H+ + 2H2 → CH3(CH2)2COO− + 2H2O ΔG0 =−107.6kJ
This process is more feasible at lower temperature than higher ones (Thauer, 1977). As
the temperature decreases the amount of free energy available increases (ΔG0) hence
making this reaction more feasible at lower temperatures (Venturoli, 1998).
Since the bioreactor is a community of bacteria, more temperature tolerant bacterial
strains/colonies are likely to survive in the bioreactor as the temperature rises. However,
this does not necessarily mean that temperature tolerant bacteria are energy efficient and
would boost up the methane production. Research conducted by Zinder et al., 1981, on
thermophilic bacteria support this point.
According to Charles’s gas law, the temperature of a system is directly proportional to
the volume of gas produced at a constant pressure (Levine, 1978). In accordance with this
law as the temperature rises so does the volume of the gas and since this is an air tight,
closed system it can be assumed that this the pressure is relatively constant. For this
reason after the new temperature was reached methane production was not recorded for
the first hour.
The temperature range maintained in the lab over the course of the chemostat project was
as a minimum of 06° and Maximum of 30°C. Since there was no means to control the
temperature of the tubing and subsequent volume changes in the methane produced, the
readings were taken at a similar time each day with the temperatures noted. This ensures
the temperature changes are approximately constant for each reading.
5.4 Volatile Fatty Acids
In a general methanogenic digester there are two main bacterial classes, those which
produce the fatty acids from feed and the methanogens which metabolise these fatty acids
into gases. These two microbes although independent form a symbiotic relationship, if
the methanogens did not metabolise the fatty acids then conditions would become too
acidic for either species to survive. (Meynell et al 1982)
Volatile fatty acids are classed as those fatty acids which have six carbon atoms or less,
in this experiment three of these were chosen; acetate, propionate and butyrate. As shown
below
O
O
O
H3C
H3C
O
acetate
H
OH
propionate
H3C
OH
butyrate
Figure 5: Showing general structures of the three VFA’s analysed. (Note as drawn
showing the H atom attached this will freely dissociate leaving a negative charge
delocalising between the two O atoms)
It has been previously noted that changes in VFA concentrations are the most important
indicator of a digester imbalance. (Holm-Neielsen et al 2007) In agreement with this
statement our bioreactor showed increased levels of VFAs as the temperature was
changed from 55ºC to 62º C and then was further heated to 70º C, this increase was
approximately linear. Our results support the theory that stress (either temperature or
organic content) is the ultimate cause of increasing acidity due to fatty acid build up.
(Meynell et al 1982)
Meynell et al also investigated how, by the addition of glycerol (3-5g/L) the bioreactor
conditions became more stable and VFA overloading did not occur. They conclude that
due to the availability of glycerol (due to the developing biodiesel industry) one possible
use for this compound is to mix it with other biomass feedstock and use it in anaerobic
digesters (Holm-Nielsen et al 2007) Findings also support a correlation between the
amount of glycerol and VFA concentrations, these were easily measured using an “OnLine” technique such as NIR spectra. The ease of analysis in beneficial to such operations
as bioreactors where quick diagnosis of problems is crucial to avoiding costly downtime.
6. Recommendations







Most importantly seal all leaks. Check all tubing and bottles under pressure.
Under water is a good way of checking for leaks
Place substrate reservoir in ice filled container to avoid fermentation in non-sterile
conditions
Only add enough substrate into the feed reservoir as needed on a daily basis to
prevent substrate overloading due to pump failure
GC analysis can be used to confirm the presence of methane production
Have a temperature sensor hooked up to the computer as well as the u tube. This
way changes in the temperature over the day can be observed and correlations
with temperature change and gas production can be inferred.
Due to the risk of explosion an emergency vent valve should be added to allow
excess gas to escape the reactor without destroying the reactor. This would
comprise a rubber bung in one of the reactor outlets that would pop out when
pressure became too high.
The effect of temperature should be measured over a longer period of time (3 days)
at each temperature with a VFA analysis conducted each day. This would give a
more accurate idea of what was happening and it would be interesting to see if
this result of optimal methane production at 62°C could be repeated.
.
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