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Proceedings 12th World Congress on Anaerobic Digestion (AD12),
Guadalajara, Mexico
October 31 – November 4, 2010

Biomethanation of synthesis gas using anaerobic granules within a closed loop gas
lift reactor

Serge R. Guiot*, Ruxandra Cimpoia and Gaël Carayon

National Research Council Canada, 6100 Royalmount, Montreal, Canada, H4P 2R2
* Corresponding author: serge.guiot@cnrc-nrc.gc.ca

Abstract Gasification of biomass produces a mixture of gas (mainly carbon monoxide, CO2 and
hydrogen) called synthesis gas, or singes, by thermal degradation without combustion. Syngas can
be used for heat or electricity production by thermochemical processes. This project aims at
developing an alternative way to bio-upgrade syngas into biogas (mainly methane), via anaerobic
fermentation. A few strains of archaea are able of carboxydotrophic methanogenesis, some
potentially present in industrial wastewater-treating anaerobic granules. An industrial granular
sludge was first characterized for its carboxydotrophic methanogenesis potential using batch tests
under various conditions. Then those granules were inoculated into a 30 liters gas-lift reactor and
supplied with a gas mixture containing carbon monoxide, to study the production of methane and
others metabolites, at different gas feeding and recirculation rates. Carbon monoxide being a
poorly soluble gas, a challenge was to reach an adequate gas-to-liquid mass transfer, without
exceeding the toxicity level.

Keywords Anaerobic; syngas; carbon monoxide; carboxydotrophic; methane; gas lift; granules

INTRODUCTION

Biomass-based energy demand is increasing. Carbon-based residues (secondary biomass)

represent one of the most promising and virtually untapped domestic renewable energy
sources. For instance, more than 150 millions metric tons of municipal solid waste
(MSW), bio-solids, agricultural, forestry and other waste products are generated in
Canada each year. Furthermore, growing perennial giant grasses (switchgrass,
miscanthus, …) on fallow or unused farmlands would add several other hundred millions
tons of non-food biomass (Wood and Layzell 2003).

Several bioconversion routes exist to turn biomass into gas or liquid fuels. A significant
portion of biomass is however difficultly and/or slowly biodegradable by
microorganisms, due to its heterogeneous and polymeric nature. When the organic
residue is relatively dry (e.g. woodchips, bug wood …) or non-biodegradable (bark,
plastics, rubber ….), it might be more appropriate to use thermo-chemical conversion
techniques such as gasification. Gasification transforms biomass at high temperature
(500-1500°C) and pressure (1-80 bars) with limited amounts of water and oxygen into a
synthetic gas (syngas) mainly composed of carbon monoxide (CO), CO2 and hydrogen.
Minor syngas components include water vapor, methane, light hydrocarbons (C2H4,
C2H6), and some volatile impurities. Syngas can be used directly to power industrial
boilers, gas turbines or fuel cells to make electricity. Syngas can also be upgraded into
methane with chemical catalysts. This includes the water-gas shift reaction for increasing
the H2/CO and nickel-catalyzed methanation of CO and CO2 into methane and water
(Ridler and Twigg 1996). After separation, methane could be used locally for energy

Guiot et al., Carboxydotrophic methanogenesis AD12 - 1/8

needs, or re-injected in the natural gas grid. In Canada and particularly Quebec, this
likely would return higher revenue as compared to electricity. Catalyzed chemical
processes are well established. They normally involve high pressure and/or temperature,
may be problematic when impurities are present and tend to have low product specificity.

To circumvent these disadvantages and use milder treatments with minimal chemical and
energy, we can harness the power of microorganisms to convert the syngas compounds
into biogas (biomethanation). A small number of microbes can reduce the CO from
syngas into methane (Methanobacterium thermoautotrophicum, Methanothermobacter
wolfeii, Methanobrevibacter arboriphilicus, Methanocaldococcus jannaschii,
Methanopyrus kandleri, Methanosaeta thermophila, Methanosarcina acetivorans, M.
barkeri, …) (Daniels et al 1977, Hammel et al 1984, Mazumder et al 1985, Klasson et al
1990, Wasserfallen et al 2000, Rother and Metcalf 2004, Sokolova et al 2009). In short,
the syngas can be a substrate for those carboxydotrophic methanogenic archaea that grow
chemolithoautotrophically on CO and H2 (Henstra et al 2007). Two direct reactions are
possible: (I) 4 CO + 2 H2O → CH4 + 3 CO2 (∆G°' = –53 kJ/mol CO, but growth might
be slight); (II) CO + 3 H2 → CH4 + H2O (∆G°' = –150 kJ/mol CO, faster growth). In
theory, as long as H2 is not limiting, the second reaction, thermodynamically more
favorable, should prevail, and the first one should take over, once H 2 becomes deficient.
Indirect reactions are also possible, with a mixed anaerobic consortium. For instance:
carboxydotrophic hydrogenogenesis (enzymatic water-gas shift reaction (Svetlichnyi et al
1991), CO + H2O → H2 + CO2, ∆G°' = –20 kJ/mol CO) followed by either the
methanogenic reduction of CO2, or carboxydotrophic methanogenesis (in either case, net
result: 4 CO + 2 H2O → CH4 + 3 CO2); either CO-homoacetogenesis (2 CO + 2 H2 →
CH3COOH) or acetogenesis (4 CO + 2 H2O → CH3COOH + 2 CO2, ∆G°' = –44 kJ/mol
CO) followed by acetoclastic methanogenesis; or carboxydotrophic production of
methanol (CO + 2 H2 → CH3OH, ∆G°' = –39 kJ/mol CO) followed by methylotrophic
methanogenesis (CH3OH → ¾ CH4 + ¼ CO2 + ½ H2O); or oxidation of CO into formic
acid (CO + H2O → HCOOH), followed by its reduction into CH4. Furthermore,
Clostridia produce, beside acetate, significant amounts of ethanol, butyrate and butanol
that can then be converted into acetate, then methane. Whatever the set of reactions, the
final CH4 yield is identical, i.e. 0.25 mol CH4 per mol CO, plus 0.25 mol CH4 per mol H2.
This would result within a treatment chain made of gasification followed by
biomethanation of syngas, into a practical CH4 yield from biomass varying between 0.2
and 0.4 m3 STP CH4/kg VS gasified, depending on the syngas composition (i.e. CO
between 30 and 60 % (vol./vol.), H2 between 60 and 25%, and CO2 between 35 and 3%).

Industrial wastewater-treating anaerobic granules could be used for achieving those

transformations, providing they have a carboxydotrophic methanogenic potential that
could be significantly enriched. This would facilitate further development, namely to
focus on the reactor design and operation optimization regardless to the microbial
constituent, and would later facilitate scale-up, as allowing for a source of microbes that
are massively available at free or low cost, and already adapted to harsh conditions that
would prevail with crude syngas. Otherwise, because the aqueous solubility of CO and
H2 is low, syngas fermentations are typically limited by the gas-to-liquid mass transfer
rate (Bredwell et al 1999). This will represent the major engineering challenge for
development of large-scale syngas fermentation facilities cost-effectively compatible
with upstream gasifier productivity. The reactor design development will have to aim at
both high density of microorganisms and minimization of gas/liquid mass transfer
limitations, which may sound antagonistic. Hence the mass transfer driving force has to

Guiot et al., Carboxydotrophic methanogenesis AD12 - 2/8

be intensified, e.g. by pressurizing the reactor gas phase, by increasing the interfacial area
by gas dispersion (e.g. using micro-spargers or bubble-free sparging), and high-rate gas
recirculation.

The objective of this work will be to assess whether carboxydotrophic methanogenic

potential is existing and can be enriched within industrial anaerobic granules, using a
closed-loop gas-lift reactor continuously fed with a gas stream of N 2-diluted CO, under a
varying operational conditions, and their sensitivity to high levels of CO in the gas phase.

MATERIAL AND METHODS

Reactor set-up
A closed-loop gas lift reactor (internal diameter 0.2 m; height 1.13 m; working liquid
volume 30 L; headspace 4 L) with an internal draft-tube of 0.6 m height and 0.1 m
diameter was used in this study. The reactor temperature was controlled by a probe (DP-
41, Omega Engineering Inc, Stamford, CT) placed just below the liquid upper level and
maintained at 35ºC by circulating water from a thermostatic bath (RK12418-30, Cole
Parmer, Niles, IL) through the reactor water jacket. The pH was measured (electrode 405-
DPAS-SC-K8S, Mettler Toledo GmbH, Urdorf, CH) and controlled between 6.9 and 7.8
using 0.1M NaOH or 0.1M HCl with a pH controller (PHP-194, Omega Engineering Inc,
Stamford, CT). The feeding gas whose CO (Praxair Canada Inc, Mississauga, ON, CA)
content was adjusted using nitrogen gas (Praxair Canada Inc) was bubbled at the center of
the reactor bottom. The gas flow rates were measured and controlled by thermal mass
flow meters (M100B, M10MB, 247D, MKS, Wilmington, MA). The gas exited the
reactor through a pressure valve (TM Swagelok, Solon, OH) to maintain the reactor
headspace at the desired pressure (manometer 0-60PSI, US Gauge, Ira Township, MI).
The gas phase was recycled with a compressor (UN036STI, Neuberger Inc, Trenton, NJ)
at a flow rate varying between 0.6 and 1.15 L/min. In the gas recycle, an empty column
was used as a condenser and foam trap. A secondary gas loop was equipped with a valve
set at about 4.3 atm in case of failure of the primary outlet and pressure buildup.

The reactor was inoculated with anaerobic granules from a full-scale UASB plant treating
fruit processing wastewater (Lassonde Inc., Rougemont, QC, Canada). The dilution water
(buffer, containing (mg/L): Na2HCO3, 2700; NaH2PO4, 1100; Ca(NO3)24H2O, 1442;
K2HPO4, 4000) was supplied (PHP-194, Omega Engineering Inc, Stamford, CT) at the
base of the reactor at an average flow rate of 0.2 L/d. The nutrient and trace metal
solutions were pumped (PHP-194, Omega Engineering Inc, Stamford, CT USA) into the
dilution water stream according to a volume ratio of 45/47/100 respectively. The nutrient
solution contained (mg/L): KH2PO4, 4040; K2HPO4, 5160; NH4HCO3, 19521. The trace
metal solution contained (mg/L): FeSO47H2O, 856; H3 BO3, 5; ZnSO47H2O, 13;
MnSO4H2O, 60; Co(NO3)26H2O, 33; CuSO4, 45; NiSO46H2O, 9; (NH4)6Mo7O244H2O,
273; AlK(SO4)212H2O, 2; Na2-EDTA, 33; MgSO47H2O 1630; Na2SeO4, 6;
Na2WO42H2O, 6.

Specific activity tests

Acetoclastic, hydrogenotrophic and carboxydotrophic specific activity tests were
performed in triplicates on the granular anaerobic inoculum as well as on granules
sampled intermittently during the reactor operation. Anaerobic activities for acetate were
determined in 120 mL serum bottles by measuring the rate of methane production and/or

Guiot et al., Carboxydotrophic methanogenesis AD12 - 3/8

substrate depletion, individually and under non-limiting conditions, as described
previously (Guiot et al 1995). Briefly once filled with the microbial biomass and buffer
solution, bottles were capped, sealed and flushed with N2/CO2 gas to establish anaerobic
conditions, and placed in a rotary shaker (New Brunswick, Edison, NJ) in a dark,
thermostatically controlled environment (35±1ºC) and gyrated at 100 rpm. The acetate
solution was injected into all bottles to obtain an initial concentration of 3 g/L. The
hydrogenotrophic activity tests were performed similarly, using as substrate instead of
acetate, a head space filled with CO2/H2 (80/20 % vol./vol.) pressurized at 2.5 atm and by
shaking at 400 rpm to ensure the gas transfer between the headspace and liquid. The
carboxydotrophic activity was assessed similarly to the hydrogenotrophic one, using as
substrate 0.20 atm CO, under conditions such as: mesophilic (35ºC) and thermophilic
(60ºC) conditions, shaking at 100 and 400 rpm, in absence of hydrogen (balance made of
N2, 0.8 atm) and in presence of hydrogen (0.64 atm, balance made of 0.16 atm CO2).

Analytical methods
Chemical oxygen demand (COD), total and suspended solids (TS and SS) and total and
suspended volatile solid (VS and VSS) according to the standard methods (APHA et al
2005). Measurement of acetic, propionic and butyric acids was made on an Agilent 6890
gas chromatograph (Wilmington, DE) equipped with a flame ionization detector (FID) on
0.2 µl samples fortified 1:1 (vol./vol.) using an internal standard of iso-butyric acid in 6%
formic acid, directly injected on a glass column of 1 m × 2 mm Carbopack C (60-80
mesh) coated with 0.3 % Carbowax 20M and 0.1% H 3PO4. The column was held at 130
°C for 4 minutes and helium was the carrier gas at a rate of 20 mL/min. The injector and
the detector were both maintained at 200°C. For alcohols, 100 µl of liquid were
transferred in 20 ml headspace vial crimped with a Teflon coated septum. The vial was
heated at 800C for 2 min, then 1000 µl of headspace gas were injected on a DB-ACL2
J&W capillary column of 30 m x 530 mm x 2 µm (Agilent). The column was held at
400C for 10 min. Helium was the carrier gas at a head pressure of 5 psi. The injector and
the detector were maintained at 2000C and 2500C, respectively. The gas composition (O2,
H2, CH4, N2, CO, CO2) was measured by injecting 300 µL of gas (model 1750 gas-tight
syringe, Hamilton, Reno, NV) into a HP 6890 gas chromatograph (Hewlett Packard, Palo
Alto, CA) equipped with a TCD and a 5 m x 2,1 mm Carboxen-1000 column (Supelco,
Bellafonte, PA) with argon as carrier gas. The column temperature was held at 60ºC for
7 min and increased to 225ºC at a rate of 60ºC per min.

RESULTS AND DISCUSSION

Methanogenic potential of inoculum

The Table 1 is showing a characterization of the inoculum in terms of specific activities
under various conditions. Acetoclastic and hydrogenotrophic methanogenic specific
activities of the inoculum, at around 300 mg substrate (either acetate or H2) consumed per
g VSS and day, were in a range such as expected. Although unadapted, the anaerobic
sludge presented also a promising carboxydotrophic potential. In mesophilic conditions,
the specific activity was around 4 mmol CO/g VSS·d in absence of H2 and 5 to 8, in
presence of H2. No lag time was observed but for the activity to be fully expressed, CO
partial pressure had to decrease down to 0.15 atm. Mass transfer did not limit
methanogenesis from CO alone while hydrogenotrophic methanogenesis increased at
higher agitation. The measured CH4 yields were in all cases close to 100 % of what
stoichiometry predicted. Volatile fatty acids (VFA) appeared to be the main co-

Guiot et al., Carboxydotrophic methanogenesis AD12 - 4/8

metabolites, although minor (between 0.24 and 5.1% of the CO input). The VFA
production was associated with the presence of H2 and accordingly increased with the
mass transfer.

Table 1. Acetoclastic, hydrogenotrophic and carboxydotrophic specific activities of the

inoculum
Substrate Conditions Specific activity CH4 yield
mmol substrate mmol CH4 % stoichiometric
/g VSS·d) /g VSS·d yield (4)
Acetate 35°C, 100rpm 6±0.3 7±0.6 102
(1)
H2/CO2 35°C, 400rpm 105±21 27±6 102
35°C, 100rpm 4±1 1.2±0.4 97±8
(1) 35°C, 400rpm 4±1.5 1±0.2 100±4
CO/N2
60°C, 100rpm 4 0.1 11±5
60°C, 400rpm 21±3 6 104
35°C, 100rpm 5±0.7 5±0.8 94±4
CO/H2/ 35°C, 400rpm 8±1.4 11±1.3 130±12
(3)
CO2 60°C, 100rpm 11 23 20±1
60°C, 400rpm 19±0.2 32±0.9 99±4
(1) 20%/80% vol./vol.; total pressure 2.5 atm.
(2) 20%/80% vol./vol.; total pressure 1 atm.
(3) 20%/64%/16% vol./vol.; total pressure 1 atm.
(4) 1 mol CH4 per mol acetate; ¼ mol CH4 per mol H2; ¼ mol CH4 per mol CO

In thermophilic conditions the metabolic pathway and respectively the methanogenic

yield depended on gas-to-liquid mass transfer. At 100 rpm and in absence of hydrogen
only 0.03 mol of methane was obtained for each mol CO consumed and high quantities of
acetate were measured. At higher mass transfer (shaking at 400 rpm), specific activity
quadrupled in absence of hydrogen added and final CH4 yields were maximal but the
process was biphasic: hydrogen is produced first from CO by water-gas shift reaction
(hydrogenogenic carboxydotrophy) and hydrogenotrophic methanogenesis started only
after CO was depleted. This means that at low transfer rate (i.e. low soluble CO
available) CO-homoacetogenesis was favored over hydrogenogenic carboxydotrophy,
while at higher soluble CO concentration (resulting from higher mass transfer)
hydrogenogenesis became more competitive. This means also that thermophilic
methanogens were more sensitive to CO than hydrogenogenic carboxydotrophs.

Reactor performance
The carboxydotrophic methanogenic potential of anaerobic granules were then tested in
the 30 L closed-loop gas-lift reactor continuously fed with a gas stream of N2-diluted CO.
Different experimental phases were carried out under various operational conditions as
shown in Table 2. Three days after the inoculation the CO loading was increased, then
kept relatively constant, at 0.35-0.41 LCO STP or 15-18 mmol/g VSS·d. Without gas
recirculation the insufficient gas holdup limited the mass transfer at 4%. At day 10, a low
gas recirculation was set on which increased the gaseous CO transfer efficiency to 51%.
At day 30, the gas recirculation-to-feeding ratio was augmented from 9:1 to 18:1. This
increased the gaseous CO transfer efficiency to 70%. Then at day 56 while the gas
recirculation ratio was returned back to 10:1, the CO partial pressure in the feeding
stream was augmented from 0.4 to 0.6 atm. This allowed for a relative conservation of
the CO transfer efficiency at 67%, while a combination of higher partial pressure and gas

Guiot et al., Carboxydotrophic methanogenesis AD12 - 5/8

recirculation ratio, i.e. 0.6 atm and 20:1 respectively, allowed for a re-increase of the gas
transfer to 75%. Those performances corresponded to methane yields of 24 to 29%
(vol./vol.), i.e. a CO conversion efficiency close to 100%. The excess of methane
production noted during the early days (first phase) could easily be explained by the
degradation of the organic matter brought in the reactor with the inoculum. The above
reactor performance, expressed in terms of methane specific carboxydotrophic
productivity, showed an increase over phases II to V from 1.7±0.9 to 3±0.3 mmol
CH4/gVSS·d. In parallel, specific activity tests performed in batch bottles on granules
sampled intermittently during the reactor operation provided values tending to slightly
increase from 1.2±0.4 mmol CH4/g VSS·d at inoculation to 1.3±0.2 at phase II and to
1.7±0.4 at phase V. There was thus a disparity between the in-reactor and batch
activities. Higher specific values found in the reactor as compared to the batch tests for
the same granules, likely indicated that the reactor conditions were less limiting as
compared to the conditions in the test bottles, notably that those ratios of gas recirculation
in the reactor (10:1 to 20:1) allowed for a better mass transfer than agitation at 100 rpm in
the bottles.

Metabolites other than methane were observed, although in traces The concentrations
accumulated in the liquid are given in Table 2. Methanol was the main co-metabolite.
This was unexpected because its formation from CO depends on H 2 and is less exergonic
than for other soluble metabolites such as acetate or ethanol (Sipma et al 2006). However
as significant concentrations of methanol only occurred until day 30, it is presumed that it
is formed from residual intragranular organics introduced together with the inoculum,
namely pectins, since granules were originating from a fruit juice wastewater plant.
Actually Clostridia have been reported to produce methanol as a major end-product
during growth on pectin (Schink and Zeikus 1980).

Table 2. Performance of reactor, as a function of the operational time

Phase I II III IV V
Period (days) 3-9 10-29 30-52 56-72 73-77
CO loading rate (mmol/g VSS·d) 17.2±0.4 16.8±0.6 12.9±5.9 17.8±1.9 17.4±1.6
Gas recirculation ratio 0 9:1 18:1 10:1 20:1
pCO in gas feeding (atm) 0.42 0.43 0.42 0.61 0.62
CO in reactor headspace (%) nd 19±5.6 11±2.2 13±0.6 8±0.6
CO transferred (%) 4±0.4 51±1.1 70±0.9 67±1.1 75±0.8
CO consumed (mmol/g VSS·d) 0.7±0.1 6.6±3.6 10±1.7 12.3±1.3 12.5±0.6
Yield CH4/CO (% mol) 74.7±9 28.6±1 24.6±3 23.8±1 23.8±1
Yield H2/CO (% mol) 0±0 0.2±0.1 0.8±1 0.4±0.1 0.1±0
Acetate (µmol/L) 64±64 746±645 125±250 366±126 147
Propionate (µmol/L) 8±15 51±42 21±19 27±0 24
Methanol (µmol/L) 1656±1042 1198±911 200±148 74±31 0.5
Ethanol (µmol/L) 33±41 27±25 4±2 0±0 0

Sensitivity to CO
The gas-to-liquid mass transfer might not be the only limitation of the process, namely
dissolved CO concentration might also not be optimal. To investigate that hypothesis, an
activity test-based kinetic study was performed, using the unadapted anaerobic sludge
(inoculum) diluted within anaerobic phosphate buffer under different initial partial
pressure of CO (pCO) varying from 0.09 to 0.9 atm corresponding to dissolved CO
concentrations from 0.08 to 0.74 mM. Carboxydotrophic specific activity results are

Guiot et al., Carboxydotrophic methanogenesis AD12 - 6/8

presented as a function of test bottle CO concentration in Figure 1. The non-acclimated
sludge presented a maximum activity of 8.15 mmol CO/g VSS·d at a CO partial pressure
of 0.19 atm (0.25 atm initial pCO). Above that maximal point the specific activity dropped
rapidly to reach 2.26 mmol CO/g VSS·d at 0.9 atm of CO. The pCO for maximal
carboxydotrophic activity corresponded to a dissolved CO concentration of 0.17 mmol/L.
This also may explain the disparity between the specific activity measured in batch and in
reactor on the same granules, as in the reactor the continuous gas recirculation insured a
dilution of the gas at a more appropriate and homogenous concentration.

7
Specific activity (mmol/gVSd)

0
0.0 0.2 0.4 0.6 0.8
CO concentration (mM)

Figure 1. Carboxydotrophic () and methanogenic () specific activity as a function of

dissolved CO

CONCLUSIONS
Industrial anaerobic granule sludge clearly has a significant carboxydotrophic
methanogenesis potential, and does not require particular acclimation. Prospects may
include enrichment and thermophilic conditions, to increase specific bioactivity, as well
as adaptation to alleviate the methanogenic inhibitory impact of CO above 0.2 mM.

As expected since the aqueous solubility of CO is low, reactor performance increased

with pressure and gas recirculation (bigger gas holdup). Further improvement of the gas-
to-liquid mass transfer rate will represent a major engineering challenge for development
of large-scale syngas fermentation facilities cost-effectively compatible with upstream
gasifier productivity. However if we hypothesize that a reactor system could be
developed without mass transfer limitation, productivity higher than 30 m3 STP CH4/m3
reactor·d could be expected in a UASB-like reactor retrofitted for syngas treatment.

Municipal solid wastes (MSW) in Canada are typically composed of compostable or

digestible organic materials (food, yard, garden) for 17% at around 30% dryness, of other
organics (paper, board, wood, plastics, non-compostable organics) for 54 % at around
45% dryness on average, and of inert wastes (metals, glass and other inerts) for 29%.
Anaerobic digestion of the first fraction, at a degradation efficiency of 60% and a yield of
0.5 m3 STP CH4/kg solid degraded, could generate 15 m3 STP CH4 per metric ton of
MSW. If this is complemented by gasification of the second fraction of other organics

Guiot et al., Carboxydotrophic methanogenesis AD12 - 7/8

 combined with biomethanation of the syngas produced, at a conservative integrated yield
of 0.3 m3 STP CH4/kg solid gasified, this would add up 73 m3 STP CH4, resulting hence
in a total of about 88 m3 STP CH4 per metric ton of MSW i.e. almost six times the
potential by anaerobic digestion alone. However the energy-intensiveness of gasification
may mitigate the net energy output.

ACKNOWLEDGMENTS
The authors wish to thank A. Corriveau, C. Beaulieu and S. Deschamps for analytical
assistance. The study was partially funded by the AAFC-NRCan-NRC National
Bioproducts Program.

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Guiot et al., Carboxydotrophic methanogenesis AD12 - 8/8

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