Environmental Microbiology Reports (2009) 1(5), 377–384
Enrichment of denitrifying anaerobic methane
oxidizing microorganisms
Shihu Hu, Raymond J. Zeng, Luke C. Burow,†
Paul Lant, Jurg Keller and Zhiguo Yuan*
Advanced Water Management Centre (AWMC), The
University of Queensland, Brisbane, Qld 4072, Australia.
Summary
The microorganisms responsible for anaerobic oxida-
tion of methane (AOM) coupled to denitrification have
not been clearly elucidated. Three recent publications
suggested it can be achieved by a denitrifying bacte-
rium with or without the involvement of anaerobic
methanotrophic archaea. A key factor limiting the
progress in this research field is the shortage of
enrichment cultures performing denitrifying anaero-
bic methane oxidation (DAMO). In this study, DAMO
cultures were enriched from mixed inoculum including
sediment from a freshwater lake, anaerobic digester
sludge and return activated sludge from a sewage
treatment plant. Two reactors, operated at 35°C and at
22°C, respectively, showed simultaneous methane
oxidation and nitrate reduction after several months of
operation. Analysis of 16S rRNA gene clone libraries
from the 35°C enrichment showed the presence of an
archaeon closely related to other DAMO archaea and a
dominated bacterium belonging to the yet unculti-
vated NC10 phylum. This culture preferred nitrite to
nitrate as the electron acceptor. The present study
suggests that the archaea are rather methanotrophs
than methanogens. The highest denitrification rate
achieved was 2.35 mmol NO3--N gVSS-1 day-1. The
culture enriched at 22°C contained the same NC10
bacterium observed in the culture enriched at 35°C but
no archaea.
Introduction
Microbially mediated anaerobic oxidation of methane
(AOM) is very important to the Earth’s climate because it
consumes methane produced from natural sediments
before it escapes to the atmosphere (DeLong, 2000). This
Received 7 May, 2009; accepted 21 August, 2009. *For correspon-
dence. E-mail zhiguo@awmc.uq.edu.au; Tel. (+61) 7 33654374; Fax
(+61) 7 33654726. †Present address: Department of Civil and Envi-
ronmental Engineering, Stanford University, Stanford, CA 94305,
USA.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
doi:10.1111/j.1758-2229.2009.00083.
emi4_83 377..384
process is estimated to oxidize up to 90% of methane
produced in anaerobic marine sediments (Reeburgh
et al., 1993). Anaerobic oxidation of methane has also
been found in freshwater environments, although its con-
tribution as a methane sink has not been quantified (Eller
et al., 2005). Despite extensive studies, no microorgan-
isms capable of the AOM have been isolated. Molecular
and biogeochemical studies have shown that for marine
environments the microorganisms responsible for AOM
consist of a consortium of methanogen-related archaea,
and sulfate-reducing bacteria (SRB) (Hinrichs et al., 1999;
Boetius et al., 2000; Niemann et al., 2006). The nature of
the cooperation between the archaea and bacteria has so
far not been elucidated. Environmental genomic studies
of anaerobic methanotrophic archea (ANME) support the
hypothesis that ANME can oxidize methane via a reverse
methanogenesis process to a substrate used by SRB
(Kruger et al., 2003; Hallam et al., 2004). The intermedi-
ates transferred between species are unknown, with
methyl sulfides proposed as the candidate by Moran and
colleagues (2008) after Nauhaus and colleagues (2002)
concluded that hydrogen, acetate, formate, methanol,
carbon monoxide and methylamines were unlikely
intermediates.
In contrast to the numerous studies about AOM coupled
to sulfate reduction mentioned above, there were only a
few publications focusing on the denitrifying anaerobic
methane oxidation (DAMO) process. Thermodynamically,
oxidized nitrogenous compounds are more favourable
electron acceptors than sulfate, where the energy
yield of AOM coupling to sulfate reduction is only -20
to -40 kJ mol-1 CH4 (Strous and Jetten, 2004). The
reactions may be described as:
5CH4 + 8NO3 − + 8H+ → 4N2 + 14H2 O + 5CO2
(1)
ΔG0′ = −765 kJmol−1 CH4
3CH4 + 8NO2 − + 8H+ → 4N2 + 10H2 O + 3CO2
(2)
ΔG0′ = −928 kJmol−1 CH4
Although methane and nitrate or nitrite often coexist in
the environment, such as anoxic sea water or freshwater
sediments receiving nitrate from agricultural run-off, the
DAMO process has not been found in nature. One pos-
sible explanation is that the process may occur within
millimetres of the oxic-anoxic interface, masking it from
geochemical detection (Raghoebarsing et al., 2006).
378 S. Hu et al.
Process engineers consider methane a potentially
inexpensive external electron donor for denitrification for
the treatment of wastewater or landfill leachate where
methane is generated on site (Islas-Lima et al., 2004;
Modin et al., 2007). However, several attempts to enrich
the DAMO microorganisms in the laboratory failed
(Mason, 1977; Thalasso et al., 1997; Eisentraeger et al.,
2001). The slow growth of these microorganisms is a
likely explanation for persistent failures to obtain DAMO
cultures.
Using freshwater canal sediment as inoculum, Raghoe-
barsing and colleagues (2006) obtained a DAMO culture
after 16 months of enrichment, with nitrate and nitrite as
electron acceptors. The culture contained a bacterium
belonging to the candidate division NC10 and an
archaeon distantly related to ANME-II. It was hypoth-
esized that DAMO was performed via reverse methano-
genesis by archaea with electron shuttling to the
denitrifying bacterium (Raghoebarsing et al., 2006).
However, the archaeal population slowly disappeared
after the reactor was scaled up and optimized to improve
the activity (Ettwig et al., 2008). Ettwig and colleagues
(2009) reported another successful enrichment of NC10
bacteria dominated culture with sediments from ditches
draining agricultural land. These studies showed that
the denitrifying anaerobic methanotrophic bacteria can
achieve DAMO with or without archaea. Ettwig and col-
leagues (2008) conjectured that the archaea in the initial
enrichment (Raghoebarsing et al., 2006) were anaerobi-
cally oxidizing methane with or without a bacterial partner,
or they could actually be methanogens performing
methanogenesis. Detailed physiological and biochemical
studies of the DAMO microorganisms are still limited by
the shortage of DAMO cultures.
The primary aim of this study was to enrich DAMO
cultures from inocula different from previous studies and
at different temperatures. To this end, inocula were pre-
pared with samples from different anoxic niches where
DAMO process may occur. After DAMO activity became
apparent, clone libraries were constructed to determine
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
the phylogenetic affiliation of the dominating archaeal and
bacterial groups and to compare them with other DAMO
organisms and ANMEs. The characteristics of the cultures
obtained are compared with previous studies and the
roles of archaea and bacteria in the DAMO cultures are
discussed.
Results and discussions
Enrichment of DAMO cultures at different temperatures
Three reactors were set up to enrich microorganisms
capable of DAMO. The inocula were a mixture of sedi-
ments from a local freshwater lake, anaerobic digester
sludge and return sludge from the Luggage Point waste-
water treatment plant (Brisbane, Australia).
Denitrification rates in all reactors decreased to lower
than 0.3 mmol NO3--N l-1 day-1 within 40 days of start-up,
suggesting organic residues contained in the inoculum
were consumed in this period (Fig. 1). The enrichment
running at 45°C was stopped on day 90, as no denitrifi-
cation activity was detected after day 40. Culture B (35°C)
showed a sharp increase in the denitrification rate after
day 220 reaching 2.0 mmol l-1 day-1 by day 312. Although
the denitrification rate of Culture A (22°C) was only about
0.11 mmol NO3--N l-1 day-1 between day 50 and 260, the
data showed that methane was consumed in this reactor
(Fig. 2).
As shown in Table 1, the measured nitrogen gas pro-
duction rates were close to the predicted values, indicat-
ing the complete conversion of nitrate to nitrogen gas.
This was also supported by the fact that no nitrite or
nitrous oxide was detected in the reactors and that neg-
ligible ammonium decrease occurred (data not shown).
Culture B (35°C) consumed 16.7% more methane than
stoichiometrically predicted. The involvement of aerobic
methane oxidation was very unlikely since the reactor was
always operated over-pressure, and oxygen was not
detected in either the liquid or the headspace. Methane
utilising sulfate reduction was not involved since no
Fig. 1. Denitrification rates of the reactors
operated at different temperatures [22°C (䉭),
35°C ( ) and 45°C (䊉)]. Each reactor has
1.6 l liquid volume and 0.4 l headspace. The
headspace was flushed periodically with
mixed gas containing nitrogen, methane and
carbon dioxide to provide methane. Nitrate
was added periodically to ensure its
availability. The reactors were operated
over-pressure (1.1–1.3 a.t.m.), and helium
was injected into the headspace to confirm
that no gas leakage occurred (data not
shown).

sulfate decrease was observed during the entire period of
the experiments. Methane assimilation into biomass and
measurement inaccuracies could have contributed to the
error.
Control experiments with Culture B (35°C) in the
absence of nitrate or methane confirmed that AOM was
linked to denitrification. In the absence of nitrate between
days 255 and 258, the partial pressure of methane
remained at 0.76 a.t.m., indicating no methane oxidation
(or production) in this period. The endogenous denitrifica-
tion rate in the absence of methane was determined to be
0.14 mmol day-1, which was only 7% of the rate when
methane was available.
To investigate the denitrification ability of Culture B
(35°C) with different nitrogenous compounds, nitrite
was added to the reactor to a concentration of
0.7 mmol NO2--N l-1 in a batch test on day 310. During the
8 h experiment, nitrite was consumed much faster than
nitrate (1.73 versus 0.46 mmol N l-1 day-1) when both
were present.
A carbon balance was performed with the use of 13C-
labelled methane in Culture B (35°C) between days 250
and 255. Briefly, 120 ml of 13C-labelled methane was
injected to the reactor on day 250. Liquid and gas
samples were analysed with GC-MS to determine the 13C
content in gas and liquid. The total amount of 13C-labelled
methane consumed after 120 h (1.8 mmol) was very
close to the amount of 13C-labelled carbon dioxide
Table 1. Nitrate and methane consumption rates and nitrogen gas production rate of cultures A (22°C) and B (35°C), determined by averaging the
rates of batch studies shown in Fig. 2.
Measurement
NO3- N2
mmol day-1 mmol day-1
Culture A (days 220–260) -0.065 0.03
Prediction errorb
Culture B (days 283–312) -3.2 ⫾ 0.4 1.7 ⫾ 0.2
Prediction errorb
a. Based on reaction (1) and the measured rate of nitrate consumption.
b. (measured value - calculated value)/measured value.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
Enrichment of DAMO microorganisms 379
Fig. 2. Denitrification and anaerobic oxidation
of methane by Culture A (A) and Culture B
(B). The consumption of methane (䊊) and
nitrate ( ), and production of nitrogen gas
(䉱) were observed. The methane and
nitrogen gas data are the total amounts of
these substances in both the gas (measured)
and liquid (calculated using Henry’s law)
phases.
13
produced (1.7 mmol), strongly suggesting that the C-
13
labelled methane consumed was converted to C-
labelled carbon dioxide.
Microbial analysis of cultures A and B
Genomic DNA from the biomass in Culture B (35°C) was
extracted and bacterial and archaeal 16S rRNA gene
libraries were constructed. A total of 48 clone inserts from
the bacterial library were grouped into eight operational
taxonomic units (OTUs) by restriction fragment length
polymorphism (RFLP). Representatives from three domi-
nant OTUs (represented 25, 15 and 3 of 48 clones) from
the bacterial clone library were chosen for sequencing.
Other OTUs from the bacterial library were not sequenced
as they were represented by only one clone each.
Sequence analysis of the two most dominant OTUs
revealed two sequences (FJ907181 and FJ907182) clus-
tering inside ‘NC10’ division (Fig. 3). They are 99.9%
similar to each other and 98% similar to other NC10
bacteria (DQ369742) (Raghoebarsing et al., 2006). The
sequence (FJ907183) retrieved from the OTU represent-
ing three clones is about 85% similar to other NC10 bac-
teria of this study and of Raghoebarsing and colleagues
(2006) and Ettwig and colleagues (2009).
The archaeal clone library consisted of only one OTU
(48 out of 48 clones). Two representatives were
sequenced. The archaeal clone library showed two
Calculateda
CH4 N2 CH4
mmol day-1 mmol day-1 mmol day-1
-0.038 0.033 -0.04
-10% -5.3%
-2.4 ⫾ 0.2 1.6 -2.0
5.9% 16.7%
380 S. Hu et al.
coal tar contaminated groundwater
(AF351217, FJ810544, AF351214)

Bootstrap values
denitrifying AOM enrichment (FJ621546)
100% - > 90%
90% - > 80% coastal aquifer
80% - > 70% (DQ837241, DQ837259)
70% - > 60%

denitrifying AOM enrichment

(DQ369742, FJ621551, FJ621552, FJ621557, FJ621559, FJ621562)

denitrifying AOM enrichment

(FJ621545, FJ621553, FJ621554, FJ621555, FJ621556, FJ621558, FJ621560, FJ621561)

denitrifying AOM enrichment

(FJ907181, FJ907182)

TCE contaminated site (AF529103)

denitrifying AOM enrichment (FJ621533)

uranium mill tailings (AJ519669)

karst water (AM991185)

vadose soil (AY177763)

TCE−contaminated site (AF529101)

saturated horizon C soil

(EU335141, EU335143, EU335206, EU335212)

seafloor lava (EU491462)

cave (AF317743)

seafloor lava
(EU491346, EU491416, EU491450) A
marine sediment clone MD2902−B8, EU048615

hydrothermal sediment clone AT−s30, AY225657

Subsurface soil
(DQ906846, DQ906849, DQ906858, DQ906859, DQ906860, DQ906861)

denitrifying AOM enrichment

(FJ621531, FJ621532, FJ621535, FJ621538, FJ621539, FJ621540, FJ621547, FJ621548, FJ621549)

denitrifying AOM enrichment

(FJ621537, FJ621541, FJ621542)

anaerobic benzene degrading enrichment (AB426238)

denitrifying AOM enrichment (FJ621543)

uranium mill tailings

(AJ519650, AJ519670)

saturated horizon C soil (EU335192)

denitrifying AOM enrichment (FJ621534)

soil around iron−manganese nodule (EF492972)

anaerobic benzene degrading enrichment (AB426228)

denitrifying AOM enrichment

(FJ621536, FJ621544)

to prairie soil
outgroup (EU134303, EU134304, EU134857)

denitrifying AOM enrichment (FJ907183)

prairie soil clone (EU132429)

prairie soil clone (EU132789)

hypersaline microbial mat

(EU245945, EU245635)
subseafloor sediment
(EU385754, EU385793)

mangrove soil (DQ811954)

mud volcano (FJ712458)
B
hypersaline microbial mat (EU245176)

hypersaline microbial mat (EU245868)

sea grass bed sediment (EU488010)

hypersaline microbial mat (EU245526)

uncultured Caldithrix spp.

(FJ902136, FJ902298, FJ902365, FJ902382, FJ902492)

Caldithrix sp.
(AJ430587, FJ999729)

0.10

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
 Enrichment of DAMO microorganisms 381
Fig. 3. Maximum likelihood (AxML) phylogenetic tree of 16S rRNA gene sequences of bacteria implicated in anaerobic oxidation of methane
(AOM) coupled to denitrification. Group A 16S rRNA clone sequences belong to the NC10 phylum. Group B clones include a sequence
obtained in this study and nearest neighbours identified using the SILVA rRNA database SINA webaligner. Clones obtained in this study are in
bold face. Sequences shorter than 1250 nucleotides were inserted into the tree using the ARB parsimony insertion tool. Bootstrap values were
obtained after 1000 resamplings. Nitrospira marina (X82559) was used as an outgroup to root the tree. The scale bar indicates the number of
changes per nucleotide position.

closely related sequences (99.8% identity), which were
98.2% similar to the only known DAMO archea enriched
by Raghoebarsing and colleagues (2006) (DQ369741)
(Fig. 4).
Both the bacterial and archaeal 16S rRNA gene
sequences were used to design specific probes for fluo-
rescence in situ hybridization (FISH). The FISH probe
S-*-NC10-1162-a-A-18 (5′-GCCTTCCTCCAGCTTGACG
CTG-3′) was designed to target NC10 phylum sequences,
and this successfully detected cells in Culture B (35°C).
Probes designed for the third bacterial sequence
(FJ907183) did not detect cells. Probes specific for
archaea sequences from Culture B were also designed.
However, they did not bind to the DAMO archaea in
Culture B. Instead, the archaeal probe DARCH-872
designed by Raghoebarsing and colleagues (2006) was
used. This probe covered a broader range than expected,
with some methanogens also targeted.

minetrophic fen clone (EU155958)

submarine permafrost sediment (FJ982666)

Bootstrap values
100% - > 90% ferromanganous micronodule (AF293017)
90% - > 80%
80% - > 70% petroleum contaminated soil (AB161352)
70% - > 60%
denitrifying AOM enrichment (DQ369741)

ferromanganous micronodule (AF293016)

submarine permafrost sediment (FJ982704)

denitrifying AOM enrichment (FJ907179)

denitrifying AOM enrichment (FJ907180) A

petroleum contaminated soil (AB161329)

limestone sinkhole (FJ902710)

coal seam groundwater (AB294257)

minetrophic fen (EU155957)

acidic peatland
(DQ301885, DQ301886)

acidic peatland (DQ301884)

deep sea hydrothermal vent

(EF644782, EF644783, EF644786)

deep sea hydrothermal vent (EF644785)

ANME-3
(AF354131, AF354133, AJ578115)

ANME-2a
(AJ578128, AJ578107, AM745238, AM745257, FJ712637, FJ555679, FJ555681)

ANME-2b B
(AF354128, AF354140, AB461391)

ANME-2c
(AF354136, AJ578119)
to
outgroup
ANME-1
(AF354126, AF354137, AF419624, AF419625, AJ578084, AJ578136)

0.10

Fig. 4. Maximum likelihood (AxML) phylogenetic tree of 16S rRNA gene sequences of archaea implicated in anaerobic oxidation of methane
(AOM) coupled to denitrification or sulfate reduction. 16S rRNA clone group A includes sequences obtained in this study and nearest
neighbours identified using the SILVA rRNA database SINA webaligner. 16S rRNA clone group B includes methanotrophic archaea associated
with sulfate reducing bacteria. Sequences shorter than 1250 nucleotides were inserted into the tree using the ARB parsimony insertion tool.
Bootstrap values were obtained after 1000 resamplings. Methanocorpusculum sp. MSP (AY260434) was used as an outgroup to root the tree.
The scale bar indicates the number of changes per nucleotide position.

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
382 S. Hu et al.

draining agricultural land

Ettwig enrichment 2009

Sediments from ditches

continuous stirred tank

0.66b (month 9)

Bacteria 70%
Archaea 0%
NO2-, NO3-

5 months
30°C

2.15
N/A
Ettwig enrichment 2008

continuous stirred tank

0.16b (month 22)

Raghoebarsing

Bacteria 70%
enrichment

Archaea 0%
NO2-, NO3-
0.0062

b. Calculated from protein data, by assuming 0.55 g protein gVSS-1 (Modin et al., 2007), and therefore representing VSS of active biomass.
30°C

N/A
0.7
Fig. 5. Fluorescence in situ hybridization of biomass from

freshwater canal
Culture B on day 297. Epifluorescence micrographs taken after

Sequencing batch
Sediments from a

0.11b (month 16)

hybridization with the archaeal probe DARCH-872 (green) and

Raghoebarsing

Archaea 10%
Bacteria 80%
specific bacterial probe NC10-1162 (red).

enrichment

NO2-, NO3-

16 months
25°C

0.18
1.07
Sludge samples collected over the enrichment period
were hybridized with these probes to show the develop-
ment of the DAMO organisms in the two cultures. Over

Same as Culture A

Sequencing batch
time, both the DAMO archaea and NC10 group bacteria

1.73 (batch test)

0.85 (month 10)
became increasingly enriched in Culture B. After 297

Archaea 40%
Bacteria 30%
days, approximately 40% of cells in Culture B (35°C) were
Culture B

8 months
archaea (based on area), all of which hybridized with

35°C
NO3-
probe DARCH-872. The remainder of the culture con-

2.0
sisted of bacteria, of which approximately 50% hybridized
with the specific probe targeting the NC10 bacteria
digester sludge and activated sludge

(Fig. 5).
Mixture of freshwater Lake sediment,

In Culture A (22°C), the NC10 bacteria became

enriched after 9 months of incubation. On day 260,
around 15% of bacteria in the culture hybridized with the
NC10 group specific probe and no archaea were detected
with the general archaeal probe. The absence of archaea
Sequencing batch

in this culture was also confirmed by PCR using universal

1.60 (month 9)

Bacteria 15%

primer for the archaeal domain.

Archaea 0%
Table 2. Comparison of different DAMO cultures.

Culture A

0.065
22°C
NO3-

N/A

N/A

Roles of archaea and bacteria in DAMO cultures and

their potential interactions
This study re-confirmed that DAMO can be achieved by
Onset of apparent DAMO activity
bacteria in all microorganisms

NC10 bacteria with or without archaea. Ettwig and col-

a. Volatile suspended solids.

leagues (2008) hypothesized that the archaea present in

Raghoebarsing enrichment may be methanotrophs or
DAMO archaea, NC10
rNO3 (mmol l-1 day-1)
rNO2 (mmol l-1 day-1)

methanogens. The results obtained in this study strongly

suggest that they were likely involved in methane oxida-
Operating mode

VSSa (gVSS l-1)

tion rather than its production. The amount of archaea

Temperature
N feeding

cells in Culture B (35°C) slowly increased over the enrich-

Inoculum

ment period, which could not have been supported by

waste products or dead cells. Furthermore, in the

© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384

absence of nitrate between days 255 and 258 (and hence
no methane oxidation in this period), the methane con-
centration in the headspace remained constant, suggest-
ing that Culture B did not produce methane.
The detailed roles of archaea and bacteria in a DAMO
process and their potential interactions remain to be elu-
cidated. However, hypotheses may be made based on the
results summarized in Table 2. Both Culture B (35°C) and
Raghoebarsing enrichment displayed about 30 times
higher nitrate reduction rates than Culture A (22°C) and
Ettwig enrichment 2008, suggesting that archaea in these
cultures may play an important role in the reduction of
nitrate to nitrite. Ettwig enrichment 2008 and Culture A,
which contained bacteria only and showed a low nitrate
reduction rate compared with Culture B and Raghoebars-
ing enrichment, had nitrite reduction rates that are com-
parable to those cultures. This observation suggests that
the bacteria in Culture B and Raghoebarsing enrichment
may contribute significantly to nitrite reduction. It is pos-
sible the DAMO archaea can only reduce nitrate to nitrite
or prefer nitrate reduction to nitrite reduction. If this is the
case, archaea would require bacteria to remove nitrite,
which is an inhibitor to a wide range of physiological
types of microorganisms (Yarbrough et al., 1980). This
may explain why all archaea-containing DAMO cultures
obtained to date also contained NC10 bacteria.
Effect of temperature on DAMO microorganisms
From the same inoculum, DAMO archaea were enriched
in Culture B at 35°C but not in Culture A at 22°C, suggest-
ing that the higher temperature may have favoured the
enrichment of the archaeal DAMO organisms. However,
Raghoebarsing and colleagues (2006) enriched archaea
in a reactor at a temperature of 25°C, although the ratio of
archaeal to bacterial cells in Raghoebarsing enrichment
(approximately 1:8) was much lower than that in Culture B
(approximately 4:3). Ettwig and colleagues (2008) further
showed that the archaea contained in Raghoebarsing
enrichment disappeared after the temperature was
increased from 25°C to 30°C.
Due to the limited number of DAMO enrichment cul-
tures, the effect of temperature on DAMO microorganisms
is uncertain at this stage and requires further research.
However, reactors inoculated with a mixture of various
sources and operated under different temperatures would
likely yield different DAMO enrichments, which can be
used for further physiological studies aimed to gain a full
understanding of the DAMO process.
Acknowledgments
We would like to thank Dr Beatrice Keller and Ms Jianguang
Li for assistance with FIA measurements. We are grateful to
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
Enrichment of DAMO microorganisms 383
Dr Greg Crocetti for the FISH probe design. This work was
funded by the Australia Research Council (ARC) through
project DP0666762.
References
Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D.,
Widdel, F., Gieseke, A., et al. (2000) A marine microbial
consortium apparently mediating anaerobic oxidation of
methane. Nature 407: 623–626.
DeLong, E.F. (2000) Microbiology – resolving a methane
mystery. Nature 407: 577–579.
Eisentraeger, A., Klag, P., Vansbotter, B., Heymann, E.,
and Dott, W. (2001) Denitrification of groundwater with
methane as sole hydrogen donor. Water Res 35: 2261–
2267.
Eller, G., Kanel, L., and Kruger, M. (2005) Cooccurrence of
aerobic and anaerobic methane oxidation in the water
column of lake Plubsee. Appl Environ Microbiol 71: 8925–
8928.
Ettwig, K.F., Shima, S., van de Pas-Schoonen, K.T., Kahnt,
J., Medema, M.H., op den Camp, H.J.M., et al. (2008)
Denitrifying bacteria anaerobically oxidize methane in the
absence of Archaea. Environ Microbiol 10: 3164–3173.
Ettwig, K.F., van Alen, T., van de Pas-Schoonen, K.T., Jetten,
M.S.M., and Strous, M. (2009) Enrichment and molecular
detection of denitrifying methanotrophic bacteria of the
NC10 phylum. Appl Environ Microbiol 75: 3656–3662.
Hallam, S.J., Putnam, N., Preston, C.M., Detter, J.C.,
Rokhsar, D., Richardson, P.M., and DeLong, E.F. (2004)
Reverse methanogenesis: testing the hypothesis with envi-
ronmental genomics. Science 305: 1457–1462.
Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G., and
DeLong, E.F. (1999) Methane-consuming archaebacteria
in marine sediments. Nature 398: 802–805.
Islas-Lima, S., Thalasso, F., and Gomez-Hernandez, J.
(2004) Evidence of anoxic methane oxidation coupled to
denitrification. Water Res 38: 13–16.
Kruger, M., Meyerdierks, A., Glockner, F.O., Amann, R.,
Widdel, F., Kube, M., et al. (2003) A conspicuous nickel
protein in microbial mats that oxidize methane anaerobi-
cally. Nature 426: 878–881.
Mason, I. (1977) Methane as a carbon source in biological
denitrification. J Water Pollut Control Fed 49: 855–857.
Modin, O., Fukushi, K., and Yamamoto, K. (2007) Denitrifica-
tion with methane as external carbon source. Water Res
41: 2726–2738.
Moran, J., Beal, E., Vrentas, J., Orphan, V., Freeman, K., and
House, C. (2008) Methyl sulfides as intermediates in the
anaerobic oxidation of methane. Environ Microbiol 10:
162–173.
Nauhaus, K., Boetius, A., Kruger, M., and Widdel, F. (2002) In
vitro demonstration of anaerobic oxidation of methane
coupled to sulphate reduction in sediment from a marine
gas hydrate area. Environ Microbiol 4: 296–305.
Niemann, H., Losekann, T., de Beer, D., Elvert, M., Nadalig,
T., Knittel, K., et al. (2006) Novel microbial communities of
the Haakon Mosby mud volcano and their role as a
methane sink. Nature 443: 854–858.
Raghoebarsing, A.A., Pol, A., van de Pas-Schoonen, K.T.,
Smolders, A.J.P., Ettwig, K.F., Rijpstra, W.I.C., et al. (2006)
384 S. Hu et al.
A microbial consortium couples anaerobic methane oxida-
tion to denitrification. Nature 440: 918–921.
Reeburgh, W.S., Whalen, S.C., and Alperin, M.J. (1993) The
role of methylotrophy in the global methane budget. In
Microbial Growth on C-1 Compounds. Murrell, J.C., and
Kelly, D.P. (eds). Andover, MA, USA: Intercept, pp. 1–14.
Strous, M., and Jetten, M.S.M. (2004) Anaerobic oxidation of
methane and ammonium. Annu Rev Microbiol 58: 99–117.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 1, 377–384
Thalasso, F., Vallecillo, A., GarciaEncina, P., and FdzPo-
lanco, F. (1997) The use of methane as a sole carbon
source for wastewater denitrification. Water Res 31:
55–60.
Yarbrough, J.M., Rake, J.B., and Eagon, R.G. (1980) Bacte-
rial inhibitory effects of nitrite: inhibition of active transport,
but not of group translocation, and of intracellular enzymes.
Appl Environ Microbiol 39: 831–834.