277Enrichment cultures consisted of 10 % archaea of the order
Methanosarcinales distantly related to ANME-2, named
ANME-2d, the rest being bacteria affiliated to the new bacte-
rial division NC10 (Raghoebarsing et al. 2006 ). Hence, it was
plausible to assume that anoxic CH 4 oxidation with nitrate
would operate similarly as with sulfate, i.e., the archaea oxidize
CH 4 to CO 2 and transfer the electrons to the denitrifiers reduc-
ing nitrate or nitrite to N 2. This hypothesis was also supported
by microscopic observations of consortia of Bacteria and
Archaea (Raghoebarsing et al. 2006 ). However, it turned out
that the oxidation of CH 4 is catalyzed exclusively by the deni-
trifiers without participation of the archaeal members (Ettwig
et al. 2008 ). Indeed, ANME disappeared after prolonged incu-
bation (Ettwig et al. 2009 ), while AOM continued suggesting
that ANME were not obligatory involved in this process. This
process, driven by members of the NC10 bacterial division,
appears to be completely different from SDMO, since it does
not seem to involve reverse methanogenesis. Complete genome
analysis of Candidatus Methylomirabilis oxyfera, the domi-
nant bacterium affiliated with NC10, and labeling experiments
pointed to a new pathway of ‘intra-aerobic denitrification’
(Ettwig et al. 2010 ). This pathway leads to the intracellular pro-
duction of O 2 by disproportionation of 2 NO coming from the
nitrite. The enzyme and genes involved in NO disproportion-
ation are not yet clearly identified. Most of the genes involved
in the classical pathway of aerobic methanotrophy were
retrieved in the genome of Candidatus M. oxyfera. Therefore,
the O 2 produced by NO disproportionation is likely to oxidize
CH 4 intracellularly. Subsequently, nitrate-dependent AOM was
also demonstrated (Eq. 16.3.) from an enrichment culture
largely dominated by a representative of the ANME-2d lin-
eage, Candidatus Methanoperedens nitroreducens Haroon
et al. 2013 ). To oxidize CH 4 , this Archaea only reduce nitrate
to nitrite which in turn is used by Methylomirabilis oxyfera or
Candidatus Kuenenia stuttgartiensis, an anaerobic ammonium
oxidizer using nitrite as electron acceptor (Haroon et al. 2013 ).
- AOM coupled to iron and manganese reduction
AOM was shown to be coupled with iron and manganese
reduction (Eqs. 16.4 and 16.5) in meCthane-seep sediments
through an enrichment culture (Beal et al. 2009 ). The com-
munities involved were dominated by an uncultured archaeal
group named Marine Benthic Group-D (MBG-D). ANME-1
and 2 were also identified in these sediments. AOM coupled
to iron and manganese reduction was suggested to largely
contribute to the global oceanic AOM. However, the process
and the identity of microorganisms involved are still unclear.16.4.2.3 Mechanisms of Anaerobic Oxidation
of Methane
In addition to the close phylogenetic affiliation between
ANMEs and methanogens, evidence for metabolic similari-
ties comes from different studies. The mcrA gene (coding for
the a-subunit of MCR, see previous Sect. 16.3.2.4) was shown
to be associated with the ANME community (see Box 16.1,
Hallam et al. 2003 ) and metagenomic analyses of ANME-
rich sediments revealed that nearly all genes involved in the
methanogenesis pathways are present in ANME-1 (Hallam
et al. 2004 ). Proteomic approaches led to isolation of an
abundant enzymatic complex with a high degree of similarity
with MCR (Kruger et al. 2003 ). This complex, named
Ni-protein I, is associated with a modified F430 cofactor
(172-methyl-thio-F430, Mayr et al. 2008 ) and may be
involved in AOM. More recently, Scheller et al. ( 2010 )
showed that an MCR extract from a methanogen was able to
cleave the strong C-H bond of CH 4 to form methyl- coenzyme
M. All these data support the hypothesis that anaerobic CH 4
oxidation is performed by a reverse methanogenesis process.16.4.2.4 Preliminary Microbiological Studies
Supporting AOM in Lake Pavin
Geochemical data and reactive transport modeling of CH 4
in Lake Pavin reveal that CH 4 produced in the monimolim-
nion is partly oxidized under anaerobic conditions (Lopes
et al. 2011 ). The water layer between 65 and 70 m, in which
environmental conditions are not favorable to the activity of
aerobic methanotrophs, may provide favorable conditions3883410
92842 222
1
4CH NO HCONHO
rGkJmol CH++→++
°=(–∆ – )^ (16.2)34 42
50343222
1
4CH NO CO NO HO
GkJmol CH+→++
°=––
(–∆ – ) (16.3)CH Fe OH HHCO Fe
HO rGkJmol CH43 3221
48158
21 572++→+
+°=() ++
()∆(16.4)CH MnOHHCOMn
HO rGkJmol CH42 3221
447 4
5 790++→+
+°=++–(–∆ – )^ (16.5)16 Methanogens and Methanotrophs in Lake Pavin