275collected in strata where an optimal activity of methanotro-
phy was observed. A wide range of growing conditions was
achieved by combining changes in temperature, concentra-
tion of nutrients and pH. Due to the dominance of
Methylobacter species in the water column of Lake Pavin,
we therefore optimized the culture conditions for the isola-
tion of Type I methanotrophs. Unfortunately, all strains iso-
lated were affiliated to Type II methanotrophs, and more
especially to the genus Methylocystis. We are currently
investigating an isolation strategy based on culture with
opposite fluxes of CH 4 and oxygen, in order to favor the
physiology of the strains dominating the methanotrophic
community of Lake Pavin.
Microcalorimetry studies performed to characterize the
physiological response of isolated Methylocystis strains to
varying concentrations of CH 4 and O 2. As illustrated in Fig.
16.5, the response of the methanotrophic strains to CH 4 input
is immediate, even after a period of deprivation, suggesting
that:
(i) The enzymes involved in the methanotrophic pathway
are continuously synthesized, even in the absence of
substrate. This hypothesis might explain the detection of
transcripts of pmoA in strata with very low CH 4 concen-
trations (Fig. 16.5),
(ii) the strain investigated has the capacity to lower its main-
tenance energy and therefore to increase its stationary
growth phase.
16.4.2 Anaerobic Methanotrophy
16.4.2.1 Brief History
From a biochemical perspective, CH 4 is relatively difficult
to activate. Its aerobic activation is catalyzed by monooxy-
genases. These enzymes that make use of high-potential
oxygen radical chemistry are unavailable to anaerobic life.
On another hand, the enrichment or detection of organisms
capable of anaerobic growth on CH 4 has remained unsuc-
cessful for a long time. This led to the idea that CH 4 was
inert under anoxic conditions, a belief that persisted among
most microbiologists throughout most of the twentieth cen-
tury. This assumption was challenged during the 1970s.
Several independent studies showed that, in marine sedi-
ments, CH 4 concentrations decreased upward while sulfate
concentrations concomitantly decreased downward. This
indicated that sulfate-reduction might be coupled to the
anaerobic oxidation of methane (AOM) with sulfate acting
as the electron acceptor according to the equation 1 (Eq.
16.1). But it took more than 30 years before the AOM in
marine sediments could be unambiguously demonstrated
thanks to the analysis of the stable carbon isotopic compo-
sition (δ^13 C) of lipid biomarkers specific to Archaea (e.g.,
isoprenoid glycerol ether lipids) and Bacteria (particular
fatty acids, Box 16.4). In the following years, a number of
publications reported the occurrence of AOM in different
sedimentary settings, highlighting the importance of this
process in the biosphere (for review, see Knittel and Boetius
2009 ).16.4.2.2 Pathways of Anaerobic Oxidation
of Methane (AOM)
Microbial communities and metabolisms involved in AOM
are diversified since at time three different processes are
described in various environments. To our knowledge,
nitrate-dependent methane oxidation (NDMO) was exclu-
sively observed in freshwater environments, whereas sulfate
dependent methane oxidation (SDMO) and iron/manganese-
dependent methane oxidation were reported from both
marine and freshwater environments.- AOM coupled to sulfate-reduction
Visual identification of cells hybridized with
fluorochrome- labeled specific oligonucleotide probes in
marine sediments revealed conspicuous aggregates of
Archaea and SRB (AOM consortia), representing > 90 % of
the total microbial community (Boetius et al. 2000 ). This
finding supported the hypothesis that CH 4 could be used as a
substrate source via the cooperation of Archaea able to acti-
vate CH 4 and SRB able to provide an electron sink (see Eq.
16.1). These consortia consist of archaea, performing a
reverse methanogenesis, which form the phylogenetically
distinct cluster ANME-2 (ANaerobic MEthanotrophs)
within the methanogenic order Methanosarcinales, and of
sulfate-reducing bacteria from the Desulfosarcina and
Desulfococcus branches of Delta-Proteobacteria.
Subsequently, similar consortia were found with Archaea of
the clusters ANME-1, which is distantly related to
Methanosarcinales and Methanomicrobiales (Orphan et al.
2001 ; Knittel et al. 2005 ), and ANME-3, which is related to
the genera Methanococoides and Methanolobus within the
Methanosarcinales (Lösekann et al. 2007 ).CH SO HCOHSHO
rGkJmol CH442
32
1(^174)
+→++
°=− −
–––
()∆
(16.1)
16 Methanogens and Methanotrophs in Lake Pavin
http://www.ebook3000.com