Biogeosciences, 9, 4969–4977, 2012
www.biogeosciences.net/9/4969/2012/
doi:10.5194/bg-9-4969-2012
© Author(s) 2012. CC Attribution 3.0 License.
Biogeosciences
Aerobic methanotrophy within the pelagic redox-zone of the
Gotland Deep (central Baltic Sea)
O. Schmale1, M. Blumenberg2, K. Kießlich1, G. Jakobs1, C. Berndmeyer2, M. Labrenz1, V. Thiel2, and G. Rehder1
1Leibniz Institute for Baltic Sea Research Warnemu¨nde (IOW), Rostock, Germany
2Geobiology Group, Geoscience Centre, Georg-August-University Go¨ttingen, Go¨ttingen, Germany
Correspondence to: O. Schmale (oliver.schmale@io-warnemuende.de)
Received: 25 June 2012 – Published in Biogeosciences Discuss.: 20 July 2012
Revised: 15 November 2012 – Accepted: 19 November 2012 – Published: 5 December 2012
Abstract. Water column samples taken in summer 2008 from
the stratified Gotland Deep (central Baltic Sea) showed a
strong gradient in dissolved methane concentrations from
high values in the saline deep water (max. 504 nM) to low
concentrations in the less dense, brackish surface water
(about 4 nM). The steep methane-gradient (between 115 and
135 m water depth) within the redox-zone, which separates
the anoxic deep part from the oxygenated surface water
(oxygen concentration 0–0.8 mL L−1), implies a methane
consumption rate of 0.28 nM d−1. The process of micro-
bial methane oxidation within this zone was evident by a
shift of the stable carbon isotope ratio of methane between
the bottom water (δ13C CH4 =−82.4 ‰) and the redox-
zone (δ13C CH4 =−38.7 ‰). Water column samples be-
tween 80 and 119 m were studied to identify the microor-
ganisms responsible for the methane turnover in that depth
interval. Notably, methane monooxygenase gene expression
analyses for water depths covering the whole redox-zone
demonstrated that accordant methanotrophic activity was
probably due to only one phylotype of the aerobic type I
methanotrophic bacteria. An imprint of these organisms on
the particular organic matter was revealed by distinctive
lipid biomarkers showing bacteriohopanepolyols and lipid
fatty acids characteristic for aerobic type I methanotrophs
(e.g., 35-aminobacteriohopane-30,31,32,33,34-pentol), cor-
roborating their role in aerobic methane oxidation in the
redox-zone of the central Baltic Sea.
1 Introduction
Methane as an atmospheric trace gas is known to have a
relevant impact on Earth’s climate. Aquatic systems repre-
sent the most significant source of atmospheric methane.
However, the importance of the marine system seems to be
marginal (Bange et al., 1994), although enormous amounts of
methane are formed in marine sediments (Reeburgh, 2007).
One effective mechanism that is limiting the flux of methane
from the sedimentary reservoir into the atmosphere is the mi-
crobial oxidation of methane in the sediment and the water
column (Reeburgh, 2007). Comprehensive studies on aquatic
sediments in different settings show that methane is micro-
bially oxidized by the use of different electron acceptors,
with oxygen being most important for the water column
and sulfate for the sedimentary turnover (Barnes and Gold-
berg, 1976; Reeburgh, 1976; Hinrichs and Boetius, 2002;
Reeburgh, 2007). Recently, anaerobic methane oxidation us-
ing iron, manganese and nitrite has also been reported (Beal
et al., 2009; Ettwig et al., 2010). Although these processes
are efficient and consume the main part of dissolved methane
before it escapes from the sediment/water interface, some
parts of the ocean are characterised by strongly elevated
methane concentrations in the water column. This holds par-
ticularly true for stagnant, oxygen-deficient basins like the
Black Sea, Cariaco Basin or central Baltic Sea (Scranton et
al., 1993; Kessler et al., 2006; Schmale et al., 2010a). Com-
pared to the number of studies on the microbial processes
of methane oxidation in sediments, water column studies
are scarce, and could to date just identify the oxidation of
methane through oxygen and sulfate (Reeburgh, 2007 and
references therein). Nevertheless, multidisciplinary studies
Published by Copernicus Publications on behalf of the European Geosciences Union.
4970 O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone
in the water column of the Black Sea could impressively
demonstrate that the flux of methane from the deep-water
reservoir into the atmosphere is effectively buffered by the
microbial oxidation of methane under anaerobic and aero-
bic conditions (Schouten et al., 2001; Schubert et al., 2006;
Wakeham et al., 2007; Blumenberg et al., 2007; Schmale et
al., 2011).
Our present investigations were carried out in the Gotland
Deep in the central part of the Baltic Sea (Fig. 1). The Baltic
Sea is a European semi-enclosed marginal sea characterised
by limnic to brackish surface water and more saline deep and
bottom water. Especially for the central deep basins of the
Baltic Sea, this results in limited vertical mixing, the devel-
opment of a prominent redox-zone with oxic to anoxic con-
ditions, and the formation of stable biogeochemical zones
(Nausch et al., 2008). In these basins, the stagnant deep wa-
ter can only be renewed by strong temporal inflow events
of saline oxygenated water from the North Sea (Reissmann
et al., 2009) or by long-term vertical transport mechanisms
mainly induced by bottom boundary mixing along the slop-
ing topography (Holtermann and Umlauf, 2012). More fre-
quent are weak inflows of North Sea water that are periodi-
cally perturbing the intermediate water column stratification
and biogeochemical zones in the central basins (Mattha¨us
et al., 2008). The Baltic Sea, like other marginal seas, is
characterised by high terrestrial inputs and production rates
of organic matter that are to a considerable extent accumu-
lated and decomposed in the sediment. Under anoxic con-
ditions, the final step of decomposition of organic matter
leads to the generation of methane within the sediment. In
the Baltic Sea, pore-water as well as acoustic investigations
demonstrated that methane is abundant in high concentra-
tions within the sediment and that in some regions methane
is also released as free or dissolved gas into the water col-
umn (Dando et al., 1994; Piker et al., 1998; Thießen et al.,
2006). Extensive water column investigations in the Baltic
Sea identified the strongest methane enrichment within the
stagnant anoxic water bodies of the deep basins (Gotland
Deep and Landsort Deep; max. 504 nM at 230 m water depth
and 1058 nM at 435 m water depth, respectively; Schmale et
al., 2010b). In contrast, surface water methane concentra-
tions in these areas are only slightly enriched compared to
the atmospheric equilibrium, indicating an effective sink that
prevents the escape of methane from the deep water into the
atmosphere (Schmale et al., 2010b). However, little is as yet
known about the processes that regulate the methane flux in
this environment. In this paper, we use a multidisciplinary ap-
proach that combines gas chemistry, molecular biology and
lipid biomarker geochemistry and present data on a microbial
methane sink within the pelagic redox-zone of the Gotland
Deep. Thus, this study aims to investigate whether aerobic
methane oxidation also plays a role in the more dynamic and
turbulent redox-zone of the central Baltic Sea.
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Figures 
 
 
 
 
 
 
Figure 1. The Baltic Sea and the location of the Gotland Deep. The study area is indicated with 
a black dot. 
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Fig. 1. The Baltic Sea and the location of the Gotland Deep. The
study area is indicated with a black dot.
2 Methods
Samples were retrieved during a scientific cruise in sum-
mer 2008 with the German research vessel Maria S. Merian
(MSM 08/3, 18 June to 18 July. The Gotland Deep (57◦18′ N,
20◦04′ E; Fig. 1) represents the deepest location in the east-
ern Gotland Basin (water depth at our water station 231 m).
The sampling strategy at this location was directed at (1)
identifying the depth interval of aerobic methane oxidation
within the redox-zone based on physical parameters and on
board gas chemistry, and (2) recovering samples from the
relevant depth interval for home-based molecular biological
and lipid biomarker studies to identify the microorganisms
involved in methane oxidation. These samples were taken
within a time frame of 3 days and with different sampling
equipment (as described below).
2.1 Physical parameters and gas chemistry
Water stations for analyses of the gas chemistry were carried
out with a rosette water sampler equipped with twenty-four
10 L Hydro-bios Free Flow bottles. For continuous CTD and
turbidity profiling a Seabird sbe911+ system, together with
a turbidity sensor (ECO FLNTU, WET Labs) were attached
to the underwater unit.
The oxygen distribution was measured according to Win-
kler’s method, whereas hydrogen sulfide was analysed col-
orimetrically with the methylene blue method (Grasshoff et
al., 1983).
Water samples (600 mL) for methane analyses were trans-
ferred directly from the sample bottle into pre-evacuated
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O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone 4971
1100 mL glass bottles. Dissolved methane was extracted us-
ing a vacuum degassing method and its mole fraction was
determined with a gas chromatograph equipped with a flame
ionisation detector (Trace GC, Thermo Electron). The aver-
age precision of this method is ±3 % (Keir et al., 2009).
For the determination of δ13C CH4 values, subsamples
of the extracted gas were analysed at the Leibniz Institute
for Baltic Sea Research Warnemu¨nde using an isotope-ratio
mass spectrometer (modified after Schmale et al., 2010a).
These subsamples were collected in 10 mL pre-evacuated
crimp-top glass vials containing 4 mL of supersaturated salt
solution (degassed Millipore water, poisoned with HgCl2)
and sealed with a butyl rubber septum. Stable carbon iso-
tope analysis involved removal of water and carbon dioxide
on a NaOH/Ascarite trap, double cryofocusing at −110 ◦C
(ethanol/nitrogen) on Hayesep D and Poraplot S columns,
gas-release by heating the traps separately to 40 ◦C and gas
separation on a MolSieve 5A Plot capillary column (Supelco,
30 m, I.D. 0.32 mm) at 30 ◦C (Trace GC Ultra, Thermo Elec-
tron), combustion to CO2 using a Ni catalyst at 1050 ◦C, re-
moval of combustion water using a Nafion trap, and injection
into a MAT 253 mass spectrometer (Thermo Electron, Bre-
men) using a continuous flow technique. The δ13C CH4 data
is expressed vs. Vienna Pee Dee Belemnite (VPDB) stan-
dard. Calibration of the system was performed daily by the
use of a CH4 standard with known isotopic composition. The
average precision of that method is ±1 ‰ .
2.2 pmoA gene expression analyses
Within the identified redox-zone filter samples were taken in
80, 100, 105 and 119 m water depth using a rosette water
sampler. 1000 mL of sample water were filtered on a Dura-
pore filter (0.2 µm pore size), frozen in liquid nitrogen and
stored at −80 ◦C.
For each sample RNA was extracted from the frozen filter
with acidic phenol (Weinbauer et al., 2002) and quantified
using a NanoDrop ND-1000 spectrometer (NanoDrop Tech-
nologies). To generate pmoA-specific cDNA, 100 ng RNA
was reverse transcribed using the iScript Select cDNA Syn-
thesis Kit (Biorad) and reverse primer mb661r (Costello and
Lidstrom, 1999). To detect potential DNA contamination one
sample was incubated without reverse transcriptase. 1 µL of
cDNA was amplified by Polymerase Chain Reaction (PCR).
For the generation of specific GC-clamped PCR products a
discontinuous PCR was applied: reactions (50 µL) containing
1×PCR buffer, 200 µM of each dNTP, 0.3 µM revere primer
mb661r, 0.1 µM forward primer A189f (Holmes et al., 1995),
0.5 mM MgCl2, 0.5 µL polymerase (Herculase II, Fusion)
and template cDNA were incubated at initial 94 ◦C for 5 min.
After 20 cycles of 60 s at 94 ◦C, 60 s at 56 ◦C and 30 s at
72 ◦C, the PCR was paused at 72 ◦C and 0.12 µM A189f GC
primer were added to each reaction. Afterwards the PCR was
resumed for another 15 cycles with conditions as described
above, followed by a final elongation step of 5 min at 72 ◦C.
Specificity of the PCR products was documented by agarose
gel electrophoresis and staining with ethidium bromide. The
described discontinuous PCR yielded more specific and dis-
tinct PCR products than a conventional PCR with GC-primer
(data not shown).
PCR products were separated by Denaturing Gradient Gel
Electrophoresis (DGGE) using a gradient of 35 % to 80 %
denaturant in a 6 % polyacrylamide gel. Electrophoresis ran
at 100 V and 60 ◦C for 16 h in 1×TAE buffer. The gel was
stained with a 1 : 5000 dilution of SYBRGold (Invitrogen)
for 30 min. All bands from each depth were excised and
reamplified in a PCR reaction containing 1×PCR buffer,
0.3 µM of A189f and mb661r each, 200 µM of each dNTP
and 0.5 µL polymerase in 30 cycles with an annealing tem-
perature of 56 ◦C. PCR products were purified with Nucle-
oSpin purification kit (Macherey-Nagel) and sequenced with
primers A189f and mb661r by AGOWA (Berlin, Germany).
Forward and reverse sequences were checked for quality
applying Seqman software (DNASTAR).
For phylogenetic analysis the ARB software package was
used (Ludwig et al., 2004). Alignment was based on par-
tial DNA sequences of pmoA and amoA genes obtained from
GenBank Database with partial sequences of amoA (Acces-
sion numbers: AF037107, AF043710, AF037108) serving as
an outgroup in the tree construction. Sequences for analysis
were reduced to unambiguously alignable positions.
Three different trees were calculated using the algorithms
maximum likelihood (PHYML), maximum parsimony and
neighbour-joining with Jukes-Cantor correction.
Nucleotide sequence accession numbers are deposited in
the GenBank database (accession number KC188735).
2.3 Lipid biomarkers
For lipid biomarker studies a sample was selected from the
centre of the redox-zone at 100 m water depth. That depth
was chosen to obtain a POM sample that reflects the in
situ microbial turnover of methane under low-oxygen con-
ditions and is not “contaminated” by external water masses
(i.e. increased oxygen concentrations or anoxic conditions)
which may also include other methane consuming microor-
ganisms (e.g. consortia performing the anaerobic oxidation
of methane). 214 L of water were filtered on glass microfiber
filters (∅ 30 cm; 0.7 µm pore size) over a time span of two
hours using a PUMP-CTD system (Strady et al., 2008). Half
of the filter was extracted in triplicate with dichloromethane
and methanol (3 : 1, v : v) in a CEM Mars 5 microwave
(Matthews, NC) at 80 ◦C and 800 W. An aliquot of the sam-
ple was acetylated with acetic acid/pyridine as described
elsewhere (Blumenberg et al., 2007) and analysed using
high performance liquid chromatography-mass spectrome-
try (LC-MS). LC-MS was performed using a Varian Prostar
Dynamax HPLC system coupled to a Varian 1200 L triple
quadrupole mass spectrometer (for analytical details see Blu-
menberg et al., 2010). Another aliquot of the extract was
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4972 O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone
 
 
Figure 2. Left: vertical distribution of salinity (black), temperature (red), and turbidity (gray). 
Right: vertical distribution of oxygen and hydrogen sulfide (expressed as negative oxygen 
equivalents, blue), methane (red), and δ13C value of methane (green). The depth interval of the 
redox-zone is displayed in gray (oxygen concentration 0 - 0.8 ml L-1). The water depths for 
molecular biological and lipid biomarker studies are indicated with colored horizontal lines 
(black = molecular biology, red = molecular biology together with lipid biomarkers). 
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Fig. 2. Left: vertical distribution of salinity (black), temperature (red), and turbidity (grey). Right: vertical distribution of oxygen and hy-
drogen sulfide (expressed as negative oxygen equivalents, blue), methane (red), and δ13C value of methane (green). The depth interval of
the redox-zone is displayed in grey (oxygen concentration 0–0.8 mL L−1). The water depths for molecular biological and lipid biomarker
studies are indicated with coloured horizontal lines (black=molecular biology, red=molecular biology together with lipid biomarkers).
separated by column chromatography into a hydrocarbon
(F1), an alcohol and ketone (F2), and a polar fraction (F3)
using a column (∅∼ 1 cm) filled with 7.5 g silica gel 60 (ac-
cording to Blumenberg et al., 2010). (F3) was transmethy-
lated using trimethylchlorosilane in methanol (1 : 8; v : v;
1.5 h at 80 ◦C). Double bond positions within unsaturated
fatty acid methyl esters were determined by derivatisation
with dimethyldisulfide (DMDS; method modified after Carl-
son et al., 1989 and Gatellier et al., 1993). The polar fraction
(F3), and the DMDS derivatized sample were analysed with
coupled gas chromatography-mass spectrometry (GC-MS)
using a Varian CP-3800 gas chromatograph equipped with
a fused silica column (Phenomenex Zebron ZB-5MS, 30 m,
I.D. 0.32 mm) coupled to a Varian 1200L mass spectrometer.
He was used as carrier gas. The temperature program was
80 ◦C (3 min) to 310 ◦C (held 25 min) at 4 ◦C min−1. Com-
pounds were identified by comparing mass spectra and reten-
tion times to published data. δ13C values of fatty acid methyl
esters from the polar fraction (F3) were measured in repli-
cate as described previously (Blumenberg et al., 2010). The
precision was generally better than 0.5 ‰ .
3 Results and discussion
3.1 Physical parameters and gas chemistry
The estuarine circulation in the Baltic Sea causes a strong
vertical salinity gradient between the surface and deep water
(Lass and Mattha¨us, 2008). This gradient is very pronounced
in the deep basins of the central Baltic Sea (e.g. Gotland and
Landsort Deep; Fig. 2) and reflects a water column stratifi-
cation that limits the vertical mixing and water renewal in
the deep strata (Reissmann et al., 2009). Oceanographic in-
vestigations, carried out at the redox-zone of the Gotland
Deep, show that this depth is periodically perturbed by in-
trusions, internal waves or eddies which can shift the ampli-
tudes of isoclines up to 10 m within time spans less than an
hour (shown for temperature and salinity in Lass et al., 2003;
Dellwig et al., 2012).
During sampling, the specific water column structure led
to oxygen deficiency below a water depth of about 80 m.
Further downward, the oxygen concentrations decreased be-
low 0.8 mL L−1, characterizing the redox-zone between the
oxic surface and anoxic deep waters. The lower boundary
of the redox-zone was located at about 138 m water depth
where the concentration of hydrogen sulphide (H2S) started
to increase. A distinct turbidity anomaly was observed at
about 120 m water depth (Fig. 2). This specific feature is
known from other anoxic basins like the Black Sea and is
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O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone 4973
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Figure 3. Unrooted maximum likelihood tree showing the phylogenetic affiliation of the partial 
pmoA DNA sequence generated from the filter samples taken in 80, 100, 105 and 119 m water 
depth (marked bold). Black circles = validation of subtree by neighbor-joining and parsimony; 
white circles = validation of subtree by parsimony; black diamond = validation of subtree by 
neighbor-joining. Scale bar represents 10 substitutions per 100 nucleotides. For tree construction 
partial amoA sequences were used as an outgroup (not shown). 
 
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Fig. 3. Unrooted maximum likelihood tree showing the phyloge-
netic affiliation of the partial pmoA DNA sequence generated from
the filter samples taken in 80, 100, 105 and 119 m water depth
(marked bold). Black circles= validation of subtree by neighbour-
joining and parsimony; white circles= validation of subtree by
parsimony; black iamond= validation of subtree by neighbour-
joining. Scale bar represents 10 substitutions per 100 nucleotides.
For tree construction partial amoA sequences were used as an out-
group (not shown).
most likely caused by the precipitation of iron and man-
ganese oxides (Kempe et al., 1991) and an enrichment of
particulate organic matter (POM) due to enhanced micro-
bial activity (Prokhorenko et al., 1994). The concentrations
of H2S and other reduced chemical species like ammonium
(NH+4 ) are constantly increasing with depth, indicating an
upward flux from the sediment or deep water towards the
redox-zone (Nausch et al., 2008). The same concentration
pattern was observed for methane (Fig. 2). Highest methane
concentrations were detected close to the seafloor (504 nM
at 230 m water depth) supporting an origin from methano-
genesis in the sediment (Piker et al., 1998). Indeed, low
δ13C CH4 values (−82.4 ‰ to −75.2 ‰, Fig. 2) observed in
the anoxic water body clearly point at a microbial methane
source (Whiticar, 1999). The methane concentration profile
shows a pronounced decrease within the redox-zone from
124 nM at 135 m water depth to 4.8 nM at 115 m water depth.
At the same time, δ13C CH4 values substantially increase
(up to −38.7 ‰ at 80 m water depth). As microbial reac-
tions favour the incorporation of 12C and thus, enrichment
in 13CH4 in the residual methane pool, this isotopic shift
clearly indicates microbial methane oxidation within that
water level (Whiticar, 1999). In a first approximation the
methane oxidation rate can be derived from the methane gra-
dient and the vertical transport velocity. Using the vertical
diffusivity (kz) of 0.95 m2 d−1 (Axell, 1998) in combination
with the methane distribution between 115 m (4.8 nM) and
at 135 m water depth (124 nM) this calculation leads to a
flux of methane of 5.7 µmol m−2 d−1. If we assume that this
Table 1. Concentrations, relative abundances and δ13C values of in-
dividual fatty acids (analysed as methyl ester derivatives) at 100 m
water depth of the Gotland Deep. Fatty acids specific for methan-
otrophic bacteria are given in bold letters.
Fatty acid Concentration % of total δ13C [‰]
[µg g−1 Corg] fatty acids
C14 : 0 37.8 0.7 −26.9
iC15 : 0 86.1 1.6 −21.5
aiC15 : 0 101.4 1.9 −26.2
C15 : 0 86.0 1.6 −25.8
iC16 : 0 29.4 0.5 −29.7
C16 : 1ω9t 31.4 0.6 −22.2
C16 : 1ω8c 9.8 0.2 −38.8
C16 : 1ω8t 33.0 0.6 −30.4
C16 : 1ω7c 231.0 4.2 −27.6
C16 : 1ω7t 57.0 1.0 –
C16 : 1ω5c 66.1 1.2 −35.7
C16 : 1ω5t 22.2 0.4 −33.8
C16 : 0 1300.1 23.7 −26.9
iC17 : 0 9.7 0.2 −29.6
aiC17 : 0 16.3 0.3 −28.6
C17 : 0 63.2 1.2 −30.9
C18 : 2 41.4 0.8 −25.4
C18 : 3 32.5 0.6 –
C18 : 1ω9c 246.4 4.5 −26.5
C18 : 1ω7c 232.6 4.2 −24.9
C18 : 1ω6c 15.0 0.3 −30.9
C18 : 1ω5c 9.0 0.2 −20.2
C18 : 0 2279.4 41.6 −27.1
iC19 : 0 49.7 0.9 −26.5
C19 : 0 47.5 0.7 –
C20 : 0 154.0 2.8 −29.6
C21 : 0 20.1 0.4 –
C22 : 0 102.7 1.9 −29.7
C24 : 0 77.2 1.4 –
flux is oxidized within the 20 m depth interval, we receive
a methane consumption rate of 0.28 nM d−1. An inverse
trend in methane carbon isotope ratios is observed above
the suboxic layer (Fig. 2; δ13C ratios between −59.9 ‰
and −48.5 ‰). This trend is probably caused by (1) the
downward ventilation of atmospheric methane (−47.4 ‰;
http://www.esrl.noaa.gov/gmd/ccgg/iadv/), and/or (2) micro-
bial methane production in shallow waters. The process of
methane formation in an oxygenated water column has been
observed in many regions (Holmes et al., 2000; Schmale
et al., 2010a) and seems to be related to the decay of
methylphosphonates, in particular under phosphate-limiting
conditions, and/or methanogenesis in the anoxic interior of
particles (Karl et al., 2008). Such methane forming processes
are also indicated in our dataset by a pronounced 13CH4 de-
pletion at 20 m water depth (δ13C=−59.9 ‰) together with
slightly elevated methane concentrations of 7 nM (surround-
ing water depths around 4 nM). However, within the surface
water, methane is only slightly enriched compared with the
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4974 O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone
Fig. 4. The relative abundances of specific bacteriohopanepolyols (BHPs) sampled in 100 m water depth, together with the chemical structure
of each compound. BHT= bacteriohopanetetrol; cycl= cyclitol.
atmospheric equilibrium (144 % saturation ratio; Schmale et
al., 2010b), indicating that the local emission of methane into
the atmosphere is rather low.
3.2 Methanotrophic microorganisms within the
redox-zone
Chemical gradients feature versatile environments and are
known to harbour enhanced microbial abundance and ac-
tivity. Within the redox-zone of the central Baltic Sea, var-
ious biogeochemical processes have been identified, such
as denitrification, ammonia oxidation, or dark CO2 fixation
(Labrenz et al., 2005; Jost et al., 2008; Glaubitz et al., 2009;
Labrenz et al., 2010) and also microbial consumption of
methane was proposed as mechanism explaining the strong
methane decrease in this water layer (Schmale et al., 2010a).
To gain information on the contribution of methanotrophic
microorganisms to the POM within the redox-zone, we per-
formed expression analyses of the methane monooxygenase
gene (pmoA), and studied concentrations and distributions of
bacteriohopanepolyols (BHPs).
The presence of methanotrophic bacteria was proved by
molecular biological studies carried out on samples obtained
from 80, 100, 105 and 119 m water depth (Fig. 2). Although
the two groups of methanotrophs, type I and type II, use dif-
ferent physiological pathways for the assimilation of carbon
from methane, namely the ribulose monophosphate pathway
and the serine pathway, the key enzyme methane monooxy-
genase responsible for the initial oxidation of methane to
methanol is present in both groups. The gene coding for the
alpha subunit of the particulate form of the enzyme (pmoA)
has been used as a marker for the detection and characteri-
sation of methanotrophic communities in different habitats
(Costello and Lidstrom, 1999; Bourne et al., 2001; Chen
et al., 2007; Chen et al., 2008). In order to identify active
methanotrophs we investigated pmoA gene expression in situ.
Based on DGGE analysis only one type of pmoA transcript,
named Uncultured GotDeep pmoA1, was present throughout
the redox-zone. Phylogenetically it is affiliated with the type
I methanotrophs and practically identical to an uncultured
bacterium found in the meromictic crater lake Lac Pavin
(Fig. 3). With a permanently anoxic monimolimnion, also
due to a halocline, elevated concentrations of CH4 and nearly
identical temperatures around 5–6 ◦C (Aeschbach-Hertig et
al., 2002) environmental conditions in Lac Pavin are in some
aspects comparable to the central Baltic Sea (Fig. 2). Thus,
activity of these identified methanotrophs could be indicative
of this kind of habitat.
To support these findings, an additional POM sample
obtained in the centre of the redox-zone was investi-
gated for lipid biomarkers. Of special biomarker value
are BHPs with an A-ring methylation at C-3 (Neunlist
and Rohmer, 1985) and/or an amino group at C-35 of
the hopanoid structure, both of which are widespread in
methanotrophic bacteria (Neunlist and Rohmer, 1985;
Talbot et al., 2001). The vast majority of BHPs was com-
posed of bacteriohopane-32,33,34,35-tetrol (BHT) and
35-aminobacteriohopane-32,33,34-triol (aminotriol), the
most common and thus unspecific BHPs (Fig. 4). C-3 methy-
lated BHPs were not observed. However, low abundances
of 35-aminobacteriohopane-31,32,33,34-tetrol (aminotetrol)
and of 35-aminobacteriohopane-30,31,32,33,34-pentol
(aminopentol) were found (Fig. 4). Whereas both these
amino-BHPs are considered indicative of methanotrophic
bacteria (Neunlist and Rohmer, 1985; Talbot and Farrimond,
2007), particularly the latter is even regarded as a biomarker
for the type I subgroup (gamma proteobacteria; Talbot and
Farrimond, 2007). Further evidence for a prominent contri-
bution of type I methanotrophs comes from the fatty acids
C16 : 1ω8c and C16 : 1ω5c which are considered as specific
Biogeosciences, 9, 4969–4977, 2012 www.biogeosciences.net/9/4969/2012/
O. Schmale et al.: Aerobic methanotrophy within the pelagic redox-zone 4975
to this group (Makula, 1978; Nichols et al., 1985; Table 1).
At the same time the lack of C18 : 1ω8c, a fatty acid specific
of type II methanotrophs (alpha proteobacteria, Bowmann et
al., 1991), indicates that these microorganisms do not play a
significant role for the methane turnover at the redox-zone
of the Gotland Deep. Biomarkers from methanotrophic
bacteria commonly show the isotopic traits of the substrate
(Summons et al., 1994). Indeed the δ13C values of the fatty
acids C16 : 1ω8c and C16 : 1ω5c (−38.8 ‰ and −35.5 ‰,
respectively; Table 1) are well within the δ13C CH4 at
80 and 105 m (=−38.7 ‰ and −50.6 ‰, respectively;
Fig. 2). Whereas biomarker indications for the presence
of methanotrophic bacteria exist, their relative abundance
among the bacterial community appears to be low. This is
indicated (i) by the low proportion of methanotroph-specific
amino-BHPs within the total BHPs (< 1.6 % of total BHPs;
note that amino-BHPs are often predominant in methan-
otrophs; Talbot et al., 2001), and (ii) by the low amounts of
type I specific fatty acids acids (C16 : 1ω8c and C16 : 1ω5c
represent 1.4 % of total fatty acids).
Thus, in contrast to studies in the redox-zone of the Black
Sea, where indications for type I, II and X were found
(Gal’chenko et al., 1988; Durisch-Kaiser et al., 2005; Blu-
menberg et al., 2007), the diversity of active aerobic methan-
otrophs in the redox-zone of the Gotland Deep seemed to
be restricted. These findings are only based on one dataset,
but it would be in line with previous studies investigating
the microbial catalysts of denitrification, nitrification, or dark
CO2 fixation in central Baltic Sea redox-zones which also re-
vealed that these pathways were actively driven by only a few
bacterial or archaeal key species (Grote et al., 2008; Glaub-
itz et al., 2009; Labrenz et al., 2010). An explanation for the
reduced diversity of active microorganisms along the central
Baltic Sea redox-zone could be the periodic perturbation of
the stratification which does not occur in the same strength
and frequency in the Black Sea. An overlap of sulfide- and
oxygen-containing waters can occur in the Gotland Basin
(Axell, 1998), and it is known that sulfide is toxic for many
organisms or at least can inhibit the activity of specific mi-
croorganisms (Erguder et al., 2009). Thus, potential sulfide
stress could inhibit other than type I methanotrophic bacteria
within the redox-zone of the Gotland Deep, but this interest-
ing aspect needs further investigation.
4 Conclusion
Using a multidisciplinary approach of gas chemistry, molec-
ular biology, and lipid geochemistry, we identified the
process of aerobic methane oxidation within the pelagic
redox-zone of the Gotland Deep (central Baltic Sea). This
was evidenced by a strong decrease in methane concen-
trations together with a 13C CH4 enrichment, the detection
of the key enzyme methane monooxygenase (pmoA), and
the occurrence of lipids specific for methanotrophic bacte-
ria (e.g., aminopentol; 16 : 1ω8c fatty acid). Phylogenetic
and biomarker data indicate that the diversity of active aer-
obic methanotrophs in the redox-zone of the Gotland Deep
was restricted to members of the type I subgroup. In con-
trast to other marine settings with a permanent stratification,
e.g. the Black Sea, the physical and biogeochemical struc-
ture of the Gotland Deep is periodically disturbed by in-
trusions, eddies, internal waves or long-term vertical trans-
port mechanisms. How this variable environment is affect-
ing the methane turnover in the water column and the micro-
bial community responsible for this process is an interesting
question that needs to be investigated in future studies. Also
the transferability of our results on a basin scale needs to be
addressed as some parts of the basin (e.g. the basin bound-
aries) are permanently influenced by intrusions and elevated
vertical mixing that might influence the processes involved
in the turnover of methane.
Acknowledgements. We thank the captain, officers and crew
aboard R/V Maria S. Merian (MSM 08/3) for their assistance
on sea. We gratefully acknowledge the efforts of G. Nausch and
S. Kru¨ger in carrying out the oxygen and hydrogen sulfide analysis
as well as the CTD work on the cruise. We also thank J. Dyckmans
(Centre for Stable Isotope Research and Analysis, University of
Go¨ttingen) for help with compound specific carbon isotope analysis
and T. Licha and K. No¨dler for support with LC-MS analyses. We
thank Marcus Elvert and an unknown reviewer for their thoughtful
reviews and comments. The study was supported by the Deutsche
Forschungsgemeinschaft (DFG) through grants SCHM 2530/2-1,
BL 971/3-1, and BL 971/1–3.
Edited by: G. Herndl
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