Introduction
After water vapor and carbon dioxide, methane is the most abundant
greenhouse gas in the atmosphere
(e.g., Hartmann et al., 2013;
Denman et al., 2007). Its atmospheric concentration has increased more than 150
% since preindustrial times, mainly through increased human activities
such as fossil fuel usage and livestock breeding
(Hartmann et
al., 2013; Wuebbles and Hayhoe, 2002; Denman et al., 2007). Determining the
natural and anthropogenic sources of methane is one of the major goals for
oceanic, terrestrial, and atmospheric scientists to be able to predict
further impacts on the world's climate. The ocean is considered to be a
modest natural source for atmospheric methane
(Wuebbles and Hayhoe, 2002; Reeburgh,
2007; EPA, 2010). However, research is still sparse on the origin of the
observed oceanic methane, which automatically leads to uncertainties in
current ocean flux estimations
(Bange
et al., 1994; Naqvi et al., 2010; Bakker et al., 2014).
Within the marine environment, the coastal areas (including estuaries and
shelf regions) are considered the major source for atmospheric methane,
contributing up to 75 % to the global ocean methane production
(Bange et al., 1994). The majority of coastal
methane is produced during microbial methanogenesis in the sediment, with
probably only a minor part originating from methane production within the
water column (Bakker et al., 2014).
However, knowledge on the magnitude, seasonality, and environmental controls
of benthic methanogenesis is still limited.
In marine sediments, methanogenesis activity is mostly restricted to the
sediment layers below sulfate reduction due to the successful competition
of sulfate reducers with methanogens for the mutual substrates acetate and
hydrogen (H2; Oremland and Polcin, 1982;
Crill and Martens, 1986; Jørgensen, 2006). Methanogens produce methane
mainly from using acetate (acetoclastic methanogenesis) or H2 and
carbon dioxide (CO2; hydrogenotrophic methanogenesis). Competition
with sulfate reducers can be relieved through the usage of noncompetitive
substrates (e.g., methanol or methylated compounds, methylotrophic
methanogenesis; Cicerone and Oremland, 1988; Oremland and
Polcin, 1982). The coexistence of sulfate reduction and methanogenesis has been
detected in a few studies from organic-rich sediments, e.g., salt-marsh
sediments (Oremland et al., 1982; Buckley et al., 2008),
coastal sediments (Holmer and
Kristensen, 1994; Jørgensen and Parkes, 2010), or sediments in upwelling
regions (Pimenov et al., 1993;
Ferdelman et al., 1997; Maltby et al., 2016), indicating the importance of
these environments for methanogenesis within the sulfate reduction zone (SRZ
methanogenesis). So far, however, environmental controls of SRZ
methanogenesis remain elusive.
The coastal inlet Eckernförde Bay (southwestern Baltic Sea) is an
excellent model environment to study seasonal and environmental controls of
benthic SRZ methanogenesis. Here, the muddy sediments are characterized by
high organic loading and high sedimentation rates (Whiticar,
2002), which lead to anoxic conditions within the uppermost 0.1–0.2 cm b.s.f. (Preisler
et al., 2007). Seasonally hypoxic (dissolved oxygen < 63 µM)
and anoxic (dissolved oxygen = 0 µM) events in the bottom water of
Eckernförde Bay
(Lennartz et al., 2014;
Steinle et al., 2017) provide ideal conditions for anaerobic processes at
the sediment surface.
Overview of processes relevant for benthic methane production,
consumption, and emission in the Eckernförde Bay. The thickness of
arrows for emissions and coupling between surface processes indicates the
strength of methane supply. Note that this figure combines existing
knowledge with results from the present study. See discussion for more
details.
Sulfate reduction is the dominant pathway of organic carbon degradation in
Eckernförde Bay sediments in the upper 30 cm b.s.f., followed by
methanogenesis in deeper sediment layers where sulfate is depleted
(≪ 30 cm b.s.f.; Whiticar,
2002; Treude et al., 2005a; Martens et al., 1998; Fig. 1). This methanogenesis
below the sulfate–methane transition zone (SMTZ) can be intense and often
leads to methane oversaturation in the porewater below 50 cm of sediment depth,
resulting in gas bubble formation
(Abegg and Anderson, 1997;
Whiticar, 2002; Thießen et al., 2006). Thus, methane is transported from
the methanogenic zone (> 30 cm b.s.f.) to the surface sediment by
both molecular diffusion and advection via rising gas bubbles
(Wever et al., 1998; Treude et al., 2005a). Although
upward-diffusing methane is mostly retained by the anaerobic oxidation of
methane (AOM; Treude et al., 2005a), a major part is
reaching the sediment–water interface through gas bubble transport
(Treude et al., 2005a; Jackson et al.,
1998), resulting in a supersaturation of the water column with respect to
atmospheric methane concentrations (Bange et al., 2010). The time series station Boknis Eck in the Eckernförde Bay is a known
site of methane emissions into the atmosphere throughout the year due to
this supersaturation of the water column (Bange et al.,
2010).
The source for benthic and water column methane was seen in methanogenesis
below the SMTZ (≪ 30 cm b.s.f.; Whiticar,
2002); however, the coexistence of sulfate reduction and methanogenesis has been
postulated (Whiticar, 2002; Treude et al., 2005a). Still,
the magnitude and environmental controls of SRZ methanogenesis are poorly
understood, even though SRZ methanogenesis may make a measurable contribution to benthic
methane emissions given the short diffusion distance to the sediment–water
interface (Knittel and Boetius, 2009). The production of methane
within the sulfate reduction zone of Eckernförde Bay sediments could
further explain the peaks in methane oxidation observed in top sediment layers,
which was previously attributed to methane transported to the sediment
surface via rising gas bubbles (Treude et al., 2005a).
In the present study, we investigated sediments from within (< 30 cm b.s.f., on a seasonal basis) and below the sulfate reduction zone
(≪ 30 cm b.s.f., on one occasion) and the water column
(on a seasonal basis) at the time series station Boknis Eck in
Eckernförde Bay to validate the existence of SRZ methanogenesis and its
potential contribution to benthic methane emissions. Water column parameters
like oxygen, temperature, and salinity together with porewater geochemistry
and benthic methanogenesis were measured over the course of 2 years. In
addition to seasonal rate measurements, inhibition and stimulation
experiments, stable isotope probing, and molecular analysis were carried out
to find out if SRZ methanogenesis (1) is controlled by environmental
parameters, (2) shows seasonal variability, and/or (3) is based on noncompetitive
substrates with a special focus on methylotrophic methanogens.
Material and methods
Study site
Samples were taken at the time series station Boknis Eck (BE;
54∘31.15 ′N, 10∘02.18 ′E; http://www.bokniseck.de) located at
the entrance of Eckernförde Bay in the southwestern Baltic Sea with a
water depth of about 28 m (map of sampling site can be found in
Hansen et al., 1999). From mid-March
until mid-September the water column is strongly stratified due to the
inflow of saltier North Sea water and warmer and fresher surface water
(Bange et al., 2011). Organic matter degradation in the deep
layers causes pronounced hypoxia (March–September) or even anoxia
(August–September; Smetacek, 1985; Smetacek et
al., 1984). The source of organic material is phytoplankton blooms that
occur regularly in spring (February–March) and fall (September–November) and
are followed by the pronounced sedimentation of organic matter
(Bange et al., 2011). To a lesser extent, phytoplankton
blooms and sedimentation are also observed during the summer months
(July–August; Smetacek et al., 1984). Sediments at BE are
generally classified as soft, fine-grained muds (< 40 µm)
with a carbon content of 3 to 5 wt % (Balzer et al.,
1986). The bulk of organic matter in Eckernförde Bay sediments
originates from marine plankton and macroalgal sources
(Orsi et al., 1996), and its degradation
leads to the production of free methane gas (Wever and
Fiedler, 1995; Abegg and Anderson, 1997; Wever et al., 1998). The oxygen
penetration depth is limited to the upper few millimeters when bottom waters
are oxic (Preisler et al., 2007). Reducing
conditions within the sulfate reduction zone lead to a dark gray or black
sediment color with a strong hydrogen sulfur odor in the upper meter of the
sediment and a dark olive-green color in the deeper sediment layers (> 1 m; Abegg and Anderson, 1997).
Sampling months with bottom water (∼ 2 m above
the seafloor) temperature (Temp.), dissolved oxygen (O2), and dissolved
methane (CH4) concentration.
Sampling month
Date
Instrument
Temp.
O2
CH4
Type of
(∘C)
(µM)
(nM)
analysis
March 2013
13.03.2013
CTD
3
340
30
WC
MUC
All
June 2013
27.06.2013
CTD
6
94
125
WC
MUC
All
September 2013
25.09.2013
CTD
10
bdl
262*
WC
MUC
All
GC
GC-All
November 2013
08.11.2013
CTD
12
163
13
WC
MUC
All
March 2014
13.03.2014
CTD
4
209
41*
WC
MUC
All
June 2014
08.06.2014
CTD
7
47
61
WC
MUC
All
September 2014
17.09.2014
CTD
13
bdl
234
WC
MUC
All
MUC: multicorer, GC: gravity corer, CTD: CTD rosette, bdl: below
detection limit (5 µM), All: methane gas analysis, porewater
analysis, sediment geochemistry, net methanogenesis analysis,
hydrogenotrophic methanogenesis analysis, GC-All: analysis for gravity
cores including methane gas analysis, porewater analysis, sediment
geochemistry, hydrogenotrophic methanogenesis analysis, WC: water column
analyses including methane analysis, chlorophyll analysis.
* Concentrations from the regular monthly Boknis Eck sampling cruises on
24 September 2013 and 5 March 2014 (www.bokniseck.de).
Water column and sediment sampling
Sampling was done on a seasonal basis during the years 2013 and 2014.
One-day field trips with either RV Alkor (cruise no. AL410), RV
Littorina, or RV Polarfuchs were conducted in March, June, and September of
each year. In 2013, additional sampling was conducted in November. In each
sampling month, water profiles of temperature, salinity, and oxygen
concentration (optical sensor RINKO III; detection limit = 2 µM)
were measured with a CTD (Hydro-Bios). In addition, water samples for
methane concentration measurements were taken at 25 m of water depth with a
Niskin bottle (4 L each) rosette attached to the CTD (Table 1).
Complementary samples for water column chlorophyll were taken at 25 m of water
depth with the CTD rosette within the same months during standardized
monthly sampling cruises to Boknis Eck organized by GEOMAR.
Sediment cores were taken with a miniature multicorer (MUC; K.U.M. Kiel),
holding four core liners (length = 60 cm, diameter = 10 cm) at once. The
cores had an average length of ∼ 30 cm and were stored at
10 ∘C in a cold room (GEOMAR) until further processing (normally
within 1–3 days after sampling).
In September 2013, a gravity core was taken in addition to the MUC cores.
The gravity core was equipped with an inner plastic bag (polyethylene;
diameter: 13 cm). After core recovery (330 cm total length), the
polyethylene bag was cut open at 12 different sampling depths, resulting in
intervals of 30 cm, and sampled directly onboard for sediment porewater
geochemistry (see Sect. 2.4), sediment methane (see Sect. 2.5), sediment
solid-phase geochemistry (see Sect. 2.6), and microbial rate measurements
for hydrogenotrophic methanogenesis as described in Sect. 2.8.
Water column parameters
In each sampling month, water samples for methane concentration measurements
were taken at 25 m of water depth in triplicates. Therefore, three 25 mL glass
vials were filled bubble free directly after CTD rosette recovery and closed
with butyl rubber stoppers. Biological activity in samples was stopped by
adding saturated mercury chloride solution followed by storage at room
temperature until further treatment.
Concentrations of dissolved methane (CH4) were determined by headspace
gas chromatography as described in Bange et al. (2010).
Calibration for CH4 was done by using a two-point calibration with known
methane concentrations before the measurement of headspace gas samples,
resulting in an error of < 5 %.
Water samples for chlorophyll concentration were taken by transferring the
complete water volume (from 25 m water of depth) from one water sampler into a
4.5 L Nalgene bottle, from which approximately 0.7–1 L (depending on
the plankton content) were filtrated back in the GEOMAR laboratory using
a GF/F filter (Whatman; 25 mm diameter, 8 µM pores size). Dissolved
chlorophyll a concentrations were determined using the fluorometric method
described
by Welschmeyer (1994) with an error of < 10 %.
Sediment porewater geochemistry
Porewater was extracted from sediment within 24 h after core retrieval
using nitrogen (N2) pre-flushed rhizons (0.2 µm; Rhizosphere
Research Products; Seeberg-Elverfeldt et al., 2005). In MUC
cores, rhizons were inserted into the sediment in 2 cm intervals through
pre-drilled holes in the core liner. In the gravity core, rhizons were
inserted into the sediment in 30 cm intervals directly after retrieval.
Extracted porewater from MUC and gravity cores was immediately analyzed for
sulfide using standardized photometric methods (Grasshoff et
al., 1999).
Sulfate concentrations were determined using ion chromatography (Metrohm
761). Analytical precision was < 1 % based on repeated analysis
of IAPSO seawater standards (dilution series) with an absolute detection
limit of 1 µM corresponding to a detection limit of 30 µM for
the undiluted sample.
For analysis of dissolved inorganic carbon (DIC), 1.8 mL of porewater was
transferred into a 2 mL glass vial, fixed with 10 µL saturated
HgCL2 solution, and crimp sealed. DIC concentration was determined as
CO2 with a multi N/C 2100 analyzer (Analytik Jena) following the
manufacturer instructions. Therefore, the sample was acidified with
phosphoric acid and the outgassing CO2 was measured. The detection
limit was 20 µM with a precision of 2–3 %.
Sediment methane concentrations
In March 2013, June 2013, and March 2014, one MUC core was sliced in 1 cm
intervals until 6 cm b.s.f. followed by 2 cm intervals until the end of the
core. In the other sampling months, the MUC core was sliced in 1 cm
intervals until 6 cm b.s.f. followed by 2 cm intervals until 10 cm b.s.f. and 5 cm
intervals until the end of the core.
Per sediment depth (in MUC and gravity cores), 2 cm-3 of sediment were
transferred into a 10 mL glass vial containing 5 mL NaOH (2.5 %) for
the determination of sediment methane concentration per volume of sediment. The
vial was quickly closed with a butyl septum, crimp sealed, and shaken
thoroughly. The vials were stored upside down at room temperature until
measurement via gas chromatography. Therefore, 100 µL of headspace
was removed from the gas vials and injected into a Shimadzu gas
chromatograph (GC-2014) equipped with a packed Haysep-D column and a flame
ionization detector. The column temperature was 80 ∘C and the
helium flow was set to 12 mL min-1. CH4 concentrations were
calibrated against CH4 standards (Scotty gases). The detection limit
was 0.1 ppm with a precision of 2 %.
Sediment solid-phase geochemistry
Following the sampling for CH4, the same cores described under Sect. 2.5 were used for the determination of the sediment solid-phase
geochemistry, i.e., porosity, particulate organic carbon (POC), and
particulate organic nitrogen (PON).
The sediment porosity of each sampled sediment section was determined by the
weight difference of 5 cm-3 of wet sediment after freeze-drying for 24 h. Dried sediment samples were then used for analysis of particulate
organic carbon (POC) and particulate organic nitrogen (PON) with a
Carlo Erba element analyzer (NA 1500). The detection limit for C and N
analysis was < 0.1 dry weight percent (%) with a precision of
< 2 %.
Sediment methanogenesis
Methanogenesis in MUC cores
In each sampling month, three MUC cores were sliced in 1 cm intervals until
6 cm b.s.f., in 2 cm intervals until 10 cm b.s.f., and in 5 cm intervals until the
bottom of the core. Every sediment layer was transferred to a separate
beaker and quickly homogenized before subsampling. The exposure time with
air, i.e., oxygen, was kept to a minimum. Sediment layers were then sampled
for the determination of net methanogenesis (defined as the sum of total methane
production and consumption, including all available methanogenic substrates
in the sediment), hydrogenotrophic methanogenesis (methanogenesis based on
the substrates CO2 and H2), and potential methanogenesis
(methanogenesis at ideal conditions, i.e., no lack of nutrients) as described
in the following sections.
Net methanogenesis
Net methanogenesis was determined with sediment slurry experiments by
measuring the headspace methane concentration over time. Per sediment layer,
triplicates of 5 cm-3 of sediment were transferred into N2-flushed
sterile glass vials (30 mL) and mixed with 5 mL of filtered bottom water. The
slurry was repeatedly flushed with N2 to remove residual methane and to
ensure complete anoxia. Slurries were incubated in the dark at in situ
temperature, which varied for each sampling date (Table 1). Headspace samples
(0.1 mL) were taken out every 3–4 days over a time period of 4 weeks and
analyzed on a Shimadzu GC-2104 gas chromatograph (see Sect. 2.5). Net
methanogenesis rates were determined by the linear increase in the methane
concentration over time (minimum of six time points; see also Fig. S1 in the Supplement).
Hydrogenotrophic methanogenesis
To determine if hydrogenotrophic methanogenesis, i.e., methanogenesis based
on the competitive substrate H2, is present in the sulfate-reducing
zone, radioactive sodium bicarbonate (NaH14CO3) was added to the
sediment.
Per sediment layer, sediment was sampled in triplicates with glass tubes (5 mL) that were closed with butyl rubber stoppers on both ends according to
Treude et al. (2005b). Through
the stopper, NaH14CO3 (dissolved in water, injection volume 6 µL, activity 222 kBq, specific activity = 1.85–2.22 GBq mmol-1) was
injected into each sample and incubated for 3 days in the dark at
in situ temperature (Table 1). To stop bacterial activity, sediment was
transferred into 50 mL glass vials filled with 20 mL of sodium hydroxide (2.5 % w/w), closed quickly with rubber stoppers, and shaken thoroughly. Five
controls were produced from various sediment depths by injecting the
radiotracer directly into the NaOH with sediment.
The production of 14C-methane was determined with the slightly modified
method by Treude et al. (2005b) used for the determination of the anaerobic
oxidation of methane. The method was identical, except no unlabeled methane
was determined by using gas chromatography. Instead, DIC values were used to
calculate hydrogenotrophic methane production.
Potential methanogenesis in manipulated experiments
To examine the interaction between sulfate reduction and methanogenesis,
inhibition and stimulation experiments were carried out. Therefore, every
other sediment layer was sampled resulting in the following examined six
sediment layers: 0–1, 2–3, 4–5, 6–8, 10–15, and 20–25 cm. From
each layer, sediment slurries were prepared by mixing 5 mL of sediment in a
1:1
ratio with an adapted artificial seawater medium (salinity 24;
Widdel and Bak, 1992) in N2-flushed, sterile glass vials before
further manipulations.
In total, four different treatments, each in triplicates, were prepared per
depth: (1) with sulfate addition (17 mM), (2) with sulfate (17 mM) and
molybdate (22 mM) addition, (3) with sulfate (17 mM) and
2-bromoethanesulfonate (BES; 60 mM) addition, and (4) with sulfate (17 mM)
and methanol (10 mM) addition. From here on, the following names are used to
describe the different treatments, respectively: (1) control treatment, (2) molybdate treatment, (3) BES treatment, and (4) methanol treatment. Control
treatments feature the natural sulfate concentrations occurring in sediments
of the sulfate reduction zone at the sampling site. Molybdate was used as an
enzymatic inhibitor for sulfate reduction (Oremland and
Capone, 1988) and BES was used as an inhibitor for methanogenic Archaea
(Hoehler et al., 1994). Methanol is a known noncompetitive
substrate, which is used by methanogens but not by sulfate reducers
(Oremland and Polcin, 1982), and thus it is suitable to examine
noncompetitive methanogenesis. Treatments were incubated similar to net
methanogenesis (see the previous paragraph about net methanogenesis) by incubating sediment slurries at the respective
in situ temperature (Table 1) in the dark for a time period of 4 weeks.
Headspace samples (0.1 mL) were taken every 3–5 days over a time period of 4 weeks and potential methanogenesis rates were determined by the linear
increase in methane concentration over time (minimum of six time points).
Potential methylotrophic methanogenesis from methanol using stable isotope probing
One additional experiment was conducted with sediments from September 2014
by adding 13C-labeled methanol to investigate the production of
13C-labeled methane. Three cores were stored at 1 ∘C after
the September 2014 cruise until further processing ∼ 3.5 months later. The low storage temperature together with the expected oxygen
depletion in the enclosed supernatant water after the retrieval of the cores
likely led to slowed anaerobic microbial activity during storage time and
preserved the sediments for potential methanogenesis measurements.
Sediment cores were sliced in 2 cm intervals and the upper 0–2 cm b.s.f.
sediment layer of all three cores was combined in a beaker and homogenized.
Then, sediment slurries were prepared by mixing 5 cm-3 of sediment with
5 mL of artificial seawater medium in N2-flushed, sterile glass vials
(30 mL). After this, methanol was added to the slurry with a final
concentration of 10 mM (see also the previous paragraph about potential methanogenesis in manipulated experiments). Methanol was enriched with
13C-labeled methanol in a ratio of 1:1000 between 13C-labeled
(99.9 % 13C) and non-labeled methanol mostly consisting of 12C
(manufacturer: Roth). In total, 54 vials were prepared for nine different
sampling time points during a total incubation time of 37 days. All vials
were incubated at 13 ∘C (in situ temperature in September 2014) in
the dark. At each sampling point, six vials were stopped: one set of
triplicates was used for headspace methane and carbon dioxide determination and a second set of triplicates was used for porewater
analysis.
Headspace methane and carbon dioxide concentrations (volume 100 µL)
were determined on a Shimadzu gas chromatograph (GC-2014) equipped with a
packed Haysep-D column, a flame ionization detector, and a methanizer. The
methanizer (reduced nickel) reduces carbon dioxide with hydrogen to methane
at a temperature of 400 ∘C. The column temperature was
80 ∘C and the helium flow was set to 12 mL min-1. Methane
concentrations (including reduced CO2) were calibrated against methane
standards (Scotty gases). The detection limit was 0.1 ppm with a precision
of 2 %.
Analyses of the 13C / 12C ratios of methane and carbon dioxide were
conducted after headspace concentration measurements by using a continuous-flow combustion gas chromatograph (Trace Ultra; Thermo Scientific), which
was coupled to an isotope ratio mass spectrometer (MAT253; Thermo
Scientific). The isotope ratios of methane and carbon dioxide given in the
common delta notation (δ13C in permill) are reported relative
to Vienna Pee Dee Belemnite (VPDB) standard. Isotope precision was ±0.5 ‰ when measuring near the detection limit of 10 ppm.
For porewater analysis of methanol concentration and isotope composition,
each sediment slurry of the triplicates was transferred into argon-flushed
15 mL centrifuge tubes and centrifuged for 6 min at 4500 rpm. Then 1 mL
of filtered (0.2 µm) porewater was transferred into N2-flushed 2 mL
glass vials for methanol analysis, crimp sealed, and immediately frozen at
-20 ∘C. Methanol concentrations and isotope composition were
determined via high-performance liquid chromatography–ion ratio mass
spectrometry (HPLC-IRMS; Thermo Fisher Scientific) at the MPI Marburg. The
detection limit was 50 µM with a precision of
0.3 ‰.
Methanogenesis in the gravity core
Ex situ hydrogenotrophic methanogenesis was determined in a gravity core taken
in September 2013. The pathway is thought to be the main methanogenic
pathway in the sediment below the SMTZ in Eckernförde Bay
(Whiticar, 2002). Hydrogenotrophic methanogenesis was determined
using radioactive sodium bicarbonate (NaH14CO3). At every sampled
sediment depth (12 depths in 30 cm intervals), triplicate glass tubes (5 mL)
were inserted directly into the sediment. Tubes were filled bubble free with
sediment and closed with butyl rubber stoppers on both ends according to
Treude et al. (2005). The methods
following sampling were identical to those described in the previous paragraph about hydrogenotrophic methanogenesis.
Molecular analysis
During the non-labeled methanol treatment of the 0–1 cm b.s.f. horizon from the
September 2014 sampling (see also the previous paragraph about potential methanogenesis in manipulated experiments), additional samples were prepared to
detect and quantify the presence of methanogens in the sediment. Therefore,
an additional 15 vials were prepared with the addition of methanol as described in
the previous paragraph about potential methanogenesis in manipulated experiments for five different time points (day 1 (= t0), day 8, day 16,
day 22, and day 36) and stopped at each time point by transferring sediment
from the triplicate slurries into whirl-paks (Nasco), which then were
immediately frozen at -20 ∘C. DNA was extracted from
∼ 500 mg of sediment using the FastDNA® SPIN Kit
for Soil (Biomedical). The quantitative real-time polymerase chain reaction
(qPCR) technique using TaqMan probes and TaqMan chemistry (Life
Technologies) was used for the detection of methanogens on a ViiA7 qPCR
machine (Life Technologies). Primer and probe sets as originally published
by Yu et al. (2005) were applied to quantify the orders Methanobacteriales, Methanosarcinales, and
Methanomicrobiales along with the two families Methanosarcinaceae and Methanosaetaceae within the
order Methanosarcinales. In addition, a universal
primer set for the detection of the domain Archaea was used (Yu et al., 2005).
Parameters measured in the water column and sediment in the
Eckernförde Bay in each sampling month in the year 2013. Net
methanogenesis (MG) and hydrogenotrophic (hydr.) methanogenesis rates are
shown in triplicates with mean (solid line).
Absolute quantification of the 16S rDNA from the groups mentioned above was
performed with standard dilution series. The standard concentration reached
from 108 to 101 copies per µL. Quantification of the
standards and samples was performed in duplicates. Reaction was performed in
a final volume of 12.5 µL containing 0.5 µL of each primer
(10 pmol µL-1; MWG), 0.25 µL of the respective probe
(10 pmol µL-1; Life Technologies), 4 µL of H2O (Roth),
6.25 µL of TaqMan Universal Master Mix II (Life Technologies), and
1 µL of sample or standard. Cycling conditions started with an initial
denaturation and activation step for 10 min at 95 ∘C followed by
45 cycles of 95 ∘C for 15 s, 56 ∘C for 30 s, and
60 ∘C for 60 s. Non-template controls were run in duplicates
with water instead of DNA for all primer and probe sets and remained
without any detectable signal after 45 cycles.
Statistical analysis
To determine the possible environmental control parameters of SRZ
methanogenesis, a principal component analysis (PCA) was applied according
to the approach described in
Gier et al. (2016). Prior to
PCA, the dataset was transformed into ranks to ensure the same data
dimensions.
In total, two PCAs were conducted. The first PCA was used to test the
relation of parameters in the surface sediment (integrated methanogenesis
(0–5 cm, mmol m-2 d-1), POC content (average value from 0–5 cm b.s.f.,
wt %), C / N (average value from 0–5 cm b.s.f., molar) and the bottom water (25 m of water depth)
oxygen (µM), temperature (∘C), salinity
(PSU), chlorophyll (µg L-1), and methane (nM). The second PCA was
applied on depth profiles of sediment SRZ methanogenesis (nmol cm-3 d-1), sediment depth (cm), sediment POC content (wt %), sediment C / N
ratio (molar), and sampling month (one value per depth profile at a specific
month, the later in the year the higher the value).
For each PCA, biplots were produced to view data from different angles and
to graphically determine a potential positive, negative, or zero correlation
between methanogenesis rates and the tested variables.
Results
Water column parameters
From March 2013 to September 2014, the water column had pronounced
temporal and spatial variability in temperature, salinity, and oxygen (Figs. 2 and 3). In 2013, the temperature of the upper water column increased from
March (1 ∘C) to September (16 ∘C), but decreased again
in November (11 ∘C). The temperature of the lower water column
increased from March 2013 (2 ∘C) to November 2013 (12 ∘C). In 2014, the lowest temperatures of the upper and lower water column were
reached in March (4 ∘C). Warmer temperatures of the upper water
column were observed in June and September (around 17 ∘C), while
the lower water column peaked in September (13 ∘C).
Salinity increased over time during 2013, showing the highest salinity of
the upper and lower water column in November (18 and 23 PSU, respectively).
In 2014, the salinity of the upper water column was highest in March and
September (both 17 PSU) and lowest in June (13 PSU). The salinity of the
lower water column increased from March 2014 (21 PSU) to September 2014
(25 PSU).
In both years, June and September showed the most pronounced vertical
gradient of temperature and salinity, featuring a pycnocline at around
∼ 14 m of water depth.
Summer stratification was also seen in the O2 profiles, which showed
O2 depleted conditions (O2 < 150 µM) in the lower
water column from June to September in both years, reaching concentrations
below 1–2 µM (detection limit of CTD sensor) in September of both
years (Figs. 2 and 3). The water column was completely ventilated, i.e.,
homogenized, in March of both years with O2 concentrations of 300–400 µM down to the seafloor at about 28 m.
Sediment geochemistry in MUC cores
Sediment porewater and solid-phase geochemistry results for the years 2013
and 2014 are shown in Figs. 2 and 3, respectively.
Sulfate concentrations at the sediment surface ranged between 15 and 20 mM.
The concentration decreased with depth in all sampling months but was never
fully depleted until the bottom of the core (18–29 cm b.s.f.; between 2 and 7 mM
sulfate). November 2013 showed the strongest decrease from ∼ 20 mM at the top to ∼ 2 mM at the bottom of the core (27 cm b.s.f.).
Opposite to sulfate, the methane concentration increased with sediment depth in
all sampling months (Figs. 2 and 3). Over the course of a year (i.e., March to
November in 2013 and March to September in 2014), the maximum methane
concentration increased, reaching the highest concentration in November 2013
(∼ 1 mM at 26 cm b.s.f.) and September 2014 (0.2 mM at
23 cm b.s.f.).
Simultaneously, methane profiles became steeper, revealing
higher methane concentrations at a shallower sediment depth late in the year.
The magnitudes of methane concentrations were similar in the respective months
of 2013 and 2014.
Parameters measured in the water column and sediment in the
Eckernförde Bay in each sampling month in the year 2014. Net
methanogenesis (MG) and hydrogenotrophic (hydr.) methanogenesis rates are
shown in triplicates with mean (solid line).
In all sampling months, the sulfide concentration increased with sediment depth
(Figs. 2 and 3). Similar to methane, sulfide profiles revealed higher sulfide
concentrations at a shallower sediment depth together with higher peak
concentrations over the course of the sampled months in each sampling year.
Accordingly, November 2013 (10.5 mM at 15 cm b.s.f.) and September 2014 (2.8 mM
at 15 cm b.s.f.) revealed the highest sulfide concentrations.
September 2014 was the only sampling month showing a pronounced decrease in
sulfide concentration from 15 to 21 cm b.s.f. of over 50 %.
DIC concentrations increased with increasing sediment depth in all sampling
months. Concomitant with the highest sulfide concentrations, the highest DIC
concentration was detected in November 2013 (26 mM at 27 cm b.s.f.). At the
surface, DIC concentrations ranged between 2 and 3 mM in all sampling months. In
June of both years, DIC concentrations were lowest at the deepest sampled
depth compared to the other sampling months (16 mM in 2013, 13 mM in 2014).
In all sampling months, POC profiles scattered around 5 ± 0.9 wt %
with depth. Only in November 2013, June 2014, and September 2014 did POC content
exceed 5 wt % in the upper 0–1 cm b.s.f. (5.9, 5.2, and 5.3 wt %,
respectively) with the highest POC content in November 2013. Also in
November 2013, the surface C / N ratio (0–1 cm b.s.f.) of the particulate organic
matter was the lowest of all sampling months (8.6). In general, the C / N ratio
increased with depth in both years with values around 9 at the surface and
values around 10–11 at the deepest sampled sediment depths.
Parameters measured in the sediment gravity core taken in the
Eckernförde Bay in September 2013. Hydrogenotrophic (hydr.)
methanogenesis rates are shown in triplicates with mean (solid line).
Sediment geochemistry in gravity cores
Results from sediment porewater and solid-phase geochemistry in the gravity
core from September 2013 are shown in Fig. 4. Please note that the sediment
depth of the gravity core was corrected by comparing the sulfate
concentrations at 0 cm b.s.f. in the gravity core with the corresponding sulfate
concentration and depth in the MUC core from September 2013 (Fig. 2). The
soft surface sediment is often lost during the gravity coring procedure.
Through this correction, the topmost layer of the gravity core was set at a
depth of 14 cm b.s.f.
Porewater sulfate concentration in the gravity core decreased with depth
(i.e., below 0.1 mM at 107 cm b.s.f.) and stayed below 0.1 mM until 324 cm b.s.f.
Sulfate increased slightly (1.9 mM) at the bottom of the core (345 cm b.s.f.).
In concert with sulfate, methane, sulfide, DIC, POC, and C / N profiles
also showed distinct alteration in the profile at 345 cm b.s.f. (see below, Fig. 4).
As fluid seepage has not been observed at the Boknis Eck station
(Schlüter et al., 2000), these alterations
could either indicate a change in sediment properties or result from a
sampling artifact from the penetration of seawater through the core catcher
into the deepest sediment layer. The latter process is, however, not
expected to considerably affect sediment solid-phase properties (POC and
C / N), and we therefore dismissed this hypothesis.
The methane concentration increased steeply with depth, reaching a maximum of 4.8 mM at 76 cm b.s.f.
The concentration stayed around 4.7 mM until 262 cm b.s.f. followed
by a slight decrease until 324 cm b.s.f. (2.8 mM). From 324 to 345 cm b.s.f.
methane increased again (3.4 mM).
Both sulfide and DIC concentrations increased with depth, showing a maximum
at 45 (∼ 5 mM) and 345 cm b.s.f. (∼ 1 mM),
respectively. While sulfide decreased after 45 cm b.s.f. to a minimum of
∼ 300 µM at 324 cm b.s.f., it slightly increased again to
∼ 1 mM at 345 cm b.s.f. In accordance, DIC concentrations showed
a distinct decrease between 324 and 345 cm b.s.f. (from 45 to 39 mM).
While POC contents varied around 5 wt % throughout the core, the C / N ratio
slightly increased with depth, revealing the lowest ratio at the surface
(∼ 3) and the highest ratio at the bottom of the core
(∼ 13). However, both POC and C / N showed a distinct increase
from 324 to 345 cm b.s.f.
Methanogenesis activity in MUC cores
Net methanogenesis
Net methanogenesis activity (calculated by the linear increase of methane
over time; see Fig. S1) was detected throughout the cores in all sampling
months (Figs. 2 and 3). Activity measured in MUC cores increased over the
course of the year in 2013 and 2014 (that is, March to November in 2013 and
March to September in 2014) with lower rates mostly < 0.1 in March and higher rates
> 0.2 nmol cm-3 d-1 in November 2013 and September 2014. In general,
November 2013 revealed the highest net methanogenesis rates (1.3 nmol cm-3 d-1 at 1–2 cm b.s.f.). Peak rates were detected at the sediment surface
(0–1 cm b.s.f.) in all sampling months except for September 2013 when the
maximum rates were situated between 10 and 15 cm b.s.f. In addition to the surface
peaks, net methanogenesis showed subsurface (= below 1 until 30 cm b.s.f.) maxima in all sampling months, but with alternating depths (between
10 and 25 cm b.s.f.).
A comparison of the integrated net methanogenesis rates (0–25 cm b.s.f.) revealed
the highest rates in September and November 2013 (0.09
and 0.08 mmol m-2 d-1, respectively) and the lowest rates in March
2014 (0.01 mmol m-2 d-1; Fig. 5). A trend of increasing areal net
methanogenesis rates from March to September was observed in both years.
Integrated net methanogenesis (MG) rates (determined by net
methane production) and hydrogenotrophic MG rates (determined by radiotracer
incubation) in the surface sediments (0–25 cm b.s.f.) of Eckernförde Bay for
different sampled time points.
Hydrogenotrophic methanogenesis
Hydrogenotrophic methanogenesis activity determined by 14C-bicarbonate
incubations of MUC cores is shown in Figs. 2 and 3. In 2013, maximum activity
ranged between 0.01 and 0.2 nmol cm-3 d-1, while in 2014 maxima ranged
only between 0.01 and 0.05 nmol cm-3 d-1. In comparison, maximum
hydrogenotrophic methanogenesis was up to 2 orders of magnitude lower
compared to net methanogenesis. Only in March 2013 did both activities reach a
similar range.
Overall, hydrogenotrophic methanogenesis increased with depth in March,
September, and November 2013 and in March, June, and September 2014. In June
2013, activity decreased with depth, showing the highest rates in the upper
0–5 cm b.s.f. and the lowest at the deepest sampled depth.
Concomitant with integrated net methanogenesis, integrated hydrogenotrophic
methanogenesis rates (0–25 cm b.s.f.) were high in September 2013, with slightly
higher rates in March 2013 (Fig. 5). The lowest areal rates of hydrogenotrophic
methanogenesis were seen in June of both years.
Hydrogenotrophic methanogenesis activity in the gravity core is shown in
Fig. 4. The highest activity (∼ 0.7 nmol cm-3 d-1) was
measured at 45 and 138 cm b.s.f. followed by a decrease with increasing
sediment depth reaching 0.01 nmol cm-3 d-1 at the deepest
sampled depth (345 cm b.s.f.).
Potential methanogenesis rates versus sediment depth in sediment
sampled in November 2013, March 2014, June 2014, and September 2014.
Presented are four different types of incubations (treatments): control (blue
symbols) describes the treatment with sediment plus artificial seawater
containing natural salinity (24 PSU) and sulfate concentrations (17 mM),
molybdate (green symbols) is the treatment with the addition of molybdate (22 mM), BES
(purple symbols) is the treatment with 60 mM BES addition, and methanol (red
symbols) is the treatment with the addition of 10 mM of methanol. Shown are
triplicates per depth interval and the mean as a solid line. Please note the
different x axis for the methanol treatment (red).
Potential methanogenesis in manipulated experiments
Potential methanogenesis rates in manipulated experiments included either
the addition of inhibitors (molybdate for the inhibition of sulfate reduction or
BES for the inhibition of methanogenesis) or the addition of a noncompetitive
substrate (methanol). Control treatments were run with neither the addition
of inhibitors nor the addition of methanol.
Controls. Potential methanogenesis activity in the control treatments was below 0.5 nmol cm-3 d-1
from March 2014 to September 2014 (Fig. 6). Only in
November 2013 did control rates exceed 0.5 nmol cm-3 d-1 below 6 cm b.s.f. While rates increased with depth in November 2013 and June 2014, they
decreased with depth in the other two sampling months.
Molybdate. Peak potential methanogenesis rates in the molybdate treatments were found
in the uppermost sediment interval (0–1 cm b.s.f.) in almost every sampling
month with rates being 3–30 times higher compared to the control treatments
(< 0.5 nmol cm-3 d-1). In November 2013, potential
methanogenesis showed two maxima (0–1 and 10–15 cm b.s.f.). The highest measured
rates were found in September 2014 (∼ 6 nmol cm-3 d-1) followed by November 2013 (∼ 5 nmol cm-3 d-1).
BES. Profiles of potential methanogenesis in the BES treatments were similar to
the controls mostly in the lower range < 0.5 nmol cm-3 d-1. Only in November 2013 did rates exceed 0.5 nmol cm-3 d-1.
Rates increased with depth in all sampling months, except for September
2014, when the highest rates were found at the sediment surface (0–1 cm b.s.f.).
Methanol. In all sampling months, potential rates in the methanol treatments were
3 orders of magnitude higher compared to the control treatments
(< 0.5 nmol cm-3 d-1). Except for November 2013, potential
methanogenesis rates in the methanol treatments were highest in the upper
0–5 cm b.s.f. and decreased with depth. In November 2013, the highest rates were
detected at the deepest sampled depth (20–25 cm b.s.f.).
Potential methanogenesis followed by 13C-methanol labeling
Total methanol concentrations (labeled and unlabeled)
in the sediment decreased sharply in the first 2 weeks from ∼ 8 mM at day 1 to 0.5 mM at day 13 (Fig. 7). At day 17, methanol was below
the detection limit. In the first 2 weeks, residual methanol was enriched
with 13C, reaching ∼ 200 ‰ at day 13.
Over the same time period, the methane content in the headspace increased
from 2 ppmv at day 1 to ∼ 66 000 ppmv at day 17 and stayed
around that value until the end of the total incubation time (until day 37; Fig. 7). The carbon isotopic signature of methane (δ13CCH4) showed a clear enrichment of the heavier isotope 13C
(Table 3) from day 9 to 17 (no methane was detectable at day 1). After day
17, δ13CCH4 stayed around 13 ‰ until
the end of the incubation. The content of CO2 in the headspace
increased from ∼ 8900 ppmv at day 1 to ∼ 29 000 ppmv at day 20 and stayed around 30 000 ppmv until the end of the incubation
(Fig. 7). Please note that the majority of CO2 was dissolved in the
porewater, and thus the CO2 content in the headspace does not show the
total CO2 abundance in the system. CO2 in the headspace was
enriched with 13C during the first 2 weeks (from -16.2 to -7.3 ‰) but then stayed around -11 ‰
until the end of the incubation.
Development of headspace gas content and isotope composition of
methane (CH4) and carbon dioxide (CO2) as well as porewater methanol
(CH3OH) concentration and isotope composition during the 13C-labeling
experiment (with sediment from the 0–2 cm b.s.f. horizon in September 2014) with
the addition of 13C-enriched methanol (13C:12C = 1:1000).
(a) Concentrations of porewater methanol (CH3OH) and headspace content of
methane (CH4) and carbon dioxide (CO2) over time. (b) Isotope
composition of porewater CH3OH, headspace CH4, and headspace
CO2 over time. Shown are means (from triplicates) with standard
deviation.
Molecular analysis of benthic methanogens
In September 2014, additional samples were run during the methanol treatment
(see Sect. 2.7.) for the detection of benthic methanogens via qPCR. The qPCR
results are shown in Fig. 8. For a better comparison, the microbial
abundances are plotted together with the sediment methane concentrations
from the methanol treatment, from which the rate calculation for the
methanol-methanogenesis at 0–1 cm b.s.f. was done (shown in Fig. 6).
Sediment methane concentrations increased over time, revealing a slow
increase in the first ∼ 10 days followed by a steep increase
between day 13 and day 20 and ending in a stationary phase.
A similar increase was seen in the abundance of total and methanogenic
Archaea. Total Archaea abundances increased sharply in the second week of
the incubation, reaching a maximum at day 16 (∼ 5000 × 106 copies g-1), and stayed around
3000 × 106–4000 × 106 copies g-1 over the course of the incubation. Similarly, methanogenic archaea,
namely the order Methanosarcinales and within this order the family Methanosarcinaceae, showed a sharp increase
in the first 2 weeks as well with the highest abundances at day 16
(∼ 6 × 108 and ∼ 1 × 106 copies g-1, respectively). Until the end of the incubation,
the abundances of Methanosarcinales and Methanosarcinaceae decreased to about one-third of their maximum
abundances (∼ 2 × 108 and ∼ 0.4 × 106 copies g-1, respectively).
Sediment methane concentrations (with sediment from the 0–1 cm b.s.f.
in September 2014) over time in the treatment with the addition of methanol (10 mM) are shown above. Shown are triplicate values per measurement. DNA copies
of Archaea, Methanosarcinales, and Methanosarcinaceae are shown below in duplicates per measurement. Please note the
secondary y axis for Methanosarcinales and Methanosarcinaceae. More data are available for methane (determined in
the gas headspace) than from DNA samples (taken from the sediment) as sample
volume for molecular analyzes was limited.
Statistical analysis
The PCA of integrated SRZ methanogenesis (0–5 cm b.s.f.; Fig. 10) showed a
positive correlation with bottom water temperature (Fig. 10a), bottom water
salinity (Fig. 10a), bottom water methane (Fig. 10b), surface sediment POC
content (0–5 cm b.s.f.; Fig. 10c), and surface sediment C / N (0–5 cm b.s.f.; Fig. 10b). A negative correlation was found with bottom water oxygen
concentration (Fig. 10b). No correlation was found with bottom water
chlorophyll.
The PCA of methanogenesis depth profiles showed positive correlations with
sediment depth (Fig. 11a) and C / N (Fig. 11b), and it showed negative
correlations with POC (Fig. 11a).
Discussion
Methanogenesis in the sulfate-reducing zone
On the basis of the results presented in Figs. 2 and 3, it is evident that
methanogenesis and sulfate reduction were concurrently active in the sulfate
reduction zone (0–30 cm b.s.f.) at Boknis Eck. Even though sulfate reduction
activity was not directly determined, the decrease in sulfate concentrations
with a concomitant increase in sulfide within the upper 30 cm b.s.f. clearly
indicated its presence (Figs. 2 and 3). Several previous studies confirmed
the high activity of sulfate reduction in the surface sediment of
Eckernförde Bay, revealing rates up to 100–10 000 nmol cm-3 d-1 in the upper 25 cm b.s.f.
(Treude et al., 2005a; Bertics
et al., 2013; Dale et al., 2013). The microbial fermentation of organic matter
was probably high in the organic-rich sediments of Eckernförde Bay (POC
contents of around 5 %; Figs. 2 and 3), providing high substrate
availability and variety for methanogenesis.
Temporal development of integrated net surface methanogenesis (0–5 cm b.s.f.) in the sediment and chlorophyll (green) and methane concentrations
(orange) in the bottom water (25 m). Methanogenesis (MG) rates and methane
concentrations are shown in means (from triplicates) with standard
deviation.
Principal component analysis (PCA) from three different angles of
integrated surface methanogenesis (0–5 cm b.s.f.) and surface particulate
organic carbon averaged over 0–5 cm b.s.f. (surface sediment POC), surface C / N
ratio averaged over 0–5 cm b.s.f. (surface sediment C / N), bottom water salinity,
bottom water temperature (T), bottom water methane (CH4), bottom water
oxygen (O2), and bottom water chlorophyll. Data were transformed into
ranks before analysis. (a) Correlation biplot of principal components 1 and
2, (b) correlation biplot of principal components 1 and 3, and (c) correlation
biplot of principal components 2 and 3. Correlation biplots are shown in a
multidimensional space with parameters shown as green lines and samples
shown as black dots. Parameters pointing in the same direction are
positively related; parameters pointing in the opposite direction are
negatively related.
The results of this study further identified methylotrophy to be a
potentially important noncompetitive methanogenic pathway in the
sulfate-reducing zone. The pathway utilizes alternative substrates, such as
methanol, to bypass competition with sulfate reducers for H2 and
acetate. The potential for methylotrophic methanogenesis within the
sulfate-reducing zone was supported by the following observations.
Hydrogenotrophic methanogenesis was up to 2 orders of magnitude lower
compared to net methanogenesis, resulting in insufficient rates to explain
the observed net methanogenesis in the upper 0–30 cm b.s.f. (Figs. 2 and 3). This
finding points towards the presence of alternative methanogenic processes in
the sulfate reduction zone, such as methylotrophic methanogenesis.
Methanogenesis increased when sulfate reduction was inhibited by molybdate,
confirming the inhibitory effect of sulfate reduction on methanogenesis with
competitive substrates (H2 and acetate; Oremland and Polcin, 1982; King
et al., 1983; Fig. 6). Consequently, the usage of noncompetitive substrates
was preferred in the sulfate reduction zone (especially in the upper
0–1 cm b.s.f.;
Fig. 6). Accordingly, hydrogenotrophic methanogenesis increased at depths
at which sulfate was depleted and thus the competitive situation was relieved
(Fig. 4).
The addition of BES did not result in the inhibition of methanogenesis,
indicating the presence of unconventional methanogenic groups using
noncompetitive substrates (Fig. 7). The unsuccessful inhibition by BES can
be explained by either incomplete inhibition or the fact that the
methanogens were insensitive to BES
(Hoehler et al., 1994; Smith and Mah,
1981; Santoro and Konisky, 1987). The BES concentration applied in the
present study (60 mM) has been shown to result in the successful inhibition of
methanogens in previous studies (Hoehler et al., 1994).
Therefore, the presence of methanogens that are insensitive to BES is more
likely. The insensitivity to BES in methanogens is explained by heritable
changes in BES permeability or the formation of BES-resistant enzymes
(Smith and Mah, 1981; Santoro and Konisky,
1987). Such BES resistance was found in Methanosarcina mutants
(Smith and Mah, 1981; Santoro and Konisky,
1987). This genus was successfully detected in our samples (for more details
see point 5) and is known for mediating the methylotrophic pathway
(Keltjens and Vogels, 1993), supporting our hypothesis on the
utilization of noncompetitive substrates by methanogens.
The addition of methanol to sulfate-rich sediments increased methanogenesis
rates by up to 3 orders of magnitude, confirming the potential of the
methanogenic community to utilize noncompetitive substrates, especially in
the 0–5 cm b.s.f. sediment horizon (Fig. 6). At this sediment depth either the
availability of noncompetitive substrates, including methanol, was highest
(derived from fresh organic matter), or the usage of noncompetitive
substrates was increased due to the high competitive situation as sulfate
reduction is most active in the 0–5 cm b.s.f. layer
(Treude et al., 2005a; Bertics et al., 2013).
It should be noted that even though methanogenesis rates were calculated
assuming a linear increase in methane concentration over the entire
incubation to make a better comparison between different treatments, the
methanol treatments generally showed a delayed response in methane
development (Figs. 8, S2). We suggest that this delayed
response was a reflection of cell growth by methanogens utilizing the
surplus methanol. We are therefore unable to decipher whether methanol plays
a major role as a substrate in the Eckernförde Bay sediments compared to
possible alternatives, as its concentration is relatively low in the natural
setting (∼ 1 µM between 0 and 25 cm b.s.f., June 2014
sampling; Zhuang, unpublished data). It is conceivable that other
noncompetitive substrates, such as methylated sulfides (e.g., dimethyl
sulfide or methanethiol), are more relevant for the support of SRZ
methanogenesis.
Methylotrophic methanogens of the order Methanosarcinales were detected in the
methanol treatment (Fig. 8), confirming the presence of methanogens that
utilize noncompetitive substrates in the natural environment (Boone et al.,
1993; Fig. 8). The delay in the growth of Methanosarcinales moreover hints towards the
predominant usage of noncompetitive substrates other than methanol (see
also point 4).
Stable isotope probing revealed highly 13C-enriched methane produced
from 13C-labeled methanol, further confirming the potential of the
methanogenic community to utilize noncompetitive substrates (Fig. 7). The
production of both methane and CO2 from methanol has been shown
previously in different strains of methylotrophic methanogens
(Penger et al., 2012). The fast conversion
of methanol to methane and CO2 (methanol was consumed completely in 17 days) hints towards the presence of methylotrophic methanogens
(e.g.,
members of the family Methanosarcinaceae, which is known for the methylotrophic
pathway;
Keltjens and Vogels, 1993). Please note, however, that the
storage of the cores (3.5 months) prior to sampling could have led to shifts
in the microbial community and thus might not reflect the in situ conditions of
the original microbial community in September 2014. The delay in methane
production also seen in the stable isotope experiment was, however, only
slightly different (methane developed earlier between day 8 and 12; data
not shown) from the non-labeled methanol treatment (between day 10 and 16;
Fig. S2), which leads us to the assumption that the storage time at
1 ∘C did not dramatically affect the methanogen community.
Similar to a previous study with arctic sediments, the addition of substrates
had no stimulatory effect on the rate of methanogenesis or on the methanogen
community structure at low temperatures (5 ∘C;
Blake et al., 2015).
Principal component analysis (PCA) from two different angles of
net methanogenesis depth profiles and sampling month (Month), sediment
depth, and depth profiles of particulate organic carbon (POC) and C / N ratio
(C / N). Data were transformed into ranks before analysis. (a) Correlation
biplot of principal components 1 and 2 and (b) correlation biplot of
principal
components 1 and 3. Correlation biplots are shown in a multidimensional
space with parameters shown as green lines and samples shown as black dots.
Parameters pointing in the same direction are positively related;
parameters pointing in the opposite direction are negatively related.
Environmental control of methanogenesis in the sulfate reduction
zone
SRZ methanogenesis in Eckernförde Bay sediments showed variations
throughout the sampling period, which may be influenced by variable
environmental factors such as temperature, salinity, oxygen, and organic
carbon. In the following, we will discuss the potential impact of those
factors on the magnitude and distribution of SRZ methanogenesis.
Temperature
During the sampling period, bottom water temperatures increased over the
course of the year from late winter (March, 3–4 ∘C) to autumn
(November, 12 ∘C; Figs. 2 and 3). The PCA revealed a positive
correlation between bottom water temperature and integrated SRZ
methanogenesis (0–5 cm b.s.f.). A temperature experiment conducted with sediment
from ∼ 75 cm b.s.f. in September 2014 within a parallel study
revealed a mesophilic temperature optimum of methanogenesis (20 ∘C; data not shown). Whether methanogenesis in the sulfate reduction zone
(0–30 cm) has the same physiology remains speculative. However, AOM
organisms, which are closely related to methanogens (Knittel and
Boetius, 2009), studied in the sulfate reduction zone from the same site
were confirmed to have a mesophilic physiology, too
(Treude et al., 2005a). The sum of these aspects leads us to
the conceivable conclusion that SRZ methanogenesis activity in the
Eckernförde Bay is positively impacted by temperature increases. Such a
correlation between benthic methanogenesis and temperature has been found in
several previous studies from different environments
(Sansone and Martens, 1981; Crill and
Martens, 1983; Martens and Klump, 1984).
Salinity and oxygen
From March 2013 to November 2013 and from March 2014 to September 2014,
salinity increased in the bottom-near water (25 m) from 19 to 23 and
from 22 to 25 PSU (Figs. 2 and 3), respectively, due the pronounced summer
stratification in the water column between saline North Sea water and less
saline Baltic Sea water (Bange et al., 2011). The PCA
detected a positive correlation between integrated SRZ methanogenesis (0–5 cm b.s.f.) and salinity in the bottom-near water (Fig. 10a). This correlation
can hardly be explained by salinity alone, as methanogens feature a broad
salinity range from freshwater to hypersaline (Zinder, 1993). It is more
likely that salinity serves as an indicator of water column stratification,
which is often correlated with low O2 concentrations in the
Eckernförde Bay (Fig. S3, Bange et
al., 2011; Bertics et al., 2013). Methanogenesis is sensitive to O2
(Oremland, 1988; Zinder, 1993), and hence conditions might be
more favorable during hypoxic or anoxic events, particularly in the sediment
closest to the sediment–water interface, but potentially also in deeper
sediment layers due to the absence of bioturbating and bioirrigating infauna
(Dale et al., 2013; Bertics et al.,
2013), which could introduce O2 beyond diffusive transport.
Accordingly, the PCA revealed a negative correlation between O2
concentration close to the seafloor and SRZ methanogenesis.
Particulate organic carbon
The supply of particulate organic carbon (POC) is one of the most important
factors controlling benthic heterotrophic processes, as it determines
substrate availability and variety (Jørgensen, 2006). In
Eckernförde Bay, the organic material reaching the seafloor originates
mainly from phytoplankton blooms in spring, summer, and autumn
(Bange et al., 2011). It has been estimated that
> 50 % in spring (February–March), < 25 % in summer
(July–August), and > 75 % in autumn (September–October) of
these blooms is reaching the seafloor (Smetacek et al.,
1984), resulting in an overall high organic carbon content of the sediment (5 wt %), which leads to high benthic microbial degradation rates including
sulfate reduction and methanogenesis
(Whiticar, 2002; Treude et al., 2005a;
Bertics et al., 2013). Previous studies revealed that high organic matter
availability can relieve competition between sulfate reducers and
methanogens in sulfate-containing marine sediments
(Oremland
et al., 1982; Holmer and Kristensen, 1994; Treude et al., 2009; Maltby et
al., 2016).
To determine the effect of POC concentration and C / N ratio (the latter as a
negative indicator for the freshness of POC) on SRZ methanogenesis, two PCAs
were conducted with (a) the focus on the upper 0–5 cm b.s.f., which is directly
influenced by freshly sedimented organic material from the water column
(Fig. 10), and (b) the focus on the depth profiles throughout the sediment
cores (up to 30 cm b.s.f.; Fig. 11).
Effect of POC and C / N ratio in the upper 0–5 cm b.s.f.
For the upper 0–5 cm b.s.f. in the sediment, a positive correlation was found
between SRZ methanogenesis (integrated) and POC content (averaged; Fig. 10c), indicating that POC content is an important controlling factor for
methanogenesis in this layer. In support, the highest bottom-near water
chlorophyll concentrations coincided with the highest bottom-near water methane
concentrations and high integrated SRZ methanogenesis (0–5 cm b.s.f.) in
September 2013, probably as a result of the sedimentation of the summer
phytoplankton bloom (Fig. 9). Indeed, the PCA revealed a positive
correlation between integrated SRZ methanogenesis rates and bottom-near
water methane concentrations (Fig. 10b) when viewed over all investigated
months. However, no correlation was found between bottom water chlorophyll
and integrated SRZ methanogenesis rates (Fig. 10). As seen in Fig. 9,
bottom-near high chlorophyll concentrations did not coincide with high
bottom-near methane concentration in June–September 2014. We explain this
result by a time lag between primary production in the water column and the
export of the produced organic material to the seafloor, which was probably
even more delayed during stratification. Such a delay was observed in a
previous study (Bange et al., 2010), revealing an enhanced water
methane concentration close to the seafloor approximately 1 month after
the chlorophyll maximum. The C / N ratio (averaged over 0–5 cm b.s.f.) also showed
no correlation with integrated methanogenesis from the same depth layer (0–5 cm b.s.f.), which is surprising as we expected that a higher C / N ratio
indicative of less labile organic carbon would have a negative effect on
noncompetitive methanogenesis. However, methanogens are not able to
directly use most of the labile organic matter due to their inability to
process large molecules (more than two C–C bondings; Zinder,
1993). Methanogens are dependent on other microbial groups to degrade large
organic compounds (e.g., amino acids) for them (Zinder, 1993).
Because of this substrate speciation and dependence, a delay between the
sedimentation of fresh, labile organic matter and the increase in
methanogenesis can be expected, which would not be captured by the applied
PCA.
Effect of POC and C / N ratio over 0–30 cm b.s.f.
In the PCA for the sediment profiles from the sulfate reduction zone (0–30 cm b.s.f.), POC showed a negative correlation with methanogenesis and sediment
depth, while C / N ratio showed a positive correlation with methanogenesis and
sediment depth (Fig. 11). Given that POC remained basically unchanged over
the top 30 cm b.s.f. with the exception of the topmost sediment layer, its
negative correlation with methanogenesis is probably solely explained by the
increase in methanogenesis with sediment depth and can therefore be
excluded as a major controlling factor. As sulfate in this zone was likely
never depleted to levels that critically limit sulfate reduction
(lowest concentration 1300 µM; compare with
Treude et al., 2014), we do not expect a significant change
in the competition between methanogens and sulfate reducers. It is therefore
more likely that the progressive degradation of labile POC into dissolvable
methanogenic substrates over depth and time had a positive impact on
methanogenesis. The C / N ratio indicates such a trend as the labile fraction
of POC decreased with depth.
Relevance of methanogenesis in the sulfate reduction zone of
Eckernförde Bay sediments
The time series station Boknis Eck in Eckernförde Bay is known for being
a methane source to the atmosphere throughout the year due to supersaturated
waters, which result from significant benthic methanogenesis and emission
(Bange et al., 2010). The benthic methane formation is
thought to take place mainly in sediments below the SMTZ
(Treude et al., 2005a; Whiticar, 2002).
In the present study, we show that SRZ methanogenesis within the sulfate
zone is present despite sulfate concentrations > 1 mM, a limit
above which methanogenesis has been thought to be negligible
(Alperin
et al., 1994; Hoehler et al., 1994; Burdige, 2006), and could thus
contribute to benthic methane emissions. In support of this hypothesis, a high
dissolved methane concentration in the water column occurred with
concomitantly high SRZ methanogenesis activity (Fig. 9). However, whether the
observed water column methane originated from SRZ methanogenesis, from gas
ebullition caused by methanogenesis below the SMTZ, or a mixture of both
remains speculative.
How much of the methane produced in the surface sediment is ultimately
emitted into the water column depends on the rate of methane consumption,
i.e., the aerobic and anaerobic oxidation of methane in the sediment
(Knittel and Boetius, 2009; Fig. 1). In organic-rich sediments,
such as in the present study, the oxygenated sediment layer is often only
millimeters thick due to the high O2 demand of microorganisms during organic
matter degradation (Jørgensen, 2006;
Preisler et al., 2007). Thus, the anaerobic oxidation of methane (AOM) might
play a more important role for methane consumption in the studied
Eckernförde Bay sediments. In an earlier study from this site, AOM
activity was detected throughout the top 0–25 cm b.s.f., which included zones
that were well above the actual SMTZ (Treude et al., 2005a).
But the authors concluded that methane oxidation was completely fueled by
methanogenesis from below sulfate penetration, as integrated AOM rates
(0.8–1.5 mmol m-2 d-1) were in the same range as the predicted
methane flux (0.66–1.88 mmol m-2 d-1) into the SMTZ.
Together with the dataset presented here we postulate that AOM above the
SMTZ (0.8 mmol m-2 d-1; Treude et al., 2005a)
could be partially or entirely fueled by SRZ methanogenesis. A similar close
coupling between methane oxidation and methanogenesis in the absence of
definite methane profiles was recently proposed from isotopic labeling
experiments with sediments from the sulfate reduction zone of the nearby
Aarhus Bay in Denmark (Xiao et al., 2017). It is
therefore likely that such a cryptic methane cycling also occurs in the
sulfate reduction zone of sediments in the Eckernförde Bay. If, in an
extreme scenario, SRZ methanogenesis represented the only methane source
for AOM above the SMTZ, then maximum SRZ methanogenesis could be on the
order of 1.6 mmol m-2 d-1 (1.5 mmol m-2 d-1 AOM +
0.09 mmol m-2 d-1 net SRZ methanogenesis).
Even though the contribution of SRZ methanogenesis to AOM above the SMTZ
remains speculative, it leads to the assumption that SRZ methanogenesis
could play a much bigger role for benthic carbon cycling in the
Eckernförde Bay than previously thought. Whether SRZ methanogenesis at
Eckernförde Bay has the potential for the direct emission of methane
into the water column goes beyond the scope of this study and should be
tested in the future.
Summary
The present study demonstrated that methanogenesis and sulfate reduction
were concurrently active within the sulfate-reducing zone in sediments at
Boknis Eck (Eckernförde Bay, SW Baltic Sea). The observed methanogenesis
was probably based on noncompetitive substrates due to the competition with
sulfate reducers for the substrates H2 and acetate. Accordingly,
members of the family Methanosarcinaceae, which are known for methylotrophic methanogenesis, were
found in the sulfate reduction zone of the sediments and are likely to be
responsible for the observed methanogenesis with the potential use of
noncompetitive substrates such as methanol, methylamines, or methylated
sulfides.
Potential environmental factors controlling SRZ methanogenesis are POC
content, C / N ratio, oxygen, and temperature, resulting in the highest
methanogenesis activity during the warm, stratified, and hypoxic months
after the late summer phytoplankton blooms.
This study provides new insights into the presence and seasonality of SRZ
methanogenesis in coastal sediments and was able to demonstrate that the
process could play an important role for the methane budget and carbon
cycling of Eckernförde Bay sediments, for example by directly fueling AOM
above the SMTZ.