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Methane production in aerobic oligotrophic surface water in the central Arctic Ocean

Methane production in aerobic oligotrophic surface water in the central Arctic Ocean Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ Biogeosciences © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Methane production in aerobic oligotrophic surface water in the central Arctic Ocean 1 1 1 1 1 2 3,4 E. Damm , E. Helmke , S. Thoms , U. Schauer , E. Nothig ¨ , K. Bakker , and R. P. Kiene Alfred Wegener Institute for Polar and Marine Research, P.O. Box 12061, 27515 Bremerhaven, Germany Royal Netherlands Institute for Sea Research, Texel, The Netherlands Department of Marine Sciences, University of South Alabama, Mobile, Alabama, USA Dauphin Island Sea Lab, Dauphin Island, Alabama, USA Received: 30 September 2009 – Published in Biogeosciences Discuss.: 11 November 2009 Revised: 25 February 2010 – Accepted: 5 March 2010 – Published: 19 March 2010 Abstract. A methane surplus relative to the atmospheric methane formation needs to be understood in order to esti- equilibrium is a frequently observed feature of ocean surface mate its global significance. water. Despite the common fact that biological processes are The role of nutrient limitation, particularly phosphate responsible for its origin, the formation of methane in aer- stress, has only recently been discussed as a possible regu- obic surface water is still poorly understood. We report on lator of methane production (Karl et al., 2008). These au- methane production in the central Arctic Ocean, which was thors suggested that under P limitation, microorganisms uti- exclusively detected in Pacific derived water but not nearby lized more organic phosphorus compounds, and they demon- in Atlantic derived water. The two water masses are distin- strated experimentally that exogenous methylphosphonate guished by their different nitrate to phosphate ratios. We can be converted to methane. show that methane production occurs if nitrate is depleted We report here that in the central Arctic Ocean methane but phosphate is available as a P source. Apparently the formation apparently occurs in phosphate-replete Pacific- low N:P ratio enhances the ability of bacteria to compete for derived water (Pdw) whereas no net methane accumulation phosphate while the phytoplankton metabolite dimethylsul- is observed in phosphate-poor Atlantic-derived water (Adw) foniopropionate (DMSP) is utilized as a C source. This was nearby. verified by experimentally induced methane production in The two water masses differ in their nitrate/phosphate re- DMSP spiked Arctic sea water. Accordingly we propose that lationship (Jones et al., 1998) due to an excess of phosphate methylated compounds may serve as precursors for methane over nitrate in Pdw (Yamamoto-Kawai et al., 2006). We dis- and thermodynamic calculations show that methylotrophic cuss the different stages of oligotrophy established in the two methanogenesis can provide energy in aerobic environments. water masses as an obvious requirement for triggering the switch from a situation of no methane production to one of methane production. 1 Introduction Complementary to the role of nutrient limitations, we discuss the potential role of DMSP (dimethylsulfoniopropi- Methane supersaturation within the oxygenated ocean sur- onate) degradation products as precursors for methane for- face mixed layer is widespread and has been known for more mation and propose methylotrophic methanogenesis as the than three decades (Lamontagne et al., 1973, Scranton and principal pathway. Brewer, 1977; Forster et al., 2008). Although biological DMSP is an abundant methylated substrate in the surface processes are considered to be responsible, the so called ocean and large amounts are produced annually by phyto- methane paradox i.e. methanogenesis in an aerobic environ- plankton (Stefels, 2000). Recently an inverse correlation ment is not yet explained. In particular, the mechanism of between DMSP and methane in polar water was observed (Damm et al., 2008). DMSP can be degraded by a cleavage Correspondence to: E. Damm and a demethylation pathway (Kiene et al., 2000). The best ([email protected]) known cleavage product of DMSP is DMS (dimethylsulfide). Published by Copernicus Publications on behalf of the European Geosciences Union. 1100 E. Damm et al.: Methane production in aerobic oligotrophic surface water DMS escapes partly to the atmosphere where it is the most of the water between the nearest shelf sea (Chukchi Sea) important natural climate-cooling gas, counterbalancing the and the study area. Although no direct near-surface velocity effect of greenhouse gases (Charlson et al., 1987). Most measurements are available indirect estimates suggest travel of the dissolved DMSP, however, is sequentially demethy- times of several months from the shelf edge to our section. lated. Aerobic as well as anaerobic demethylation path- Sea ice takes more than two years to cross the Arctic in the ways are known to provide bacteria with energy and carbon fastest passage, the Transpolar Drift, and it is expected to sources for biosynthesis (Kiene et al., 2000). An interme- drive rather than to slow down the surface waters. Woodgate diate product of both pathways is methanethiol. Its anaer- et al. (2005) estimate that Pdw takes several months to transit obic metabolism to methane was described by Tallant and the Chukchi Sea and we assume that this speed holds also for Kryzcki (1997). In addition, a new metabolic pathway for the passage of the deep ocean. anaerobic methane oxidation was recently proposed, where methane serves as electron donor and methanethiol is created as intermediate product (Moran et al., 2008). 3 Data and methods We discuss this type of metabolism in its reverse direction and calculate the energy yield associated with this pathway. Two transects were sampled in the central Arctic during an expedition with the research vessel “Polarstern” in Septem- ber 2007 (Schauer et al., 2008). Both transects cross from 2 Regional setting ◦ ◦ ◦ 130 E to 130 W roughly along the 85 N latitude (Fig. 1). The Atlantic-derived water was detected between approx- The Arctic Ocean circulation comprises both low salinity and ◦ ◦ imately 130 E and 180 . On the stations further west, nutrient-rich Pacific Ocean water and relatively nutrient-poor ◦ ◦ i.e. from 180 to 130 W, the proportion of Pacific-derived and more saline Atlantic Ocean water (Pabi et al., 2008). The water increases. In the following, methane concentration and Atlantic water enters through the Fram Strait and the Barents ancillary data will be categorized as part of either Atlantic- Sea and is distributed via counterclockwise currents in the derived water (Adw) or Pacific-derived water (Pdw) (Fig. 1). Eurasian sector. Pacific water enters the Arctic basin through Salinity and temperature were measured with a Seabird the Bering Strait via the Chukchi Sea and exits through the SBE 911 plus CTD. Water samples from up to eight different Canadian Archipelago and the western Fram Strait. Because depths were collected during the upcast at each CTD station the broad shelves of the Bering and Chukchi Seas are denitri- with 10 L Niskin bottles mounted on a rosette sampler. fication sites, water of Pacific origin is continuously depleted Estimates of phytoplankton biomass and abundances of in nitrate relative to phosphate during its journey from the dominant unicellular organisms were obtained from samples entrance at the Bering Strait via the central Arctic to its out- out of the 10 m water depth Niskin bottle. For the chlorophyll flows (Yamamoto-Kawai et al., 2006). In the early nineties, a determination, 0.5–2.0 L of water were filtered through the front separating the Atlantic and the Pacific water has Whatman GF/F glass-fiber filters and stored at −18 C and been located over the Alpha-Mendeleev Ridge (Carmack et later analyzed in the home laboratory. The filters were ex- al., 1997, Pabi et al., 2008). Hence, in the central Arctic tracted in 90% acetone and analyzed with a spectrophotome- Ocean (north of 85 N) the influence of Pacific water is re- ter for higher values and with a Turner-Design fluorome- stricted to the region west of the 180 longitude. ter for lower values according to the methods described in On the Arctic shelves huge amounts of submarine methane Edler (1979) and Evans and O’Reily (1984). The values are stored in hydrates, source rocks and permafrost sediments from the fluorometer were calibrated with a spectrophotome- and when released causes excess methane in shelf water, as ter, using authentic chlorophyll a (Sigma). Phyto- and pro- was observed in the Beaufort Sea (Mac Donald, 1976; Kven- tozooplankton samples were preserved in hexamine-buffered volden et al., 1993), in the Laptev and East Siberian Sea formalin in brown glass bottles. A minimum of 50–100 cells (Shakhova and Semiletov, 2007) as well as in the Barents Sea of the dominant species or groups were counted in aliquots (Lammers et al., 1994; Damm et al., 2005). Plume spread- of 50 ml settled for 48 h, under an inverted microscope. Here ing in the stratified shelf water transports dissolved methane we only present cell counts for species or groups which were mainly along isopycnals into the deeper and dense shelf wa- easy to recognize in the samples. For the diatoms only veg- ter but methane also escapes into surface water by vertical etative cells were used for the calculations since resting cells mixing (Jeong et al., 2004; Damm et al., 2005). However, have a fairly low metabolism. offshore transports of dissolved methane are rapidly reduced by open ocean dispersion, sea to air flux and methane oxi- Sampling for nutrient analyses was carried out directly dation processes. Thus, the central Arctic, which is nearly from the Niskin bottles. The samples were kept cool and dark 1000 km away from the shelves, is not influenced by excess and analyzed within 10 h using a Technicon TRAACS 800, methane from the shelves (T. D. Lorenson, personal commu- continuous flow auto-analyzer. Phosphate was measured ac- nication, 2009). Shelf sources of the elevated methane val- cording to Murphy and Riley (1962) and nitrate according to ues in Pdw are unlikely because of the long transfer times Grasshoff et al. (1983). Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ E. Damm et al.: Methane production in aerobic oligotrophic surface water 1101 Methane concentration was analyzed within hours of developed in the bottles spiked with DMSP was determined sampling. The dissolved gas was extracted from water after 6, 8, and 10 days by means of CARD-FISH as well as by vacuum-ultrasonic treatment and subsequently measured DGGE-analysis. The DGGE analysis was conducted at the with a gas chromatograph (Chrompack 9003, GC) with flame start of the experiment and after 8 and 10 days using 600 ml ionization detector (FID). For gas chromatographic separa- of each sample. tion we used a packed column (Porapac Q 80/100 mesh). The Total bacterial counts were determined by epifluorescence GC oven was operated isothermally (60 C) and the heated microscopy of acridine orange–stained cells after Hobbie et zone of the FID was held at a temperature of 250 C. Two al. (1977). sets of standard gas mixtures (10 and 100 ppmv) were used Denaturing gradient gel electrophoresis (DGGE) analysis for calibration. The standard deviation of duplicate analyses based on the 16S rRNA gene was used to examine the di- was 5%. This high overall error is almost exclusively due to versity of natural bacterioplankton assemblages as well as the gas extraction procedure and not to GC precision, which the bacterial consortia grown in the microcosm experiments. had an error of only 1%. After GC analyses, the remainder Plankton cells concentrated by centrifugation were freeze- of the gas was transferred into evacuated glass containers for dried and stored deep frozen until DNA analyses. Bacterial analysis of the carbon isotopic signature on shore. cells of the microcosm experiments were sampled on Nu- The δ C-CH values were determined by a Delta XP clepore filters (0.2 μm pore size). Total community DNA plus, Finnigan mass spectrometer. The extracted gas was was extracted using the Ultraclean soil DNA kit (MoBio purged and trapped with PreCon equipment (Finnigan) to Laboratories, USA). To increase the sensitivity of PCR- pre-concentrate the sample. Depending on the concentration amplification and DGGE-analysis, a non-specific amplifica- of methane, the reproducibility derived from duplicates was tion step was applied with the natural bacterial community 0.5–1‰. Isotopic ratios are reported relative to the Pee Dee using the GenomiPhi DNA Amplification Kit (GE Health- Belemnite (PDB) standard using conventional delta notation care) as specified by the manufacturer. One μl product of (Craig, 1957). the GenomiPhi reaction and 1 to 5 μl original DNA extract DMSPt (DMSPtotal) samples were collected directly from of the microcosm experiments were applied as template in Niskin sample bottles into 50 ml centrifuge tubes that con- the 16S rRNA gene specific PCR with GM5 plus GC-clamp tained 167 μl of 50% H SO . The tubes were sealed and the as forward primer and 907RM as reverse primer. PCR con- 2 4 samples stored for later analysis on shore. DMSPt is sta- ditions were as, described by Gerdes et al. (2005). PCR- ble for months in acid solution (Curran et al., 1998). DM- products were analyzed by DGGE, based on the protocol of SPt in the stored samples was analyzed as DMS after alka- Muyzer et al. (1993) using a gradient-chamber. Significant line cleavage. A subsample of the solution was pipetted into bands from the DGGE-pattern were selected and, after ex- a 14 ml serum vial and treated with 1 ml of 5 N NaOH and cision from the gel, re-amplified by PCR as described by quickly sealed. The released DMS was purged into a cryo- Gerdes et al. (2005). The 16S rRNA gene amplicons were trap and quantified with a gas chromatograph equipped with sequenced and the sequences compared to those deposited in a Chromosil 330 column and a flame photometric detector. GenBank using the BLAST algorithm. The oven temperature was 100 C and Helium was used as Amplification of nifH gene fragments was performed to the purge and carrier gas. The analytical system was cali- screen for dinitrogen fixing bacteria. The DNA-products of brated for DMS with standards. The detection limit was 0.5 the GenomiPhi amplification reaction and the extracts of the to 1 pmol DMS injected, which yielded DMSP detection lim- microcosm-experiment respectively were employed in the its of 0.17 to 0.33 nM in a 3-mL sample. nifH gene specific PCR-reaction using the forward primer A microcosm experiment was set up to study the ability nifH-f (5’-TAYGGNAARGGNGGNATYGGNAARTC-3’), of natural Arctic bacterial communities to produce methane designed by Boulygina et al. (2002), and the reverse from DMSP. Sea water sampling, incubation and methane primer nifH-r (5’-ADNGCCATCATYTCNCC-3’), designed measurements were conducted in the Greenland Sea in 2008 by Zehr and McReynolds (1989). Details of the amplification ◦ 0 ◦ 0 (77 59.9 N 12 0.19 W). Seawater was transferred from 10 L conditions are given by Dedysh et al. (2004). Niskin bottles into sterile 1 liter glass bottles with silicone Catalyzed reporter deposition (CARD) – Fluorescence in membranes. Immediately after water sampling five bottles situ hybridization (FISH) was employed to characterize the were spiked with 50 μM DMSP. Five bottles served as con- structure of the bacterial communities, which developed in trols which were free of DMSP supplements. The methane the microcosm-experiments. Water samples were fixed with concentration was measured as described above. Measure- buffered paraformaldehyde solution. After about 2 h the ments were conducted at the beginning of the experiment and cells were immobilized on white polycarbonate filters and after 3, 6, 8, 10 and 12 days by sacrificing one bottle spiked rinsed with phosphate-buffered saline and distilled water. with DMSP and one control bottle at each time point. The in- Air-dried filters were stored at −20 C until further process- cubation was carried out at approximately in situ temperature ing. CARD-FISH analysis was conducted according to the (about 1 C) in artificial daylight to simulate the polar sum- method of Pernthaler et al. (2002) using horseradish peroxi- mer situation. The composition of the bacterial communities dase (HRP)-labeled oligonucleotide probes (ThermoHybaid; www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 150°E 0° 180° 30°E 150°W 60°E 1102 E. Damm et al.: Methane production in aerobic oligotrophic surface water 120°W D E FG Fig. 1. Map of the central Arctic Ocean showing the spread of Pacific/Atlantic water as blue and colorless areas, respectively. Dots indicate stations along two transects. Profiles for red transect running from east to west are shown in diagrams (A) to (G). (A) shows the potential density in sigma θ units, (B), the concentration of oxygen (μmol/L), (C), NO (μmol/L), (D), PO (μmol/L) (E), DMSPt nM), (F), CH 3 4 ( 4 (nM) and (G), δ C-CH as (‰ PDB). Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ 90°E 90°W 60°W 120°E 30°W E. Damm et al.: Methane production in aerobic oligotrophic surface water 1103 Ulm, Germany) with Alexa546 as the reporter signal. The 4.2 Phytoplankton bloom and nutrient availability standard set of oligonucleotide probes were applied specific Primary production in the central Arctic is essentially reg- for the kingdoms of Eubacteria and Archaea, for the do- ulated by sea ice dynamics. The melting ice exposes the main Bacteroidetes and the subclasses alpha- and gamma- nutrient-rich surface waters to more light, which triggers the proteobacteria. The probe Non338 (Wallner et al., 1993) was phytoplankton bloom. However, nutrients in near-surface used to test for non-specific probe binding. Hybridization water are rapidly exhausted as the density stratification in- temperature was 35 C. Air-dried hybridized samples were hibits an upward transport and replenishment of nutrients counterstained with 4’, 6’-diamidino-2-phenylindole (DAPI; −1 (Sakshaug, 2003). High phosphate and nitrate concentra- final concentration 1 μg ml ). Samples were evaluated un- tions are therefore present in subsurface water (>50 m water der an Axioplan2 epifluorescent microscope equipped with depth). appropriate filter sets for Alexa546 and DAPI fluorescence. Between 800 and 2000 DAPI-stained objects were counted In near-surface water nitrate becomes depleted and even- tually exhausted in the top layers. Phosphate, however, is per probe and sample. only limited in Adw, while in Pdw excess phosphate remains available up to the sea surface (Fig. 1). Nitrate and phosphate are the primary regulators of phytoplankton growth and con- 4 Results and discussion sequently varying availability induces different bloom stages 4.1 Excess methane in Pacific derived surface water in the two water masses. Low chlorophyll a concentrations were generally en- Fresh water anomalies traced in the past decade between the countered, with those in the Pdw 0.11 μg/L (range 0.06– East Siberian Sea (Jones et al., 2008) and Fram Strait (Rabe 0.22 μg/L) being lower than in Adw 0.33 μg/L (range 0.19– et al., 2009) suggest that several years are needed for sur- 0.48 μg/L). The composition and the size of unicellular face waters to cross the deep Arctic Basin. Additional sea-ice plankton organisms also varied in the two water masses. In cover in the central Arctic impedes the gas exchange between Adw, diatoms (55%) were the dominant species followed atmosphere and ocean. Hence, under-ice methane consump- by nanoflagellates (31%), ciliates and dinoflagellates (12%) tion is the main process which determines the methane inven- while in Pdw small nanoflagellates (85%) clearly dominated. tory in the central Arctic Ocean. As a result concentration The plankton composition in Adw represented a late spring becomes more and more depleted relative to the atmospheric to summer phase of new production, while the nitrate and equilibrium concentration while at the same time the resid- phosphate depletion in near-surface water reflected an ex- ual methane becomes increasingly enriched in C in rela- hausting bloom (Fig. 1). tion to the atmospheric carbon isotopic signature of methane In comparison, the composition and size of unicellular (Damm et al., 2007). plankton organisms in Pdw reflect an impoverished phyto- The isolation from the atmosphere and ongoing consump- plankton community. As phosphate excess is available the tion are clearly reflected in subsurface water (water depth phytoplankton growth is clearly limited by nitrate depletion >100 m) where the effects of under-ice consumption remain (Fig. 1). conserved due to the density of the highly stratified wa- ter column, which restricts vertical mixing during summer 4.3 Nutrient limitations and bacterioplankton (Fig. 1). In comparison, near-surface water (water depth composition <100 m) is influenced by air-sea gas exchange during sum- mer and as a result, methane tends to equilibrate with the During the phytoplankton growth the global mean ratio of atmospheric background. In polar water, methane concen- 16:1 between nitrogen and phosphorous remains preserved tration in equilibrium with atmosphere is estimated to range if both are consumed and released in the same constant between 3.5 and 4 nM. Actually, in Adw, the concentration ratio, while shifts of this ratio are induced by increasing is close to atmospheric equilibrium and reflects an air-sea oligotrophy (Redfield, 1958). A perturbation of the Red- methane exchange, which is also confirmed by the δ C-CH field ratio is expressed in the definition of N* (N*=(N- values (from −41 to −43‰ PDB). These correspond to a 16P+2.9 μmol/kg)·0.87), a quasi-conservative tracer (Gruber two component mixing between reservoirs with different iso- and Sarmiento, 1997). N* is about zero if the Redfield ratio topic compositions i.e. the δ C-CH value of the local ma- is retained, while positive and negative values of N* are as- rine background (−38‰ PDB in subsurface water) and the sociated with deviations from the conservative behavior. Al- δ C-CH value of the atmospheric reservoir (−47‰ PDB) though deviations from the global mean Redfield ratio are (Fig. 1). However, in Pdw, the methane concentration is evident in both water masses, N* remains constant about clearly elevated relative to the equilibrium level. Further- 1.8 μmol/kg in Adw, in contrast to Pdw where N* decreases more, the methane is more enriched in C compared to that from 1 to −1.5 μmol/kg reflecting increasing perturbation in Adw (up to −46‰ PDB). These two features combined, (Fig. 2). Methane excess increases where nitrate is the only are indicative of methane production in Pdw (Fig. 1). growth-limiting nutrient and N* is decreasing. www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 1104 E. Damm et al.: Methane production in aerobic oligotrophic surface water 2 ples suggesting that nitrogen depletion may in this case be compensated by dinitrogen fixation. Nitrogenases are gener- ally oxygen sensitive and therefore often accommodated in separate cell compartments (Gallon, 1992). In such an envi- ronment in which hydrogen is produced by nitrogenases, we suggest that methane may be formed from methylated com- pounds by eubacteria e.g. alpha-proteobacteria. 4.4 DMSP as precursors for methane formation in nitrate-stressed environment -1 y = 0.457x - 1.6359 R = 0.7856 In oligotrophic water growth rates for the bacterioplankton are limited, either by mineral nutrients or by organic car- -2 bon (Thingstad et al., 2008). An important component for 0 2468 10 12 the bacterial carbon demand is DMSP (dimethylsulfoniopro- DMSPt/methane pionate) (Simo et al, 2002) and in microcosm experiments, a significant increase in bacterial biomass production is in- Fig. 2. Relationship between N* and the DMSPt to methane ratio duced by the addition of DMSP as a C source while phos- for water samples of the central Arctic Ocean. Circles and squares phate was the second limiting nutrient once C limitation correspond to Pacific and Atlantic derived surface water (<50 m), was alleviated (Pinhassi et al., 2005). Consequently, phos- respectively. N* is a quasi conservative tracer concerning the N:P phate availability may influence the utilization of DMSP as ratio. N* of about zero reflects a constant ratio while deviations C source for bacterial biomass production. from zero are induced by perturbations of the Redfield ratio (Gru- In Pdw, excess phosphate is available for bacterial biomass ber and Sarmiento 1997, see text). In Pacific derived water, the production. The inverse correlation found between phos- DMSPt/methane ratio correlates with increasing deviations from the phate and DMSPt (r = 0.7), may be indicative of the fol- conservative N:P ratio. lowing: First, the correlation simply indicates the increas- ing degree of oligotrophy. Second, the decreasing DMSPt During phytoplankton senescence, the activity of bacteri- concentration reflects an increasing utilization of DMSPt as oplankton is stimulated by the release of nutrients and dis- carbon source in the Pdw where phosphate is available for solved organic matter. In the central Arctic where only low the bacteria (Fig. 3). The following features corroborate this bacterial biomass is detected (in Pdw on average 4.8×10 assumption. In Pdw, phosphate is also significantly corre- and in Adw on average 17.7×10 bacterial counts per ml) lated with methane (r = 0.9), (Fig. 3) and a correlation be- the quantity differences correspond to those of the chloro- tween DMSPt decrease and methane production has previ- phyll between the two water masses. ously been found (Damm et al., 2008). The composition of the bacterioplankton in selected sam- The differences in methane, DMSPt and nutrient cycling ples of the Adw and Pdw was examined by means of DGGE between Pdw and Adw are illustrated in the correlation/non- based on the phylogenetically relevant 16S rRNA gene. In correlation between N* vs. the DMSPt/methane ratio respec- all water samples DGGE-bands of eukaryotic chloroplast tively. We find a coupling of DMSPt decrease with methane DNA were found while only few bands could be assigned formation on the one hand, while on the other the decreasing to prokaryotic taxa. A preponderance of sequences in the DMSPt/methane ratio is correlated with an increasing pertur- Pdw samples belonged to the alpha-proteobacteria subclass bation of the N:P ratio (i.e., more negative N*, Fig. 2). Thus, and these had very high similarities (99%) to sequences the coupling of several environmental factors is apparently of uncultured Sulfitobacter, Roseobacter, and Phaeobacter obligatory for methane to be formed as a by-product of a yet types within the family Rhodobacteraceae. Bacteria of the unexplained metabolic pathway. Rhodobacter/Roseobacter group are frequent in oligotrophic ocean surface waters but mostly uncultured. Cultured rep- 4.5 Implications for a potential pathway resentatives of this group are known for their highly diverse and flexible metabolism. A survey of available Roseobac- 4.5.1 Methane formation in DMSP spiked sea water ter genomes by Moran et al. (2007) revealed that 50% of the genomes contained genes for DMSP demethylation, 92% for In a microcosm experiment using Arctic surface water we phosphonate use, 25% for aerobic anoxygenic phototrophy, could show a microbial degradation of DMSP to methane and 92% for carbon monoxide oxidation. Further, different (Fig. 4). After 12 days considerable amounts of methane strains of Rhodobacter sphaeroides are able to fix dinitro- were formed in the water supplemented with DMSP while gen and possess the nifH (nitrogenase) gene (Moran et al., methane concentrations in the control bottles remained un- 2007). We could also amplify the nifH gene in the Pdw sam- changed. DGGE- as well as FISH-analyses revealed a clear Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ N*[µmol/kg] E. Damm et al.: Methane production in aerobic oligotrophic surface water 1105 Fig. 4. Formation of methane during a microcosm experiment with Fig. 3. Relationship between phosphate and DMSPt (left panel) Arctic surface water supplemented with DMSP (red line, closed cir- and phosphate and methane (right panel) for water samples from cles) and without DMSP (green line, closed rectangles). A change the central Arctic Ocean. Circles and squares correspond to Pa- in the bacterial community structure was followed up by CARD- cific and Atlantic derived surface water (<50 m), respectively. In FISH using general probes to detect Bacteria, alpha- and gamma- Pacific-derived water, phosphate is inversely correlated with DM- Proteobacteria, Bacteroidetes. and Archaea, Cyanobacteria were SPt and correlates with methane, while in Atlantic-derived water, determined by their morphology and yellow fluorescence at UV- no correlations exist. light. alteration of the bacterial communities in the DMSP-spiked 4.5.2 Thermodynamic calculations bottles. After 8 days the originally diverse DGGE-pattern with several light bands, had reduced to 3 dominant strong Moran et al. (2008) proposed a new metabolic pathway for bands resembling the sequences found in the Pdw and closely the anaerobic methane oxidation where methanethiol acts as related to sequences of uncultered Rhodobacter, Sulfitobac- an intermediate. The methanethiol is formed via two re- ter, and Mesorhizobium types. The CARD-FISH analysis actions, one dealing with oxidation of methane, the other, (Fig. 4) complemented this result showing that Archaea re- with reduction of CO . These reactions are coupled in such mained negligible in the DMSP supplemented approaches a way that the reduction equivalents (like H ) released by while Bacteria (hybridizing with the general bacterial probe) methane oxidation are immediately used by CO reduction. became nearly 100% of the community. After 8 days, alpha- Therefore, in the overall equation for methanethiol produc- and gamma-proteobacteria together accounted for more than tion, the contributions from the reduction equivalents cancel 75% of the DAPI-stained cells with the yet unsolved puzzle each other out. This tight coupling of the two half reactions that no dominant gamma-proteobacterium sequence could be minimizes the loss of reduction equivalents to competing ox- isolated. As in the Pdw samples, the nifH gene could be idative reactions. The latter is an important issue, especially amplified from all three DMSP supplemented samples show- under aerobic environmental conditions. Here, we suggest ing a clear increase over the course of the experiment. This that the pathway of methanethiol formation (Moran et al., further corroborates a close link between nitrogen fixation, 2008) operating in its reverse direction might also explain DMSP degradation, and methane production by Eubacteria. the production of methane. Hence, we propose the following It was shown experimentally, in a phosphate-free environ- methane formation reaction: ment, that methylphosphonate (MPn) acts as a precursor for 4 1 5 4 − + − methane production (Karl et al., 2008). We postulate that H CSH+ H O → CH + HCO + H + HS (1) 3 2 4 3 3 3 3 degradation products of DMSP may serve as precursors of The Gibbs free energy change (1G) for reaction (1) can be methane if phosphate is available for bacteria. Although in calculated using the following equation: phosphate-replete and phosphate-free environments respec- tively, bacteria utilize different P sources, methyl groups are 1 4 − / − / 3 3 a common feature of the molecule structure of MPn and 0 [CH ]· HCO · HS 0 3 1G = 1G +R·T · ln (2) DMSP degradation products. Thus, methane formation by [H CSH] both precursors is likely to occur in oligotrophic sea wa- 3 ter with methylotrophic methanogenesis being the potential (T is the Temperature in Kelvin, R is the universal gas con- pathway. A potential degradation product of DMSP and a po- − − stant, [H CSH], [CH ], [HCO ] and [HS ] designate con- 3 4 tential direct precursor for methane is methanthiol which is centrations in mol/L). For a reaction under physiological produced by the demethylation and cleavage pathway (Kiene conditions it is more appropriate to define the reference [H ] et al., 2000). differently from the standard concentration of 1 M (pH=0). The Gibbs free energy change at the appropriate neutral pH www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 1106 E. Damm et al.: Methane production in aerobic oligotrophic surface water and with all reaction partners, except protons, kept at stan- A bacterial cell needs a minimum of about −20 KJ per dard concentrations is labeled 1G . It is calculated for re- mol to exploit the 1G in a metabolic reaction (“biologi- action (1) from cal energy quantum”, Schink, 1997). Hence, our result for −1 1G of −35.7 KJ mol indicates that methane production 0 5 0 0 1G = 1G −2.3026·R·T · · pH (3) via reaction (1) is consistent with the constraints of ther- (T ) modynamics and the biological energy quantum. It should be noted here, that 1G of reaction (1) calculated for the where 1G is the free energy change at standard concen- (T ) high intracellular [HCO ] and [HS ] represents a conser- trations of all reaction partners corrected for temperature. 3 vative estimate. The methane production via reaction (1) be- The 1G at an actual temperature (T ) can be calculated (T ) comes more favorable for seawater concentrations at pH=8.2 from the free energy change at the standard reference temper- −1 (1G < −35.7 KJ mol ). ature (T = 298.15 K) by means of the van’t Hoff equation ref The pathway for the reaction (1) suggested by Moran (Atkins, 1990) et al. (2008) involves CoM which is so far only found in T T −T ref 0 0 0 anaerobic methanogenic Archeabacteria. There is no proof 1G = 1G · +1H · (4) (T ) (T ) (T ) ref ref T T ref ref for its presence in aerobic seawater. However, there is a structural similarity between CoM and MPA (Mercaptopro- with 1H being the standard free enthalpy change at tem- (T ) ref pionate), which is a further product of aerobic demethylation perature T . The derivation of Eq. (4) assumes that 1H ref (T ) ref of DMSP. Hence, the MPA might substitute for CoM in the is not greatly changed from 1H within a (physiologically formation of methane. (T ) reasonable) temperature interval T –298.15 K. It is usually true that 1H for reactions varies rather slowly with T, (T ) 5 Conclusions provided no phase changes occur in the temperature inter- 0 0 0 val. 1G and 1H and in turn 1G can be cal- (T ) (T ) (T ) ref ref In the central Arctic Ocean a shift from a phytoplankton culated with the tabulated values of standard free energies bloom situation to oligotrophic conditions occurs during and enthalpies of formation from the elements for H O , 2 (l) − summer both in Atlantic and Pacific derived water where ni- H CSH , CH , HCO , and HS (Lide, 1999). Since re- 3 (g) 4(g) trate limits primary production. Nitrate depletion appears to action (1) proceeds in the aqueous phase, the value of 1G (T ) be a primary requirement for methane production in aerobic is corrected for the dissolution of H CSH and CH in 3 (g) 4(g) surface water. A second pre-condition in the high latitudes is water using the Henry’s law constant (k ), which describes the phosphate excess, which may be utilized by bacteria as the equilibrium partitioning of H CSH and CH between the 3 4 a P source. Where phosphate is available as a source of P, gas and the aqueous phase: 1 G = −R · T ln k . The soln H (T ) methylated compounds like DMSP and its degradation prod- temperature dependence of k is given by the equation: ucts may serve as the bacterial C source. When a combina- −1 H 1 1 soln tion of these conditions exists, methane may be a metabolic k = k · exp · − (5) H H,T ref R T T by-product and its production could yield energy under aer- ref obic conditions. However, to prove the proposed mechanism where 1 H is the enthalpy of dissolution. The val- soln ongoing research is required. ues of the Henry’s law constants at standard conditions Methane production may occur as a rapid response to envi- (k ) and of 1 H are available from Sander (1999) H,T soln ref ronmental perturbations during the shift from a phytoplank- −3 (methane: k = 1.3×10 M/atm and −1/R × 1 H H,T soln ref ton bloom to an oligotrophic system, induced by a switch −1 = 1800 K; methanethiol: k = 2.0×10 M/atm and H,T ref in the utilization of phosphate and methylated compounds. −1/R × 1 H = 2800 K). At the environmental tempera- soln Hence, methane production in aerobic surface water is di- ture of 2 C it follows from Eqs. (3–5) that reaction (1) is rectly linked to the N, P and C cycles. Recent change in the exergonic under standard conditions at the intracellular pH 0 0 Arctic has altered seasonal ice coverage and density stratifi- 0 −1 0 of 7 (1G = −33 KJ mol ). Using our result for 1G , the cation of surface water, which may have profound effects on Gibbs free energy change (1G) for the actual concentrations these biogeochemical cycles. Thus, feedback effects on cy- of the reaction partners can be calculated by means of Eq. (2). cling pathways of the climatically relevant biogases methane We simulate respiration within the cell by assuming [HCO ] and DMS are likely, with DMSP catabolism in high latitudes = 30 mM and [HS ]=1 mM. From the field data we estimate possible contributing to a warming effect on the earth’s cli- [H CSH] = 1 nM and [CH ]=10 nM. Then, the Gibbs free 3 4 mate through production of the greenhouse gas, methane. energy change that derives from Eq. (2) is given by 1G = −1 − 35.7 KJ mol . Thus, for the assumed concentrations of the reaction partners energy is released during methane for- mation. Since N -fixation is a highly energy consuming pro- cess, methane production could contribute additional free en- ergy for bacterial growth. Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ E. Damm et al.: Methane production in aerobic oligotrophic surface water 1107 ◦ ◦ Acknowledgements. We are grateful to Ingrid Stimac and the Atlantic Ocean between 50 N and 52 S: Latitudinal distri- Jutta Jur ¨ gens who participated in the work at sea and in the home bution and sea-to-air flux, Deep-Sea. Res. Pt. II, 56, 964–976, lab. We thank the scientific party and crew of RV Polarstern for doi:10.1016/jdsr2.2008.12.002, 2008. their support at sea. The participation of R. P. Kiene was supported Gallon, J. R.: Tansley Review No. 44, Reconciling the incompati- by grants OPP-0230497 and OCE-0724017 from the US National ble: N fixation and O , New Phytol., 122, 571–609, 1992. 2 2 Science Foundation. 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Methane production in aerobic oligotrophic surface water in the central Arctic Ocean

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Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ Biogeosciences © Author(s) 2010. This work is distributed under the Creative Commons Attribution 3.0 License. Methane production in aerobic oligotrophic surface water in the central Arctic Ocean 1 1 1 1 1 2 3,4 E. Damm , E. Helmke , S. Thoms , U. Schauer , E. Nothig ¨ , K. Bakker , and R. P. Kiene Alfred Wegener Institute for Polar and Marine Research, P.O. Box 12061, 27515 Bremerhaven, Germany Royal Netherlands Institute for Sea Research, Texel, The Netherlands Department of Marine Sciences, University of South Alabama, Mobile, Alabama, USA Dauphin Island Sea Lab, Dauphin Island, Alabama, USA Received: 30 September 2009 – Published in Biogeosciences Discuss.: 11 November 2009 Revised: 25 February 2010 – Accepted: 5 March 2010 – Published: 19 March 2010 Abstract. A methane surplus relative to the atmospheric methane formation needs to be understood in order to esti- equilibrium is a frequently observed feature of ocean surface mate its global significance. water. Despite the common fact that biological processes are The role of nutrient limitation, particularly phosphate responsible for its origin, the formation of methane in aer- stress, has only recently been discussed as a possible regu- obic surface water is still poorly understood. We report on lator of methane production (Karl et al., 2008). These au- methane production in the central Arctic Ocean, which was thors suggested that under P limitation, microorganisms uti- exclusively detected in Pacific derived water but not nearby lized more organic phosphorus compounds, and they demon- in Atlantic derived water. The two water masses are distin- strated experimentally that exogenous methylphosphonate guished by their different nitrate to phosphate ratios. We can be converted to methane. show that methane production occurs if nitrate is depleted We report here that in the central Arctic Ocean methane but phosphate is available as a P source. Apparently the formation apparently occurs in phosphate-replete Pacific- low N:P ratio enhances the ability of bacteria to compete for derived water (Pdw) whereas no net methane accumulation phosphate while the phytoplankton metabolite dimethylsul- is observed in phosphate-poor Atlantic-derived water (Adw) foniopropionate (DMSP) is utilized as a C source. This was nearby. verified by experimentally induced methane production in The two water masses differ in their nitrate/phosphate re- DMSP spiked Arctic sea water. Accordingly we propose that lationship (Jones et al., 1998) due to an excess of phosphate methylated compounds may serve as precursors for methane over nitrate in Pdw (Yamamoto-Kawai et al., 2006). We dis- and thermodynamic calculations show that methylotrophic cuss the different stages of oligotrophy established in the two methanogenesis can provide energy in aerobic environments. water masses as an obvious requirement for triggering the switch from a situation of no methane production to one of methane production. 1 Introduction Complementary to the role of nutrient limitations, we discuss the potential role of DMSP (dimethylsulfoniopropi- Methane supersaturation within the oxygenated ocean sur- onate) degradation products as precursors for methane for- face mixed layer is widespread and has been known for more mation and propose methylotrophic methanogenesis as the than three decades (Lamontagne et al., 1973, Scranton and principal pathway. Brewer, 1977; Forster et al., 2008). Although biological DMSP is an abundant methylated substrate in the surface processes are considered to be responsible, the so called ocean and large amounts are produced annually by phyto- methane paradox i.e. methanogenesis in an aerobic environ- plankton (Stefels, 2000). Recently an inverse correlation ment is not yet explained. In particular, the mechanism of between DMSP and methane in polar water was observed (Damm et al., 2008). DMSP can be degraded by a cleavage Correspondence to: E. Damm and a demethylation pathway (Kiene et al., 2000). The best ([email protected]) known cleavage product of DMSP is DMS (dimethylsulfide). Published by Copernicus Publications on behalf of the European Geosciences Union. 1100 E. Damm et al.: Methane production in aerobic oligotrophic surface water DMS escapes partly to the atmosphere where it is the most of the water between the nearest shelf sea (Chukchi Sea) important natural climate-cooling gas, counterbalancing the and the study area. Although no direct near-surface velocity effect of greenhouse gases (Charlson et al., 1987). Most measurements are available indirect estimates suggest travel of the dissolved DMSP, however, is sequentially demethy- times of several months from the shelf edge to our section. lated. Aerobic as well as anaerobic demethylation path- Sea ice takes more than two years to cross the Arctic in the ways are known to provide bacteria with energy and carbon fastest passage, the Transpolar Drift, and it is expected to sources for biosynthesis (Kiene et al., 2000). An interme- drive rather than to slow down the surface waters. Woodgate diate product of both pathways is methanethiol. Its anaer- et al. (2005) estimate that Pdw takes several months to transit obic metabolism to methane was described by Tallant and the Chukchi Sea and we assume that this speed holds also for Kryzcki (1997). In addition, a new metabolic pathway for the passage of the deep ocean. anaerobic methane oxidation was recently proposed, where methane serves as electron donor and methanethiol is created as intermediate product (Moran et al., 2008). 3 Data and methods We discuss this type of metabolism in its reverse direction and calculate the energy yield associated with this pathway. Two transects were sampled in the central Arctic during an expedition with the research vessel “Polarstern” in Septem- ber 2007 (Schauer et al., 2008). Both transects cross from 2 Regional setting ◦ ◦ ◦ 130 E to 130 W roughly along the 85 N latitude (Fig. 1). The Atlantic-derived water was detected between approx- The Arctic Ocean circulation comprises both low salinity and ◦ ◦ imately 130 E and 180 . On the stations further west, nutrient-rich Pacific Ocean water and relatively nutrient-poor ◦ ◦ i.e. from 180 to 130 W, the proportion of Pacific-derived and more saline Atlantic Ocean water (Pabi et al., 2008). The water increases. In the following, methane concentration and Atlantic water enters through the Fram Strait and the Barents ancillary data will be categorized as part of either Atlantic- Sea and is distributed via counterclockwise currents in the derived water (Adw) or Pacific-derived water (Pdw) (Fig. 1). Eurasian sector. Pacific water enters the Arctic basin through Salinity and temperature were measured with a Seabird the Bering Strait via the Chukchi Sea and exits through the SBE 911 plus CTD. Water samples from up to eight different Canadian Archipelago and the western Fram Strait. Because depths were collected during the upcast at each CTD station the broad shelves of the Bering and Chukchi Seas are denitri- with 10 L Niskin bottles mounted on a rosette sampler. fication sites, water of Pacific origin is continuously depleted Estimates of phytoplankton biomass and abundances of in nitrate relative to phosphate during its journey from the dominant unicellular organisms were obtained from samples entrance at the Bering Strait via the central Arctic to its out- out of the 10 m water depth Niskin bottle. For the chlorophyll flows (Yamamoto-Kawai et al., 2006). In the early nineties, a determination, 0.5–2.0 L of water were filtered through the front separating the Atlantic and the Pacific water has Whatman GF/F glass-fiber filters and stored at −18 C and been located over the Alpha-Mendeleev Ridge (Carmack et later analyzed in the home laboratory. The filters were ex- al., 1997, Pabi et al., 2008). Hence, in the central Arctic tracted in 90% acetone and analyzed with a spectrophotome- Ocean (north of 85 N) the influence of Pacific water is re- ter for higher values and with a Turner-Design fluorome- stricted to the region west of the 180 longitude. ter for lower values according to the methods described in On the Arctic shelves huge amounts of submarine methane Edler (1979) and Evans and O’Reily (1984). The values are stored in hydrates, source rocks and permafrost sediments from the fluorometer were calibrated with a spectrophotome- and when released causes excess methane in shelf water, as ter, using authentic chlorophyll a (Sigma). Phyto- and pro- was observed in the Beaufort Sea (Mac Donald, 1976; Kven- tozooplankton samples were preserved in hexamine-buffered volden et al., 1993), in the Laptev and East Siberian Sea formalin in brown glass bottles. A minimum of 50–100 cells (Shakhova and Semiletov, 2007) as well as in the Barents Sea of the dominant species or groups were counted in aliquots (Lammers et al., 1994; Damm et al., 2005). Plume spread- of 50 ml settled for 48 h, under an inverted microscope. Here ing in the stratified shelf water transports dissolved methane we only present cell counts for species or groups which were mainly along isopycnals into the deeper and dense shelf wa- easy to recognize in the samples. For the diatoms only veg- ter but methane also escapes into surface water by vertical etative cells were used for the calculations since resting cells mixing (Jeong et al., 2004; Damm et al., 2005). However, have a fairly low metabolism. offshore transports of dissolved methane are rapidly reduced by open ocean dispersion, sea to air flux and methane oxi- Sampling for nutrient analyses was carried out directly dation processes. Thus, the central Arctic, which is nearly from the Niskin bottles. The samples were kept cool and dark 1000 km away from the shelves, is not influenced by excess and analyzed within 10 h using a Technicon TRAACS 800, methane from the shelves (T. D. Lorenson, personal commu- continuous flow auto-analyzer. Phosphate was measured ac- nication, 2009). Shelf sources of the elevated methane val- cording to Murphy and Riley (1962) and nitrate according to ues in Pdw are unlikely because of the long transfer times Grasshoff et al. (1983). Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ E. Damm et al.: Methane production in aerobic oligotrophic surface water 1101 Methane concentration was analyzed within hours of developed in the bottles spiked with DMSP was determined sampling. The dissolved gas was extracted from water after 6, 8, and 10 days by means of CARD-FISH as well as by vacuum-ultrasonic treatment and subsequently measured DGGE-analysis. The DGGE analysis was conducted at the with a gas chromatograph (Chrompack 9003, GC) with flame start of the experiment and after 8 and 10 days using 600 ml ionization detector (FID). For gas chromatographic separa- of each sample. tion we used a packed column (Porapac Q 80/100 mesh). The Total bacterial counts were determined by epifluorescence GC oven was operated isothermally (60 C) and the heated microscopy of acridine orange–stained cells after Hobbie et zone of the FID was held at a temperature of 250 C. Two al. (1977). sets of standard gas mixtures (10 and 100 ppmv) were used Denaturing gradient gel electrophoresis (DGGE) analysis for calibration. The standard deviation of duplicate analyses based on the 16S rRNA gene was used to examine the di- was 5%. This high overall error is almost exclusively due to versity of natural bacterioplankton assemblages as well as the gas extraction procedure and not to GC precision, which the bacterial consortia grown in the microcosm experiments. had an error of only 1%. After GC analyses, the remainder Plankton cells concentrated by centrifugation were freeze- of the gas was transferred into evacuated glass containers for dried and stored deep frozen until DNA analyses. Bacterial analysis of the carbon isotopic signature on shore. cells of the microcosm experiments were sampled on Nu- The δ C-CH values were determined by a Delta XP clepore filters (0.2 μm pore size). Total community DNA plus, Finnigan mass spectrometer. The extracted gas was was extracted using the Ultraclean soil DNA kit (MoBio purged and trapped with PreCon equipment (Finnigan) to Laboratories, USA). To increase the sensitivity of PCR- pre-concentrate the sample. Depending on the concentration amplification and DGGE-analysis, a non-specific amplifica- of methane, the reproducibility derived from duplicates was tion step was applied with the natural bacterial community 0.5–1‰. Isotopic ratios are reported relative to the Pee Dee using the GenomiPhi DNA Amplification Kit (GE Health- Belemnite (PDB) standard using conventional delta notation care) as specified by the manufacturer. One μl product of (Craig, 1957). the GenomiPhi reaction and 1 to 5 μl original DNA extract DMSPt (DMSPtotal) samples were collected directly from of the microcosm experiments were applied as template in Niskin sample bottles into 50 ml centrifuge tubes that con- the 16S rRNA gene specific PCR with GM5 plus GC-clamp tained 167 μl of 50% H SO . The tubes were sealed and the as forward primer and 907RM as reverse primer. PCR con- 2 4 samples stored for later analysis on shore. DMSPt is sta- ditions were as, described by Gerdes et al. (2005). PCR- ble for months in acid solution (Curran et al., 1998). DM- products were analyzed by DGGE, based on the protocol of SPt in the stored samples was analyzed as DMS after alka- Muyzer et al. (1993) using a gradient-chamber. Significant line cleavage. A subsample of the solution was pipetted into bands from the DGGE-pattern were selected and, after ex- a 14 ml serum vial and treated with 1 ml of 5 N NaOH and cision from the gel, re-amplified by PCR as described by quickly sealed. The released DMS was purged into a cryo- Gerdes et al. (2005). The 16S rRNA gene amplicons were trap and quantified with a gas chromatograph equipped with sequenced and the sequences compared to those deposited in a Chromosil 330 column and a flame photometric detector. GenBank using the BLAST algorithm. The oven temperature was 100 C and Helium was used as Amplification of nifH gene fragments was performed to the purge and carrier gas. The analytical system was cali- screen for dinitrogen fixing bacteria. The DNA-products of brated for DMS with standards. The detection limit was 0.5 the GenomiPhi amplification reaction and the extracts of the to 1 pmol DMS injected, which yielded DMSP detection lim- microcosm-experiment respectively were employed in the its of 0.17 to 0.33 nM in a 3-mL sample. nifH gene specific PCR-reaction using the forward primer A microcosm experiment was set up to study the ability nifH-f (5’-TAYGGNAARGGNGGNATYGGNAARTC-3’), of natural Arctic bacterial communities to produce methane designed by Boulygina et al. (2002), and the reverse from DMSP. Sea water sampling, incubation and methane primer nifH-r (5’-ADNGCCATCATYTCNCC-3’), designed measurements were conducted in the Greenland Sea in 2008 by Zehr and McReynolds (1989). Details of the amplification ◦ 0 ◦ 0 (77 59.9 N 12 0.19 W). Seawater was transferred from 10 L conditions are given by Dedysh et al. (2004). Niskin bottles into sterile 1 liter glass bottles with silicone Catalyzed reporter deposition (CARD) – Fluorescence in membranes. Immediately after water sampling five bottles situ hybridization (FISH) was employed to characterize the were spiked with 50 μM DMSP. Five bottles served as con- structure of the bacterial communities, which developed in trols which were free of DMSP supplements. The methane the microcosm-experiments. Water samples were fixed with concentration was measured as described above. Measure- buffered paraformaldehyde solution. After about 2 h the ments were conducted at the beginning of the experiment and cells were immobilized on white polycarbonate filters and after 3, 6, 8, 10 and 12 days by sacrificing one bottle spiked rinsed with phosphate-buffered saline and distilled water. with DMSP and one control bottle at each time point. The in- Air-dried filters were stored at −20 C until further process- cubation was carried out at approximately in situ temperature ing. CARD-FISH analysis was conducted according to the (about 1 C) in artificial daylight to simulate the polar sum- method of Pernthaler et al. (2002) using horseradish peroxi- mer situation. The composition of the bacterial communities dase (HRP)-labeled oligonucleotide probes (ThermoHybaid; www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 150°E 0° 180° 30°E 150°W 60°E 1102 E. Damm et al.: Methane production in aerobic oligotrophic surface water 120°W D E FG Fig. 1. Map of the central Arctic Ocean showing the spread of Pacific/Atlantic water as blue and colorless areas, respectively. Dots indicate stations along two transects. Profiles for red transect running from east to west are shown in diagrams (A) to (G). (A) shows the potential density in sigma θ units, (B), the concentration of oxygen (μmol/L), (C), NO (μmol/L), (D), PO (μmol/L) (E), DMSPt nM), (F), CH 3 4 ( 4 (nM) and (G), δ C-CH as (‰ PDB). Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ 90°E 90°W 60°W 120°E 30°W E. Damm et al.: Methane production in aerobic oligotrophic surface water 1103 Ulm, Germany) with Alexa546 as the reporter signal. The 4.2 Phytoplankton bloom and nutrient availability standard set of oligonucleotide probes were applied specific Primary production in the central Arctic is essentially reg- for the kingdoms of Eubacteria and Archaea, for the do- ulated by sea ice dynamics. The melting ice exposes the main Bacteroidetes and the subclasses alpha- and gamma- nutrient-rich surface waters to more light, which triggers the proteobacteria. The probe Non338 (Wallner et al., 1993) was phytoplankton bloom. However, nutrients in near-surface used to test for non-specific probe binding. Hybridization water are rapidly exhausted as the density stratification in- temperature was 35 C. Air-dried hybridized samples were hibits an upward transport and replenishment of nutrients counterstained with 4’, 6’-diamidino-2-phenylindole (DAPI; −1 (Sakshaug, 2003). High phosphate and nitrate concentra- final concentration 1 μg ml ). Samples were evaluated un- tions are therefore present in subsurface water (>50 m water der an Axioplan2 epifluorescent microscope equipped with depth). appropriate filter sets for Alexa546 and DAPI fluorescence. Between 800 and 2000 DAPI-stained objects were counted In near-surface water nitrate becomes depleted and even- tually exhausted in the top layers. Phosphate, however, is per probe and sample. only limited in Adw, while in Pdw excess phosphate remains available up to the sea surface (Fig. 1). Nitrate and phosphate are the primary regulators of phytoplankton growth and con- 4 Results and discussion sequently varying availability induces different bloom stages 4.1 Excess methane in Pacific derived surface water in the two water masses. Low chlorophyll a concentrations were generally en- Fresh water anomalies traced in the past decade between the countered, with those in the Pdw 0.11 μg/L (range 0.06– East Siberian Sea (Jones et al., 2008) and Fram Strait (Rabe 0.22 μg/L) being lower than in Adw 0.33 μg/L (range 0.19– et al., 2009) suggest that several years are needed for sur- 0.48 μg/L). The composition and the size of unicellular face waters to cross the deep Arctic Basin. Additional sea-ice plankton organisms also varied in the two water masses. In cover in the central Arctic impedes the gas exchange between Adw, diatoms (55%) were the dominant species followed atmosphere and ocean. Hence, under-ice methane consump- by nanoflagellates (31%), ciliates and dinoflagellates (12%) tion is the main process which determines the methane inven- while in Pdw small nanoflagellates (85%) clearly dominated. tory in the central Arctic Ocean. As a result concentration The plankton composition in Adw represented a late spring becomes more and more depleted relative to the atmospheric to summer phase of new production, while the nitrate and equilibrium concentration while at the same time the resid- phosphate depletion in near-surface water reflected an ex- ual methane becomes increasingly enriched in C in rela- hausting bloom (Fig. 1). tion to the atmospheric carbon isotopic signature of methane In comparison, the composition and size of unicellular (Damm et al., 2007). plankton organisms in Pdw reflect an impoverished phyto- The isolation from the atmosphere and ongoing consump- plankton community. As phosphate excess is available the tion are clearly reflected in subsurface water (water depth phytoplankton growth is clearly limited by nitrate depletion >100 m) where the effects of under-ice consumption remain (Fig. 1). conserved due to the density of the highly stratified wa- ter column, which restricts vertical mixing during summer 4.3 Nutrient limitations and bacterioplankton (Fig. 1). In comparison, near-surface water (water depth composition <100 m) is influenced by air-sea gas exchange during sum- mer and as a result, methane tends to equilibrate with the During the phytoplankton growth the global mean ratio of atmospheric background. In polar water, methane concen- 16:1 between nitrogen and phosphorous remains preserved tration in equilibrium with atmosphere is estimated to range if both are consumed and released in the same constant between 3.5 and 4 nM. Actually, in Adw, the concentration ratio, while shifts of this ratio are induced by increasing is close to atmospheric equilibrium and reflects an air-sea oligotrophy (Redfield, 1958). A perturbation of the Red- methane exchange, which is also confirmed by the δ C-CH field ratio is expressed in the definition of N* (N*=(N- values (from −41 to −43‰ PDB). These correspond to a 16P+2.9 μmol/kg)·0.87), a quasi-conservative tracer (Gruber two component mixing between reservoirs with different iso- and Sarmiento, 1997). N* is about zero if the Redfield ratio topic compositions i.e. the δ C-CH value of the local ma- is retained, while positive and negative values of N* are as- rine background (−38‰ PDB in subsurface water) and the sociated with deviations from the conservative behavior. Al- δ C-CH value of the atmospheric reservoir (−47‰ PDB) though deviations from the global mean Redfield ratio are (Fig. 1). However, in Pdw, the methane concentration is evident in both water masses, N* remains constant about clearly elevated relative to the equilibrium level. Further- 1.8 μmol/kg in Adw, in contrast to Pdw where N* decreases more, the methane is more enriched in C compared to that from 1 to −1.5 μmol/kg reflecting increasing perturbation in Adw (up to −46‰ PDB). These two features combined, (Fig. 2). Methane excess increases where nitrate is the only are indicative of methane production in Pdw (Fig. 1). growth-limiting nutrient and N* is decreasing. www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 1104 E. Damm et al.: Methane production in aerobic oligotrophic surface water 2 ples suggesting that nitrogen depletion may in this case be compensated by dinitrogen fixation. Nitrogenases are gener- ally oxygen sensitive and therefore often accommodated in separate cell compartments (Gallon, 1992). In such an envi- ronment in which hydrogen is produced by nitrogenases, we suggest that methane may be formed from methylated com- pounds by eubacteria e.g. alpha-proteobacteria. 4.4 DMSP as precursors for methane formation in nitrate-stressed environment -1 y = 0.457x - 1.6359 R = 0.7856 In oligotrophic water growth rates for the bacterioplankton are limited, either by mineral nutrients or by organic car- -2 bon (Thingstad et al., 2008). An important component for 0 2468 10 12 the bacterial carbon demand is DMSP (dimethylsulfoniopro- DMSPt/methane pionate) (Simo et al, 2002) and in microcosm experiments, a significant increase in bacterial biomass production is in- Fig. 2. Relationship between N* and the DMSPt to methane ratio duced by the addition of DMSP as a C source while phos- for water samples of the central Arctic Ocean. Circles and squares phate was the second limiting nutrient once C limitation correspond to Pacific and Atlantic derived surface water (<50 m), was alleviated (Pinhassi et al., 2005). Consequently, phos- respectively. N* is a quasi conservative tracer concerning the N:P phate availability may influence the utilization of DMSP as ratio. N* of about zero reflects a constant ratio while deviations C source for bacterial biomass production. from zero are induced by perturbations of the Redfield ratio (Gru- In Pdw, excess phosphate is available for bacterial biomass ber and Sarmiento 1997, see text). In Pacific derived water, the production. The inverse correlation found between phos- DMSPt/methane ratio correlates with increasing deviations from the phate and DMSPt (r = 0.7), may be indicative of the fol- conservative N:P ratio. lowing: First, the correlation simply indicates the increas- ing degree of oligotrophy. Second, the decreasing DMSPt During phytoplankton senescence, the activity of bacteri- concentration reflects an increasing utilization of DMSPt as oplankton is stimulated by the release of nutrients and dis- carbon source in the Pdw where phosphate is available for solved organic matter. In the central Arctic where only low the bacteria (Fig. 3). The following features corroborate this bacterial biomass is detected (in Pdw on average 4.8×10 assumption. In Pdw, phosphate is also significantly corre- and in Adw on average 17.7×10 bacterial counts per ml) lated with methane (r = 0.9), (Fig. 3) and a correlation be- the quantity differences correspond to those of the chloro- tween DMSPt decrease and methane production has previ- phyll between the two water masses. ously been found (Damm et al., 2008). The composition of the bacterioplankton in selected sam- The differences in methane, DMSPt and nutrient cycling ples of the Adw and Pdw was examined by means of DGGE between Pdw and Adw are illustrated in the correlation/non- based on the phylogenetically relevant 16S rRNA gene. In correlation between N* vs. the DMSPt/methane ratio respec- all water samples DGGE-bands of eukaryotic chloroplast tively. We find a coupling of DMSPt decrease with methane DNA were found while only few bands could be assigned formation on the one hand, while on the other the decreasing to prokaryotic taxa. A preponderance of sequences in the DMSPt/methane ratio is correlated with an increasing pertur- Pdw samples belonged to the alpha-proteobacteria subclass bation of the N:P ratio (i.e., more negative N*, Fig. 2). Thus, and these had very high similarities (99%) to sequences the coupling of several environmental factors is apparently of uncultured Sulfitobacter, Roseobacter, and Phaeobacter obligatory for methane to be formed as a by-product of a yet types within the family Rhodobacteraceae. Bacteria of the unexplained metabolic pathway. Rhodobacter/Roseobacter group are frequent in oligotrophic ocean surface waters but mostly uncultured. Cultured rep- 4.5 Implications for a potential pathway resentatives of this group are known for their highly diverse and flexible metabolism. A survey of available Roseobac- 4.5.1 Methane formation in DMSP spiked sea water ter genomes by Moran et al. (2007) revealed that 50% of the genomes contained genes for DMSP demethylation, 92% for In a microcosm experiment using Arctic surface water we phosphonate use, 25% for aerobic anoxygenic phototrophy, could show a microbial degradation of DMSP to methane and 92% for carbon monoxide oxidation. Further, different (Fig. 4). After 12 days considerable amounts of methane strains of Rhodobacter sphaeroides are able to fix dinitro- were formed in the water supplemented with DMSP while gen and possess the nifH (nitrogenase) gene (Moran et al., methane concentrations in the control bottles remained un- 2007). We could also amplify the nifH gene in the Pdw sam- changed. DGGE- as well as FISH-analyses revealed a clear Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ N*[µmol/kg] E. Damm et al.: Methane production in aerobic oligotrophic surface water 1105 Fig. 4. Formation of methane during a microcosm experiment with Fig. 3. Relationship between phosphate and DMSPt (left panel) Arctic surface water supplemented with DMSP (red line, closed cir- and phosphate and methane (right panel) for water samples from cles) and without DMSP (green line, closed rectangles). A change the central Arctic Ocean. Circles and squares correspond to Pa- in the bacterial community structure was followed up by CARD- cific and Atlantic derived surface water (<50 m), respectively. In FISH using general probes to detect Bacteria, alpha- and gamma- Pacific-derived water, phosphate is inversely correlated with DM- Proteobacteria, Bacteroidetes. and Archaea, Cyanobacteria were SPt and correlates with methane, while in Atlantic-derived water, determined by their morphology and yellow fluorescence at UV- no correlations exist. light. alteration of the bacterial communities in the DMSP-spiked 4.5.2 Thermodynamic calculations bottles. After 8 days the originally diverse DGGE-pattern with several light bands, had reduced to 3 dominant strong Moran et al. (2008) proposed a new metabolic pathway for bands resembling the sequences found in the Pdw and closely the anaerobic methane oxidation where methanethiol acts as related to sequences of uncultered Rhodobacter, Sulfitobac- an intermediate. The methanethiol is formed via two re- ter, and Mesorhizobium types. The CARD-FISH analysis actions, one dealing with oxidation of methane, the other, (Fig. 4) complemented this result showing that Archaea re- with reduction of CO . These reactions are coupled in such mained negligible in the DMSP supplemented approaches a way that the reduction equivalents (like H ) released by while Bacteria (hybridizing with the general bacterial probe) methane oxidation are immediately used by CO reduction. became nearly 100% of the community. After 8 days, alpha- Therefore, in the overall equation for methanethiol produc- and gamma-proteobacteria together accounted for more than tion, the contributions from the reduction equivalents cancel 75% of the DAPI-stained cells with the yet unsolved puzzle each other out. This tight coupling of the two half reactions that no dominant gamma-proteobacterium sequence could be minimizes the loss of reduction equivalents to competing ox- isolated. As in the Pdw samples, the nifH gene could be idative reactions. The latter is an important issue, especially amplified from all three DMSP supplemented samples show- under aerobic environmental conditions. Here, we suggest ing a clear increase over the course of the experiment. This that the pathway of methanethiol formation (Moran et al., further corroborates a close link between nitrogen fixation, 2008) operating in its reverse direction might also explain DMSP degradation, and methane production by Eubacteria. the production of methane. Hence, we propose the following It was shown experimentally, in a phosphate-free environ- methane formation reaction: ment, that methylphosphonate (MPn) acts as a precursor for 4 1 5 4 − + − methane production (Karl et al., 2008). We postulate that H CSH+ H O → CH + HCO + H + HS (1) 3 2 4 3 3 3 3 degradation products of DMSP may serve as precursors of The Gibbs free energy change (1G) for reaction (1) can be methane if phosphate is available for bacteria. Although in calculated using the following equation: phosphate-replete and phosphate-free environments respec- tively, bacteria utilize different P sources, methyl groups are 1 4 − / − / 3 3 a common feature of the molecule structure of MPn and 0 [CH ]· HCO · HS 0 3 1G = 1G +R·T · ln (2) DMSP degradation products. Thus, methane formation by [H CSH] both precursors is likely to occur in oligotrophic sea wa- 3 ter with methylotrophic methanogenesis being the potential (T is the Temperature in Kelvin, R is the universal gas con- pathway. A potential degradation product of DMSP and a po- − − stant, [H CSH], [CH ], [HCO ] and [HS ] designate con- 3 4 tential direct precursor for methane is methanthiol which is centrations in mol/L). For a reaction under physiological produced by the demethylation and cleavage pathway (Kiene conditions it is more appropriate to define the reference [H ] et al., 2000). differently from the standard concentration of 1 M (pH=0). The Gibbs free energy change at the appropriate neutral pH www.biogeosciences.net/7/1099/2010/ Biogeosciences, 7, 1099–1108, 2010 1106 E. Damm et al.: Methane production in aerobic oligotrophic surface water and with all reaction partners, except protons, kept at stan- A bacterial cell needs a minimum of about −20 KJ per dard concentrations is labeled 1G . It is calculated for re- mol to exploit the 1G in a metabolic reaction (“biologi- action (1) from cal energy quantum”, Schink, 1997). Hence, our result for −1 1G of −35.7 KJ mol indicates that methane production 0 5 0 0 1G = 1G −2.3026·R·T · · pH (3) via reaction (1) is consistent with the constraints of ther- (T ) modynamics and the biological energy quantum. It should be noted here, that 1G of reaction (1) calculated for the where 1G is the free energy change at standard concen- (T ) high intracellular [HCO ] and [HS ] represents a conser- trations of all reaction partners corrected for temperature. 3 vative estimate. The methane production via reaction (1) be- The 1G at an actual temperature (T ) can be calculated (T ) comes more favorable for seawater concentrations at pH=8.2 from the free energy change at the standard reference temper- −1 (1G < −35.7 KJ mol ). ature (T = 298.15 K) by means of the van’t Hoff equation ref The pathway for the reaction (1) suggested by Moran (Atkins, 1990) et al. (2008) involves CoM which is so far only found in T T −T ref 0 0 0 anaerobic methanogenic Archeabacteria. There is no proof 1G = 1G · +1H · (4) (T ) (T ) (T ) ref ref T T ref ref for its presence in aerobic seawater. However, there is a structural similarity between CoM and MPA (Mercaptopro- with 1H being the standard free enthalpy change at tem- (T ) ref pionate), which is a further product of aerobic demethylation perature T . The derivation of Eq. (4) assumes that 1H ref (T ) ref of DMSP. Hence, the MPA might substitute for CoM in the is not greatly changed from 1H within a (physiologically formation of methane. (T ) reasonable) temperature interval T –298.15 K. It is usually true that 1H for reactions varies rather slowly with T, (T ) 5 Conclusions provided no phase changes occur in the temperature inter- 0 0 0 val. 1G and 1H and in turn 1G can be cal- (T ) (T ) (T ) ref ref In the central Arctic Ocean a shift from a phytoplankton culated with the tabulated values of standard free energies bloom situation to oligotrophic conditions occurs during and enthalpies of formation from the elements for H O , 2 (l) − summer both in Atlantic and Pacific derived water where ni- H CSH , CH , HCO , and HS (Lide, 1999). Since re- 3 (g) 4(g) trate limits primary production. Nitrate depletion appears to action (1) proceeds in the aqueous phase, the value of 1G (T ) be a primary requirement for methane production in aerobic is corrected for the dissolution of H CSH and CH in 3 (g) 4(g) surface water. A second pre-condition in the high latitudes is water using the Henry’s law constant (k ), which describes the phosphate excess, which may be utilized by bacteria as the equilibrium partitioning of H CSH and CH between the 3 4 a P source. Where phosphate is available as a source of P, gas and the aqueous phase: 1 G = −R · T ln k . The soln H (T ) methylated compounds like DMSP and its degradation prod- temperature dependence of k is given by the equation: ucts may serve as the bacterial C source. When a combina- −1 H 1 1 soln tion of these conditions exists, methane may be a metabolic k = k · exp · − (5) H H,T ref R T T by-product and its production could yield energy under aer- ref obic conditions. However, to prove the proposed mechanism where 1 H is the enthalpy of dissolution. The val- soln ongoing research is required. ues of the Henry’s law constants at standard conditions Methane production may occur as a rapid response to envi- (k ) and of 1 H are available from Sander (1999) H,T soln ref ronmental perturbations during the shift from a phytoplank- −3 (methane: k = 1.3×10 M/atm and −1/R × 1 H H,T soln ref ton bloom to an oligotrophic system, induced by a switch −1 = 1800 K; methanethiol: k = 2.0×10 M/atm and H,T ref in the utilization of phosphate and methylated compounds. −1/R × 1 H = 2800 K). At the environmental tempera- soln Hence, methane production in aerobic surface water is di- ture of 2 C it follows from Eqs. (3–5) that reaction (1) is rectly linked to the N, P and C cycles. Recent change in the exergonic under standard conditions at the intracellular pH 0 0 Arctic has altered seasonal ice coverage and density stratifi- 0 −1 0 of 7 (1G = −33 KJ mol ). Using our result for 1G , the cation of surface water, which may have profound effects on Gibbs free energy change (1G) for the actual concentrations these biogeochemical cycles. Thus, feedback effects on cy- of the reaction partners can be calculated by means of Eq. (2). cling pathways of the climatically relevant biogases methane We simulate respiration within the cell by assuming [HCO ] and DMS are likely, with DMSP catabolism in high latitudes = 30 mM and [HS ]=1 mM. From the field data we estimate possible contributing to a warming effect on the earth’s cli- [H CSH] = 1 nM and [CH ]=10 nM. Then, the Gibbs free 3 4 mate through production of the greenhouse gas, methane. energy change that derives from Eq. (2) is given by 1G = −1 − 35.7 KJ mol . Thus, for the assumed concentrations of the reaction partners energy is released during methane for- mation. Since N -fixation is a highly energy consuming pro- cess, methane production could contribute additional free en- ergy for bacterial growth. Biogeosciences, 7, 1099–1108, 2010 www.biogeosciences.net/7/1099/2010/ E. Damm et al.: Methane production in aerobic oligotrophic surface water 1107 ◦ ◦ Acknowledgements. We are grateful to Ingrid Stimac and the Atlantic Ocean between 50 N and 52 S: Latitudinal distri- Jutta Jur ¨ gens who participated in the work at sea and in the home bution and sea-to-air flux, Deep-Sea. Res. Pt. II, 56, 964–976, lab. We thank the scientific party and crew of RV Polarstern for doi:10.1016/jdsr2.2008.12.002, 2008. their support at sea. The participation of R. P. Kiene was supported Gallon, J. R.: Tansley Review No. 44, Reconciling the incompati- by grants OPP-0230497 and OCE-0724017 from the US National ble: N fixation and O , New Phytol., 122, 571–609, 1992. 2 2 Science Foundation. 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