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Applied and Environmental Microbiology, May 2005, p. 2303-2309, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2303-2309.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Contribution of Archaea to Total Prokaryotic Production in the Deep Atlantic Ocean

Department of Biological Oceanography,¹ Department of Physical Oceanography, Royal Netherlands Institute for Sea Research, 1790 AB Den Burg, The Netherlands,² Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany³

Received 9 September 2004/ Accepted 29 November 2004

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Prokaryotic plankton are the major driver of biogeochemical cycles in the ocean. While the importance of bacteria in the oceanic biogeochemical cyles is well established, particularly for surface waters, our knowledge of the metabolically active fraction of the prokaryotic community in the mesopelagic and bathypelagic realms of the ocean, comprising about 70% of the ocean's volume, is rather rudimentary. Using fluorescence in situ hybridization (FISH) and polynucleotide probes, it has been shown recently that planktonic Archaea, consisting of two major groups, the Crenarchaeota and Euryarchaeota, might account for about one-third of all prokaryotic cells in the global ocean (15). In the Antarctic Circumpolar Current, however, the abundance of archaea was found to be generally lower than at the Hawaiian Ocean Time-Series (HOT) station (4). In the deep mesopelagic and bathypelagic realm of the HOT station in the Pacific gyre, the abundance of Crenarchaeota is about equal to that of bacteria (15). In this paper, we present the distribution of bacteria, Crenarchaeota, and Euryarchaeota in the mesopelagic and bathypelagic waters of the North Atlantic, following the path of the North Atlantic Deep Water (NADW) over a transect of more than 4,000 km using a modified catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes (33).

Up to now, planktonic archaea have resisted all attempts at culture. Consequently, nothing is known about their metabolic requirements. Compound specific stable isotope analysis on membrane lipids specific for Crenarchaeota indicates that they might be chemoautotrophic, fixing inorganic carbon (25, 37). However, it was also shown that planktonic archaea might take up amino acids (22).

There are only a few estimates of bulk prokaryotic activity in deep waters, none of them at a single-cell level. Thus, the fraction of metabolically active cells among the deepwater prokaryotic community taking up specific compounds remains unknown. We used the CARD-FISH method combined with microautoradiography (MICRO-CARD-FISH) to determine the fraction of the bacteria, Crenarchaeota, and Euryarchaeota metabolically active in the deep waters of the North Atlantic.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Sample collection.
Sampling of mesopelagic and bathypelagic waters was carried out along a >4,000-km-long transect from Bermuda to east of Greenland, following the western branch of the NADW to its origin during the TRANSAT-2 cruise with R/V Pelagia in May-June 2003 (Fig. 1). Samples were taken from the 100-m layer, the oxygen minimum, the Labrador Seawater (LSW), the NADW, and, if present, the Denmark Strait Overflow Water (DSOW). For the depth range and the physical characteristics of the sampled water masses, see Table 1. Additionally, samples were collected in the southern Atlantic Ocean at the Polar Front between depths of 200 and 3,000 m during the Eifex cruise on board R/V Polarstern between January and March 2004. Along the transect in the North Atlantic, water masses were identified based on their salinity-temperature characteristics (35, 36). Water from the distinct water masses was collected with Neox bottles mounted on a conductivity-temperature-depth (CTD) frame during the TRANSAT-2 cruise and with Niskin bottles during the Eifex cruise. Samples were taken for the enumeration of total picoplankton, bacteria, and archaea and fixed instantly with formaldehyde (2% final concentration).

View larger version (62K): [in this window] [in a new window]   FIG. 1. Map of stations occupied during the TRANSAT-2 cruise (indicated by dots) in May-June 2003 in the North Atlantic following the NADW.

MICRO-CARD-FISH.
To 30- to 40-ml samples, [³H]leucine (specific activity [SA], 157 Ci mmol^–1; Amersham) at a final concentration of 20 nM or [¹⁴C]bicarbonate (100 µCi; SA, 54.0 mCi mmol^–1; Amersham) was added, and the samples were incubated in the dark at in situ temperatures for 8 to 10 h and 60 to 72 h, respectively. Controls were fixed with paraformaldehyde, 2% final concentration. Incubations were terminated by the addition of paraformaldehyde (2% final concentration), and the samples were stored in the dark at 4°C for 12 to 18 h. The autoradiographic development was conducted by transferring previously hybridized filter sections onto slides coated with photographic emulsion (type NTB-2, melted at 43°C for 1 h). Subsequently, the slides were placed in a dark box with a drying agent and exposed at 4°C for 36 to 48 h. The slides were developed and fixed using Kodak specifications (Dektol developer [1:1 dilution with Milli-Q water] for 2 min, a rinse with Milli-Q water for 10 s, and in fixer for 5 min, followed by a Milli-Q water rinse for 2 min). Cells were counterstained with a DAPI mixture (5.5 parts of Citifluor, 1 part of Vectashield, and 0.5 parts of phosphate-buffered saline amended with DAPI at a final concentration of 1 µg ml^–1). The silver grains in the autoradiographic emulsion were detected by switching to the transmission mode of the microscope. More than 700 DAPI-stained cells were counted per sample.

Bulk prokaryotic and archaeal production measurements.
Bulk prokaryotic activity was measured by incubating 30 ml of water in duplicate and one formaldehyde-killed blank with 10 nM [³H]leucine (final concentration, SA 157 Ci mmol^–1; Amersham) in the dark at in situ (±1°C) temperatures for 4 to 7 h. Thereafter, the incubation was terminated by the addition of formaldehyde (2% final concentration) to the samples; the samples were then filtered through 0.22-µm cellulose nitrate filters (25-mm filter diameter; Millipore). Subsequently, the filters were rinsed with 5% ice-cold trichloroacetic acid, placed in scintillation vials, and stored at –20°C until being counted with a liquid scintillation counter. The disintegrations per minute (DPM) of the formalin-fixed blank were subtracted from the samples, and the resulting DPM were converted into leucine incorporation rates. These incorporation rates were further converted into bacterial carbon production values using the theoretical conversion factor with and without the intracellular dilution factor (x2) (30) and the empirically determined factors as described below.

Archaeal production was estimated via the incorporation of [¹⁴C]bicarbonate (100 µCi; SA, 54.0 mCi mmol^–1; Amersham) in 40-ml samples in duplicate and one formaldehyde-fixed blank incubated in the dark at in situ temperatures for 60 to 72 h. Subsequently, the incubations were terminated by the addition of formaldehyde (2% final concentration) to the samples, filtered onto 0.22-µm filters (Millipore, cellulose nitrate), and rinsed three times with 10 ml of ultrafiltered seawater (30-kDa molecular mass cutoff); the filters were exposed to a fume of concentrated HCl for 12 h. Then, the filters were placed in scintillation vials and stored in the dark at –20°C until the radioactivity of the filters was counted. The resulting DPM of the samples were corrected for the DPM of the abiotic control and converted into organic carbon produced over time in three different ways. The most conservative estimate of archaeal production assumes that inorganic carbon is the sole carbon source. We also determined empirical conversion factors by following the increase in archaeal abundance and the corresponding increase in carbon fixation in unamended seawater batch cultures as described below.

Seawater batch cultures to empirically determine conversion factors.
On specific locations, water from the NADW was filtered through rinsed 0.8-µm polycarbonate filters to remove potential grazers. Two-liter seawater batch cultures were established in duplicate and incubated at in situ water temperatures (3.1°C) in the dark for 6 to 8 days. At irregular intervals, samples were taken to determine picoplankton, bacterial, and archaeal abundance and prokaryotic and archaeal production as described above. Empirical conversion factors were determined from the observed increase in cell abundance and the respective incorporation rates. For total prokaryotes, the increase in DAPI-stainable cells was related to the [³H]leucine incorporation rates by integrative (18) and modified derivative (8) approaches. For archaea, the increase in archaeal cells over time (Crenarchaeota and Euryarchaeota) was related to the radiolabeled inorganic carbon incorporated by integrative (18) and cumulative (3) approaches.

Prior to terminating the batch culture incubations, samples were taken to determine the number of cells taking up leucine and bicarbonate by using MICRO-CARD-FISH as described above.

For estimating C biomass production, the calculated prokaryotic and archaeal cell production was converted into C production by assuming a C content of 10 fg C cell^–1 (7).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial and archaeal abundance.
Bacterial and archaeal abundance lacked latitudinal trends in cell abundance along the transect in the North Atlantic; therefore, averages for the individual water masses are given in Fig. 2. Generally, the contribution of bacteria to picoplankton abundance was rather constant throughout the water column (at a depth of 100 m, 32% of DAPI counts; in the NADW, 30% of DAPI counts), as was that of the Euryarcheota (17% of picoplankton abundance). The contribution of Crenarchaeota, however, increased with depth from 18.5% in the 100-m layer to 26.4% of the picoplankton abundance in the NADW. A similar distribution pattern was obtained from depth profiles (down to a depth of 3,000 m) in the Antarctic Circumpolar Current (Fig. 3). However, while archaea comprised >50% of the prokaryotic abundance below a depth of 100 m in the North Atlantic, archaea never exceeded bacterial abundance in the Antarctic Circumpolar Current (Fig. 3). Church et al. (4) also reported a lower abundance of archaea than bacteria in the Antarctic Circumpolar Current with rRNA-targeted polynucleotide probes as also used in another study at the HOT station (15). Thus, our data confirm previous observations that archaea are apparently less abundant in the Antarctic Circumpolar Current waters than in both the open Pacific and the Atlantic. Generally, euryarchaeotal abundance reported by Church et al. (4) was <10% of the total picoplankton abundance, while Euryarchaeota in this study comprised 10 to 20% of picoplankton abundance between 500 m and 3,000 m (Fig. 3). Thus, our estimated euryarchaeotal abundance is about twice as high as reported previously (4, 15), probably due to the improved permeabilization procedure of the archaeal cell wall applied here. We used proteinase K instead of lysozyme, allowing a more efficient penetration of the oligonucleotide probe into the archaeal cell, as shown recently (33). An archaeal dominance of the picoplankton, as we found throughout the aphotic layer of the North Atlantic (Fig. 2), was also found in coastal waters and the Mediterranean Sea (9, 19, 22).

View larger version (65K): [in this window] [in a new window]   FIG. 2. Abundance of bacteria and archaea (Crenarchaeota and Euryarchaeota) enumerated with CARD-FISH in the different water masses of the North Atlantic (O2-min, O2 minimum). Horizontal bars indicate errors of the mean. Numbers on the right indicate the number of samples.

View larger version (56K): [in this window] [in a new window]   FIG. 3. Distribution of bacteria, Crenarchaeota, and Euryarchaeota in the water column of the Antarctic Circumpolar Current (at 49°08' to 49°24'S, 1°52' to 2°15'E) as percentages of DAPI counts. Columns indicate means, and bars indicate SDs of three depth profiles.

Uptake of specific compounds by bacteria and archaea assessed by MICRO-CARD-FISH.
MICRO-CARD-FISH of samples collected throughout the water column of the Antarctic Circumpolar Current revealed that the percentage of bacteria taking up leucine decreased from 42% at a depth of 200 m to 16% at a depth of 3,000 m (Fig. 4A). However, the percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend with depth, ranging from 6 to 35% and 3 to 18%, respectively (Fig. 4A). Bacteria taking up bicarbonate ranged from 0 to 2% at depths of 500 to 1,000 m; at a depth of 200 m, 4% of the bacteria took up bicarbonate (Fig. 4B). The percentage of Euryarchaeota and Crenarchaeota incorporating bicarbonate ranged from 7 to 23% and 6 to 18%, respectively (Fig. 4B). While the percentage of Crenarchaeota taking up inorganic carbon was rather constant between depths of 200 and 2,000 m and varied only between 6 to 8%, at a depth of 3,000 m about 18% of the Crenarchaeota took up bicarbonate. In contrast, the percentage of the Euryarchaeota taking up inorganic carbon decreased with depth (Fig. 4B).

View larger version (27K): [in this window] [in a new window]   FIG. 4. Percentage of bacteria, Crenarchaeota, and Euryarchaeota taking up leucine (A) and bicarbonate (B) at different depth horizons of the Antarctic Circumpolar Current as determined by MICRO-CARD-FISH. Horizontal bars indicate the errors of the mean of three depth profiles.

Bacterial uptake of inorganic carbon could indicate the presence of chemoautotrophic bacteria or the anaplerotic metabolism of bacteria responsible for the uptake of CO₂. It has been shown in laboratory studies that the anaplerotic uptake of CO₂ might be responsible for up to 5% of the cellular carbon in bacteria if easily metabolizable substrate is available (2, 29, 32). Whether anaplerotic metabolism of bacteria and/or archaea could be responsible for the observed bicarbonate uptake was tested in batch cultures with NADW processed through a 0.8-µm filter (Fig. 5).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Development of bacterial and archaeal abundance and DI14C uptake in unamended seawater batch cultures processed through a 0.8-µm filter. Water was collected from the NADW at 47.62°W, 53.45°N. Symbols represent means of duplicate cultures. Bars indicate the range; if not visible, the range was smaller than the symbol. The increase in archaea was almost exclusively due to Crenarchaeota.

Based on the increase in archaeal abundance over time in the seawater batch cultures and the bicarbonate incorporated, we can calculate the minimum carbon content of newly produced archaeal cells. Based on the assumption that inorganic carbon is the sole carbon source for archaea, we arrive at an average C content of 8.39 fg C archaeon^–1 (standard deviation [SD] = 2.01; n = 5). This is at the lower end of the C content of similar-sized open ocean bacteria (10 fg C bacterium^–1) (7) but provides evidence that it is unlikely that the incorporation of inorganic carbon as observed by MICRO-CARD-FISH is due to anaplerotic metabolism. Rather, it provides evidence that a substantial fraction of the carbon requirements of planktonic archaea might be derived from the uptake of inorganic carbon, supporting previous reports based on compound-specific stable isotope analysis (25, 37). As shown by microautoradiography combined with FISH in this work (Fig. 4A) and a previous study (22), archaea also take up amino acids. Therefore, bicarbonate might not be the only carbon source for archaea. As shown in Fig. 4B, dark inorganic carbon uptake by the picoplankton is largely restricted to archaea particularly in layers below 500 m; they might therefore be considered as, at least partially, chemoautotrophs (20, 25). As archaea numerically dominate the prokaryotic community in the North Atlantic from a depth of 100 m to the NADW (Fig. 2), archaea might represent a hitherto-unrecognized sink for inorganic carbon in the largest oceanic subsystem, the mesopelagic and bathypelagic realm.

Estimating bulk prokaryotic and archaeal production.
Our results obtained by MICRO-CARD-FISH suggest that the commonly used leucine incorporation method to estimate bacterial biomass production (17, 30) might actually be considered, at least to some extent, an estimate of total prokaryotic (bacteria plus archaea) biomass production. As bicarbonate uptake is largely restricted to archaea (Fig. 4B), it might serve as a proxy for archaeal biomass production.

The measured leucine incorporation was converted into prokaryotic production using different conversion factors (Table 2) (8, 30). To convert bicarbonate incorporation into archaeal biomass production, it was assumed that inorganic carbon is the sole carbon source of archaea and that the carbon dioxide uptake by bacteria via their anaplerotic pathway is insignificant, due to the low percentage of bacteria taking up bicarbonate (Fig. 4B) and their lower relative abundance compared to archaea below a depth of 100 m (Fig. 2). Additionally, the seawater batch culture-derived relation between the increase in archaeal abundance versus DPM of the radiolabeled bicarbonate incorporated (Fig. 5) was used to calculate the conversion factors by two approaches (3, 18). The five different conversion factors used to estimate prokaryotic production in the different water layers of the mesopelagic and bathypelagic North Atlantic resulted in prokaryotic production estimates differing by up to 1 order of magnitude (Table 2). The conservative estimates of prokaryotic production using the theoretical conversion factor with and without the intracellular isotope dilution taken into account (30) agree well with the few prokaryotic production data available from mesopelagic and bathypelagic realms of the global ocean (11, 21, 23).

Similar to bacterial and archaeal abundance, prokaryotic and archaeal production in the mesopelagic and bathypelagic water column of the North Atlantic did not show any latitudinal trends. Thus, the data were pooled according to the water masses. The most likely estimate of the actual prokaryotic and archaeal production of the main mesopelagic and bathypelagic water masses of the North Atlantic is shown in Fig. 6A. Mean prokaryotic production declined from the 100-m layer to the oxygen minimum layer at a depth of around 400 m from 20 µmol C m^–3 day^–1 to about 5 µmol C m^–3 day^–1, declining further to 1.2 µmol C m^–3 day^–1 in the NADW. Archaeal production generally followed the same decreasing trend with depth as prokaryotic production (Fig. 6A) and comprised, depending on calculation of the production rates, from 13 to 27% of total prokaryotic production in the oxygen minimum layer, from 41 to 84% in the Labrador Seawater, from 10 to 20% in the NADW, and from 12 to 24% in the Denmark Strait Overflow Water (Table 3).

View larger version (21K): [in this window] [in a new window]   FIG. 6. Prokaryotic and archaeal production (A) and turnover rate (production/biomass day–1) (B) in the different water masses of the North Atlantic. For abbreviations, see Table 1. In panel A, numbers on the right indicate the number of samples. Prokaryotic production is based on the theoretical conversion factor with an intracellular isotope dilution factors of 2 (30) (i.e., 3.1 kg C mol–1 leucine incorporated); archaeal production was calculated with the empirical conversion factor obtained from the batch culture incubations using the integrative approach (18).

As all our production measurements were made under surface pressure conditions, decompression of the cells could have influenced the production measurements. It might be that the contrasting patterns in the growth rates between archaea and bacteria observed in the short-term incubations (archaea exhibiting lower growth rates than bacteria) and in the batch cultures where archaea grew at higher rates than bacteria reflect differences in sensitivity to changing pressure conditions (Fig. 5). Clearly, more work has to be done on the influence of pressure on deep-sea prokaryotes.

Organic versus inorganic carbon utilization by planktonic archaea.
We presented evidence that archaea are actively growing in the oxygenated water column using, at least to some extent, bicarbonate or CO₂ as a carbon source as described previously for some Crenarchaeota (20, 25). Archaea numerically dominate the prokaryotic community in the North Atlantic below a depth of 100 m to the NADW. Therefore, archaea might represent a hitherto-unrecognized sink for inorganic carbon in the largest oceanic subsystem, the mesopelagic and bathypelagic realm. Combining the depth-integrated mean C fixation rate (derived from the conservative estimate that inorganic carbon is the sole carbon source for archaea) of 0.014 fmol C archaeon^–1 day^–1 and assuming that approximately 1.3 x 10²⁸ archaeal cells inhabit the global ocean (15), we arrive at a global inorganic carbon fixation in the dark ocean by archaea of 6.55 x 10¹³ mol C year^–1. Current estimates of oceanic methane formation, as a major sink of CO₂ in the dark ocean, amount to about 8 x 10¹¹ mol C year^–1 (16). Thus, archaeal dark C fixation might be 2 orders of magnitude higher than oceanic methane formation and is in the same range as global methane formation (approximately 4.5 x 10¹³ mol C year^–1) (16).

The metabolic requirements of planktonic archaea remain a major unsolved issue. CO₂ and O₂ and nitrate, as potential electron acceptors, are generally readily available throughout the dark ocean. Archaeal and bacterial growth rates remained rather constant throughout the water column from the oxygen minimum layer to the DSOW (Fig. 6B), reflecting the similar decrease in prokaryotic abundance and production with depth. Commonly, the availability of the energy source for prokaryotes is assumed to decrease with water column depth. While this is certainly true for heterotrophic bacteria, the energy source for planktonic archaea is unknown. A previous study suggested that archaea might use hydrogen as electron donor to reduce nitrate (31). Besides hydrogen, all the known electron donors for chemolithotrophic metabolism are only available at trace concentrations in the ocean's interior. A fraction of the required energy might also be derived from the uptake of dissolved organic matter (DOM), or some archaea utilize DOM while others live on an autotrophic mode. The uptake of amino acids as indicated by microautoradiography in this study could be an indication of the utilization of DOM as an energy source. Since archaea exhibited lower growth rates than bacteria in the short-term incubations, it might well be that some archaea are utilizing different fractions of the generally refractory DOM in the ocean's interior than bacteria. However, the question about the nature of the electron donor responsible for fuelling cell growth and CO₂ fixation in pelagic archaea remains to be answered.

	ACKNOWLEDGMENTS

 
We thank the captain and crew of the R/V Pelagia and R/V Polarstern for their help on the research cruises. Jan Hegeman performed the bicarbonate radioactivity work at sea. Victor Smetacek provided helpful comments on a previous version of the manuscript.

This work was funded by a grant from the Dutch Science Foundation-Earth and Life Sciences NWO-ALW (TRANSAT, project 811.33.004) to G.J.H. and a European Community Marie Curie Fellowship to E.T.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, 1790 AB Den Burg, The Netherlands. Phone: 31 222 369 507. Fax: 31 222 319 674. E-mail: herdl{at}nioz.nl.

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