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Dissolved organic matter and its utilization by bacteria during spring in the Southern Ocean
Deep-Sea
Pergamon
II, Vol. 44,No. l-2, pp. 341-353, 1997 ScienceLtd 1997 Published by Elsevier Printedin GreatBritain.All rights reserved 09674645/97 $17.00 + 0.00
Research 0
PII: SO967-0645(96)00071-9
Dissolved organic matter and its utilization by bacteria during spring in the Southern Ocean PAUL
KAHLER,*
PETER
K. BJBRNSEN,f AVAN ANTIAS
(Received 20 November 1995; in revisedform
KARIN
LOCHTE*
and
30 January 1996; accepted 13 June 1996)
AbstractXoncentrations of dissolved organic carbon (DOC) and nitrogen (DON) were measured during early austral Spring 1992 at a number of stations along the 6”W meridian between 47”and 60”s. This included the Polar Front in the north, the zone of melting sea-ice in the south, and waters of the Antarctic Circumpolar Current in between. Concentrations of DOC were low in deep water (34-38 PM) with generally similar or slightly higher values in the surface mixed layer (38-Z pM). DOC:DON ratios are wider in surface water than in deep water, i.e. surface accumulations contain relatively C-rich dissolved organic matter. The highly variable distribution of the surface DOC was not related to hydrographic or biotic features (fronts, plankton development) indicating the lability and transient occurrence of this material. Growth rates of bacteria were determined in subsamples from 5 1 0.8~pm-filtered batches of seawater incubated in the dark at in-situ temperature. Thymidine and leucine uptake and bacterial biomass change as well as changes in dissolved organic carbon in the batches, and oxygen consumption in parallel incubations correlated linearly over 2 weeks of incubation which allowed extrapolation to in-situ conditions. Bacterial growth in these experiments depended strongly on the amount of initial DOC. Growth in water from greater depth (1000 m) containing 38 PM DOC was minimal, as were DOC-decrease and oxygen consumption. Higher rates were observed in surface water slightly enriched with DOC, and highest rates in surface water amended with DOC-rich melted sea ice. Bacterial growth efficiencies (biomass C-increase vs DOC consumed) were about 30%. The experiments showed that at least 4& 60% of the DOC in excess of deep water concentrations was available to bacteria. 0 1997 Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION Little is known about the quantity and quality of dissolved organic matter and its significance for carbon cycling in the ocean. After a period of confusion about the level of dissolved organic carbon (DOC) measured by high temperature catalytic oxidation techniques (Hedges et al., 1993; Suzuki, 1993), this problem has been resolved by establishing a proper blank correction procedure. Recent international intercalibration exercises proved that a number of workers could reproduce DOC-measurements in oceanic samples (Sharp, 1993, 1994). Although recent concentration measurements (with blank correction) are in the lower range of pre-Suzuki values, oceanic DOC is one of the largest carbon reservoirs on earth, and its behaviour is of interest in the context of global carbon
* Institut fiir Ostseeforschung, Seestr. 15, D-181 19 Wamemiinde, Germany. t Marine Biological Laboratory, Strandpromenaden 5, DK-3000, Helsingsr, Denmark. $ Sonderforschungsbereich 313, Universitlt Kiel, Olshausenstr. 40, D-24123 Kiel, Germany. 341
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cycling. However, a large pool size is not in itself a reason to assume oceanic DOC to be a relevant component of oceanic, and global, carbon fluxes. Whether there is a pathway of CO2 removal from the atmosphere (and the upper ocean that interacts with it) involving oceanic DOC is largely a question of its reactivity and the timescale of its turnover. A pool of chemically and biologically inert DOC would obviously not interact with other carbon pools or contribute to any carbon fluxes, no matter how much of this material was present. A pool of reactive DOC on the other hand, respired soon after its production, would only transiently affect the carbon budget with no lasting effect on the fluxes in question. Carbon export from the surface via DOC is only possible by accumulation and subsequent deep mixing or downwelling, by eddy diffusion, or by its (efficient) transformation into sinkable particulate material. Any DOC respired in the surface mixed layer can be returned to the atmosphere as CO;?. Kirchmann et al. (1991) reported rapid consumption by bacteria of as much as one-third of a high apparent ambient oceanic DOC-level in the North Atlantic. Their DOCmeasurements, however, were made with an instrumental setup that was later (Suzuki, 1993) shown to produce erroneously high and inconsistent values. Bacterial carbon production amounted to only a few percent of the DOC apparently consumed in their study. In other studies in marine environments, the incorporation of DOC into bacterial biomass was found to be more efficient: 1 l-54% (Baltic Sea, Zweifel et al., 1993) and 40% (Weddell Sea, Bjornsen and Kuparinen, 199 1). Here, we report on the levels of DOC- and DON measured Antarctic waters during Spring 1992 in the Southern Ocean, its availability to bacteria as a food source, the efficiency with which it is transformed into bacterial biomass, and how much is respired.
MATERIAL
AND
METHODS
The results presented are from transects along the 6”W meridian between the Polar Front near 47”s and the ice edge near 6O”S, taken in October and November 1993 during the R.V. Polar-stern expedition ANT X/6. For the positions of the transects and general background, see Smetacek et al. (1997). For the DOC and DON analyses, lo-ml samples were drawn into clean sealed and freshly opened glass ampoules directly from the Niskin bottles, acidified with 200 ~1 42% phosphoric acid, resealed, and stored refrigerated until analysis on board (odd station numbers) or later on land (even numbers). The samples were not filtered to avoid a source of contamination; hence total organic carbon (TOC) and total nitrogen (TN) were measured, DOC and DON were obtained by subtracting particulate, and in the case of nitrogen also dissolved inorganic, C and N, respectively. The magnitude of this correction was 0.5-10 PM POC (see also Fig. 2), 0.1-2 PM PON and 2s-35 PM DIN. The acidified samples were stripped of inorganic carbon by passing a stream of CO*-free gas (oxygen or argon) through them immediately prior to analysis; TOC and TN were then measured by a high temperature catalytic combustion technique with a modified “Dimatoc 100” (Dimatec) total carbon analyser where they were oxidized to CO2 and NO, respectively. Aliquots of 100 ,~l of seawater were injected into a quartz tube containing 50 g 5% platinum-on-alumina catalyst (Degussa) heated to 900°C covered with 0.5 g of platinum wool. The carrier gas was 5% 02 in argon at a flow rate of 100 ml min- ‘. The high temperature and the low oxygen content of the carrier gas were necessary for the complete recovery of nitrogen as NO and did not affect CO2 yield as compared with pure oxygen as
Dissolved organic matter
343
the carrier gas. Glass tubes filled with zinc and bronze, or 10 g of Sulfix (Wake Chemicals) at 500°C served to scrub HCl and SO2 from the combustion gas and ice/water and Mg(ClO& traps to remove moisture. Carbon dioxide was measured in a “Binos 100” (Rosemount) non-dispersive infra-red detector and NO in an “Antek 720” (Antek) chemoluminescence detector in line behind, and the area of the resulting peaks determined with a chromatography program (“Boreal”, Flowtech). At least four injections were made per sample. Calibration was against seawater spiked with glucose and urea (five concentrations) and a standard gas mixture of CO2 and NO in nitrogen. Blanks were determined with “Milli-Q” water twice distilled from persulphate/phosphoric acid according to Benner and Strom (1993). A number of ampoules were filled from a bottle of one of the first casts and the measurement of this water included in each calibration to check the stability of the measurements. This reference water was later measured against water from an international intercalibration (Sharp, 1994) to ensure general comparability of the data. Experience with the combustion unit used had shown that the blank and the response were variable over time with both the DOC and TN measurements. We achieved significant improvements in the reproducibility and long-term stability of calibrations through the use of a catalyst precombusted at 1250°C and the alternate injection of low-carbon deionized water to strip traces of CO* and NO absorbed by sea-salt residues in the cold portion of the combustion tube. Calibrations and measurements of reference water were made before and after each run of 12 samples. The precision of both the TOC and TN measurements was between 0.3 and 3 PM (standard deviation of replicate injections given in Fig. 1). Since this relatively high variability stems from the fact that CO* and NO are deposited and remobilized in the cold portions of the combustion tube, a mean value of four injections is closer to the accurate value than indicated by the variability of the single measurements. The overall error for DOC and DON should include the uncertainties in the determinations of POC, PON, nitrate and ammonia used to calculate them, which are not considered here. For the incubation experiments, water was filtered through 14-cm-diameter/O.8-pm-poresize polycarbonate filters (Costar) with precombusted GF/C (Whatman) prefilters using a peristaltic pump. This was to exclude the bulk of particulate matter and predators of bacteria, but to include bacteria in the filtrate. To avoid clogging of the filters and the buildup of backpressure, the filters were changed frequently during filtration. A third to half of the bacterial count passed into the filtrate. The water used was deep water (from 1000 m depth), surface water (from 20 m depth), and surface water amended with l/3 melted brown sea ice. Two sets of experiments were conducted (at 56”3O’S;6”W, starting 23 October and 13 November). In each, two parallels of each batch were incubated at 1“C in the dark in wellleached 5-l polyethylene bottles, and a number of 100-ml glass bottles for the oxygen determinations. They were subsampled over a period of 2 weeks as indicated in Fig. 3. For the analysis of particulate organic matter in the incubation experiments, 2-l subsamples were filtered on to precombusted GF/F-filters at the beginning and end of the experiments. Although GF/F-filters are not much different in pore size from the polycarbonate filters that we used to filter the water before setting up the experiments (in order to include a seeding population of bacteria in the filtrate), we were able to recover bacteria on these filters. This was because bacteria growing during the incubations were large, and, probably, the small area of the filters (2.5 cm in diameter) caused efficient clogging to trap particles smaller than the nominal pore size. Microscopic inspection of GF/ F-filtrates of the batches showed that only a few percent of the bacteria passed the filters,
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which means that bacterial growth was only slightly underestimated. Particulate organic carbon (POC) and nitrogen (PON) were measured by combustion of the filters in a Carlo Erba Elemental Analyser. Oxygen was measured in triplicate by a Winkler technique with automatic photometric endpoint detection following WOCE recommendations (Manuels, 1994), nutrients by standard procedures (Grasshoff et al., 1983). Growth rates of bacteria were determined by
60
Dissolved
organic
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the thymidine and leucine uptake method in lo-ml subsamples as described by Lochte et al. (1997). Bacteria were counted by epifluorescence microscopy of subsamples fixed in 2% formaldehyde solution, filtered on to 0.2~pm nucleopore filters and stained with DAPI; bacterial size was determined by comparison with fluorescent beads (Bjornsen and Kuparinen, 1991; Lochte et al., 1997).
RESULTS DOC and DON inventory Figure 1 shows the distribution of DOC and DON with depth for several stations. Note the relative uniformity of the profiles with no or only small increases of DOC, and in some profiles even DON minima, in the surface water. Low values at or slightly below 38 pmol DOC 1-l are found from 300 m down to the greatest depths measured (>4000 m); similarly, DON is constant at 8 PM in deep water, i.e. most variability is near the surface. Figure 2 shows the mean DOC accumulation in the top 100 m (depth-weighted averages) over the background value at 300 m depth along three transects taken between 11 October and 21 November along the 6”W meridian from 47” to 60”s. The surface DOC enrichment is extremely variable at a low level between 0 and 20 PM. There is no trend in the DOC level over the nearly 6-week period between the first and last transects. For comparison, the values of particulate organic carbon are more uniform and show the buildup of phytoplankton stocks in the Polar Frontal region.
Bacterial growth on DOM The experiments were designed to investigate bacterial growth with dissolved organic matter as the sole carbon source excluding mortality by predation which allows the measurement of bacterial production by biomass increase. They were also used as test systems for the calibration of bacterial production measurements (determination of conversion factors; Lochte et al., 1997) by the thymidine and leucine incorporation techniques (cf. Bjornsen and Kuparinen, 1991). Figure 3 shows the time course of the bacterial growth as measured by different parameters and changes in carbon and dissolved oxygen during the incubations. Clearly, the different sources of water support bacterial growth to different degrees. The higher the original content of dissolved organic matter, the higher are growth rates and bacterial biomass yield. The richest substrate is surface water amended with melted sea ice; deep water is the poorest substrate. The two sets of growth experiments (set 2 is a replicate of set 1, set up 3 weeks later at the same location) behaved similarly, with the exception that, in the meltice/surface-water variant of set 2, there was a decline in bacterial numbers and total volume in the last interval of the incubation period, with thymidine and leucine uptake still indicating growth. This odd behaviour may be caused by viral infection and lysis, which also may explain the higher thymidine:leucine uptake ratio. Correlations between the measured parameters are linear, except for the last interval of the second meltice-enriched surface water variant. Correlation coefficients (r’) are generally well above 0.9. Regression coefficients [conversion factors, CF determined following the procedure described by Bjornsen and Kuparinen (1991)] are, however, quite different for each batch. The CF from thymidine incorporation to cell number (1.2 x 1018 cells mol-‘) and from leucine uptake to
346
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et al.
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degr. S Fig. 2. Average contents of DOC in the upper 100 m in excess of that present at 300 m depth (= surface DOC accumulation, closed circles) and POC (lines) between 47% and 60”s along 6”W. Bottom: 11-22 October; middle: 24-31 October; top: l&21 November. The arrow marks the position of the ice edge; the polar front is at 49”s.
bacterial volume production (4.4 x 10” pm3 mol-‘), based on all 12 experimental units are consistent with literature values (Bjornsen and Kuparinen, 1991). The CF from bacterial volume to POC of Table 1 provide a carbon and nitrogen balance of the experiments. The differences between days 1 and 13 (means of two parallels) in DOC, POC, PON, bacterial volume, oxygen, and dissolved inorganic nitrogen are listed. In the deep water, the precision of the TOC and POC measurements was not sufficient to explain the observed low oxygen consumption and bacterial growth in a meaningful way. Deep water DOM was expected to be resistant to bacterial breakdown though (see below), so the small changes observed in the deep water probably give an indication of the maximum
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Fig. 3. Bacterial growth determined by 3H-thymidine and 3H-leucine incorporation (cumulative) and change in bacterial numbers and total bacterial volume (two parallels), and oxygen consumption (means of three parallels) in 0.8-pm filtered seawater from 20-m (circles), from 1000 m (squares), and from 20 m enriched with melted brown ice (triangles) filtered through 0.8 pm. Left and right panels represent two sets of experiments conducted (left: growth experiment 1; right: growth experiment 2).
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Table 1. Losses and gains of carbon, nitrogen, oxygen and bacterial total volume during the growth of bacteria on natural dissolved organic matter in surface water, deep water and meltice-enriched surface water (growth experiment I) Changes Initial DOC
TOC*
86
-20
52 38
-6 0
Ice-enriched Surface water Surface water Deep water *TOC (total organic
carbon);
particulate
POC (pmol I-‘)
in PON
Oxygen
DIN
10.5
2.45
-26.7
-1.4
493
1.8 1.1
0.55 0.19
-7.5 -2.0
-0.5 -
77 20
organic
carbon
formed during incubation
Bacterial
volume (pm3 I- ‘)
is included
level of organic contamination during incubation and handling of the samples. These data are given only to show the difference in scale regarding the changes observed in surface water. The other two variants (surface water and meltice-enriched surface water) show significant changes for every parameter measured. An increase in mean cell size was observed during the incubations (from 0.06 to 0.09 pm3 in the 20-m batches, and from 0.09 to 0.15 and 0.25 pm3 in the ice-enriched batches) which we interpret as being caused by selection for small bacteria by the filtration at the start and an effect of the absence of predators during the growth phase. This size shift did not affect growth characteristics; the relative proportions of thymidine and leucine incorporation, oxygen consumption and bacterial (volumetric) growth stayed the same. From this, we conclude that they should also apply to the natural bacterial community. DISCUSSION In-situ
DOC and DON concentrations
Deep-water concentrations of DOC are in the range of values encountered elsewhere in the deep ocean; concentrations of 34-38 PM are slightly lower than deep-water concentrations measured in the North Atlantic (42-43 PM, Bodungen and Kshler, 1994; Carlson et al., 1994) and similar to those in the Pacific (35-39 PM, Carlson and Ducklow, 1995; Peltzer and Hayward, 1996), pointing to a very slow breakdown of this material at timescales of ocean mixing along the main routes of deep-water circulation, a process that is almost complete in the region of our study. Hence, this material can be considered extremely recalcitrant under deep-sea conditions. The old age of such material (Williams and Druffel, 1987) is a consequence of this. The occurrence of surface minima of DON in some profiles suggests that a portion of deep-water dissolved organic matter (DOM) has been broken down, i.e. it is not stable under surface conditions. There is evidence that photodestruction and subsequent consumption by bacteria of some of its products is a sink of old DOM (Kieber et al., 1989; Mopper et al., 1991). Photobleaching of gelbstoff (Determann, 1995) also shows that some of this material is reactive under conditions encountered at the sea surface. Figures 1 and 2 show surface maxima though, of DON in some, and of DOC, in most cases. Lower DOC:DON ratios (up to 8) than in deep water (4-5) show that the additional
Dissolved organic matter
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material is relatively enriched in carbon. Possible sources of dissolved organic matter in surface water include a fraction of primary production (phytoplankton exudates, Bjornsen, 1988) and waste from zooplankton activities (inefficient feeding, leaching and excretion) (Lee and Henrichs, 1993). Addition of viral matter is considered insignificant in terms of carbon (Proctor and Fuhrman, 1991). Under the special conditions of the Southern Ocean, melting sea ice is an additional source of DOM to surface water (see below). Marked differences and patterns, in both space and time, were detected in the occurrence and production of phytoplankton and zooplankton during the cruise. Three zones of different hydrographic conditions (ice edge, Polar Frontal region, and water masses of the Antarctic Circumpolar Current in between, Veth et al., 1997) differed significantly in their biological development. Primary production was enhanced in the Polar Frontal region leading to the buildup of high phytoplankton stocks there, a lesser increase of productivity was observed near the retreating ice edge, and productivity stayed low in the waters in between (Mathot et al., 1994; Jochem et al., 1995). Stocks and productivity of zooplankton closely followed those of phytoplankton (Bathmann et al., 1997). The distribution and development of POC (Fig. 2) give an indication of this overall pattern. The observed differences in DOC concentrations in surface water, however, show no relationship with these biological stocks and activities, which are potential suppliers of DOC. At the ice edge, with ice melt initialising the seasonal cycle of pelagic production, the variability of DOC concentration is already very high. Situations with no enrichment at all, moderate enrichment, and exceptionally high concentrations in surface waters (ice-edge station 931, Fig. 1; not included in Fig. 2) were encountered in this region and under ice cover. This implies that at least part of the additional DOC in surface water is not connected with pelagic production, but is released from sea ice. Except for the extreme value mentioned, variability of the surface DOC enrichment was similar in the other hydrographic zones. Since there was no trend in DOC concentrations over the time between the transects (Fig. 2) we conclude that buildup and breakdown of DOC were generally coupled and in balance. Uncoupling seems to occur only transiently, reflected in higher concentrations at some stations, but the time scale of these accumulations must be much shorter than those of phytoplankton and zooplankton development. Similar results were obtained in a study by Skoog and Wedborg (1994), who measured TOC (total organic carbon) in the Weddell Sea later in the season and found concentrations in the same range as we did. In their study as well, concentrations at stations with high biological activity were not notably different from those at other stations. Bacterial
utilization
of DOC
The magnitude of DOC turnover is not evident from the in-situ concentrations but was determined experimentally. By eliminating larger phytoplankton and zooplankton, and incubating in the dark, the major sources of DOC were removed in the experiments, but bacteria passed the filter. Since heterotrophic bacteria are able to take up dissolved material and to respond quickly to available substrates, they are the most likely agents of DOC breakdown. In Table 2, various parameters derived from the data of Table 1, characterizing bacterial growth and substrate utilization are listed. These parameters show that bacterial growth with natural DOC being the only substrate is not exceptional in any way. The volume:carbon ratios of bacteria grown at 0.26 (surface water) and 0.28 (ice-enriched)
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P. KLhler et al. Table 2.
Parameters describing bacterial growth and carbon utilization of growth experiment 1 Bacterial
Ice-enriched surface water Surface water
C/vol.
(pg pm-3)
C/N
0.26 0.28
4.3 3.3
Grown
efficiency* 0.30 0.26
RQ 0.75 0.80
Fraction of additional DOC usedt 0.63 0.56
*Delta POC/delta TOC + POC. tAdditiona1 DOC = excess over deep water DOC.
agree well with recently published figures (Fry, 1988; Simon and Azam, 1989); their molar C:N-ratio is about 4. Growth efficiencies, i.e. bacterial carbon gained from DOC consumed are 0.25 and 0.3, respectively, which are within the range observed for many natural substrates (Lignell, 1990 and references therein). These observations are in contrast to those of Pomeroy et al. (199 1) who found that, at low temperatures (1 “C in our case) and at dilute substrate concentrations, bacteria have particularly low conversion efficiencies. The efficiencies that we observed are similar to those observed in temperate marine environments (Bjornsen, 1986; Zweifel et al., 1993). A conversion efficiency of as low as 2%, reported by Kirchmann et al. (1991) for temperate oceanic waters, is considered an artifact. The experiments show that a substantial fraction of the DOM in surface water is biologically labile and can be efficiently transformed into bacterial carbon. Half, or more than half, of the dissolved organic carbon in excess of the deep-water concentration was consumed over the period of 2 weeks. Since growth was still going on when the experiments were terminated, the potentially usable fraction should be even greater. The net consumption of inorganic nitrogen is probably due to the relative C-richness of the surface-accumulated dissolved organic matter. Growth rates of bacteria, which vary between 0.2 and 2 pg C per 1-l day- ’(Lochte et al., 1997), follow the trend of primary productivity, i.e. high rates are connected with the phytoplankton bloom in the Polar Frontal region. Hence, the bacteria can be assumed not to rely on the accumulated DOC, but on a substrate provided directly or indirectly from ongoing primary production. Given a growth efficiency of 0.3, the carbon demand of bacteria can be estimated by multipying their growth rates by 3.3; this results in a C-demand of 0.055-0.55 pmol per 1-l day-‘. If the supply of fresh substrate were cut off and the bacteria were to grow with the accumulated DOC as their sole carbon source, a mean concentration of 10 PM (Fig. 2) could support them for 18 days at the polar frontal zone to a maximum of 180 days near the ice edge, where minimal growth rates were observed. In summary, bacteria can consume enough DOC added from primary production to prevent seasonal accumulation, and they have the potential to cope with the small accumulations observed (whatever their source is) in a matter of months. Implications
for oceanic DOM dynamics
Many findings point to the stability of a substantial fraction of DOC: the old 14C age of DOC (Williams and Druffel, 1987), the absence of seasonal variability of deep-water DOC, and the very low variability of deep-water DOC worldwide (Bodungen and Kahler, 1994; Peltzer and Hayward, 1996; Carlson and Ducklow, 1995). On the other hand, there are
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reports on the lability of part of the surface water DOC (Amon and Benner, 1994) and of DOC freshly produced by algae (Lignell, 1990; Chen and Wangersky, 1993). The high variability of surface DOC concentrations observed in this study and elsewhere in the ocean (Seltzer and KHhler, in prep.), and the marked seasonality of oceanic surface mixed-layer concentrations of DOC (Bodungen and Kahler, 1994; Carlson et al., 1994) also point to the lability of a large DOC fraction in surface water. From these findings, it can be concluded that dissolved organic matter in the ocean is a mixture of compounds that span a wide range of persistence, from extremely labile to extremely recalcitrant. So far, the magnitude of the recalcitrant fraction and a fraction persistent for less than a year have been discussed. Whether there is a third fraction of intermediate stability is difficult to recognize. Such a component, formed in surface water and stable for years to centuries, should form a permanent gradient below the maximum depth of the mixed layer. In well-stratified waters, this has been observed (Peltzer and Hayward, 1996; equatorial Pacific; Kahler and Peltzer, in prep., North Atlantic). In waters of the Southern Ocean with their high mixing rates in greater water depth, such a gradient, should it exist, would be less steep and probably not detectable with the precision of the DOC measurements. This shows that such a DOM fraction is of minor importance only. It is only such material that would be available for export. Although only a few studies of DOC in the Southern Ocean have been performed, there exists a wide variety of opinions concerning its importance there. The reported levels span a wide range, some of which may reflect analytical difficulties, and some high concentrations are from special marine environments (Dawson et al., 1985). Dafner (1992) found a strong correlation of DOC content and primary productivity and reported DOC concentrations as high as 700 PM in the Polar Frontal Zone, i.e. more than 10 times higher than those that we found during the spring. Moriarty and Bianchi (1994) believe in a source of organic matter (dissolved or suspended) at, or south of, the Polar Frontal Zone, which is advected at intermediate depths to regions far North, in order to explain high bacterial productivity in intermediate waters there. They revive a hypothesis by Sorokin (1971) who had made similar observations: measured bacterial production requires more carbon than supplied from local primary production. This hypothesis has been refuted by Banse (1974), but has since found new attention during the period of confusion regarding the validity of wetchemical DOC determinations following the publication of Sugimura and Suzuki (1988) on the measurement of DOC by high temperature catalytic oxidation (see Karl, 1993 for a recent discussion). Other authors observed particularly low concentrations of dissolved organic matter in oceanic waters around Antarctica (Jackson and Williams, 1985; le Jehan and Treguer, 1985). Prego (199 1) only found substantial DOC accumulations near the pack-ice zone and very low concentrations elsewhere during austral summer in the Weddell Sea. Billen and Becquevort (1991) observed a time lag between primary and secondary production in southern polar waters of Prydz Bay and postulated the temporary accumulation of bioavailable DOC over the period of a month only. It has already been mentioned that the low DOC observed by us during spring and the lack of a relationship between DOC concentrations and biological activity continued later in the year (Skoog and Wedborg, 1994). This is in contrast to equatorial regions where stable accumulations of DOC were observed (Peltzer and Hayward, 1996) and temperate regions where larger transient accumulations occur seasonally (Bodungen and Kahler, 1994; Carlson et al., 1994).
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Hence, the Southern Ocean appears to be a region where DOC is not available for export, either vertically or horizontally, and a region that is poorer in DOC than other oceans. Acknowledgements-We acknowledge the help of Rinus Manuels (dissolved oxygen), Peter Fritsche (nutrients) Laetitia Teissier and Heike Siegmund (POC, PON), and Alexandra Nielsen (bacteria counts). Part of this work (P.K.) was supported by a grant of the German Research Foundation (DFG) and grant No. 03FOlOSF of the German Ministry of Research and Technology.
REFERENCES Amon
R. M. W. and R. Benner (1994) Rapid cycling of high-molecular-weight dissolved organic matter in the ocean. Nature, 369, 5499552. Banse K. (1974) On the role of bacterioplankton in the tropical ocean. Marine Biology, 24, l-5. Bathmann U. V., R. Scharek C. Klaas, C. D. Dubischar and V. Smetacek (1997) Spring development of phytoplankton biomass and composition in major water masses in the Atlantic sector of the Southern Ocean. Deep-Sea Research II, 44, 51-67. Benner R. and M. Strom (1993) A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation. Marine Chemistry, 41, 1533160. Billen G. and S. Becquevort (1991) Phytoplankton-bacteria relationship in the Antarctic marine ecosystem. Polar Research, 10, 245-253. Bjornsen P. K. (1986) Bakterioplankton growth yield in continuous seawater cultures. Marine Ecology Progress Series, 30, 1057-1075. Bjornsen P. K. (1988) Phytoplankton exudation of organic matter: why do healthy cells do it? Limnology and Oceanography, 33, 15 l-l 54. Bjornsen P. K. and J. Kuparinen (1991) Determination of bacterioplankton biomass, net production, and growth efficiency in the Southern Ocean. Marine Ecology Progress Series, 71, 185-194. Bodungen B. and P. Kahler (1994) Dissolved organic carbon in the Atlantic-seasonal and regional patterns. Eos, Transactions, American Geophysical Union, Supplement, 75, 106. Carlson C. A. and H. W. Ducklow (1995) Dissolved organic carbon in the upper ocean of the central equatorial Pacfic Ocean, 1992: Daily and tinescale vertical variations. Deep-Sea Research ZZ,42, 639-656. Carlson C. A., H. W. Ducklow and A. F. Michaels (1994) Annual flux of dissolved organic carbon from the euphotic zone in the northwestern Sargasso Sea. Nufure, 371, 405408. Chen W. and P. J. Wangersky (1993) High-temperature combustion of dissolved organic carbon produced in phytoplankton cultures. Marine Chemistry, 41, 167-172. Dafner E. V. (1992) Dissolved organic carbon in waters of the Polar Frontal Zone of the Atlantic Antarctic in the spring-summer season of 1988-1989. Marine Chemistry, 37, 275-283. Dawson R., W. Schramm and M. Bolter (1985) Factors influencing the production, decomposition and distribution of organic and inorganic matter in Admiralty Bay, King George Island. In: Antarctic nutrient cycles andfood webs, W. R. Siegfried, P. R. Condy and R. M. Laws, editors, Springer, Berlin, pp. 109114. Determann S. (1995) Analyse biologischer und biochemischer Prozesse im Meer mit Fluoreszenzspektroskopie. Ph.D. Thesis, University of Oldenburg, 111 pp. Fry C. (1988) Determination of biomass. In: Methods in aquatic microbiology, B. Austin, editor, Wiley, Chichester, pp. 27-72. Grasshoff K., M. Ehrhardt and K. Kremling (1983) Methods ofseawater analysis. Chemie, Weinheim, 419 pp. Hedges J., B. A. Bergamaschi and R. Benner (1993) Comparative analyses of DOC and DON in natural waters. Marine Chemistry, 41, 121-134. Jackson G. A. and P. M. Williams (1985) Importance of dissolved organic nitrogen and phosphorus to biological nutrient cycling. Deep-Sea Research, 32, 223-235. le Jehan S. and P. Treguer (1985) The distribution of inorganic nitrogen, phosphorus, silicon, and dissolved organic matter in surface and deep waters of the Southern Ocean. In: Antarctic nutrient cycles andfood webs, W. R. Siegfried, P. R. Condy and R. M. Laws, Springer, Berlin, pp. 22-29. Jochem F. J., S. Mathot and B. Queguinier (1995) Size-fractionated primary production in the open Southern Ocean in austral spring. Polar Biology, 15, 381-392. Klhler P. and Peltzer E. (in prep.) Carbon export in the ocean: the role of DOC.
Dissolved
organic
matter
353
Karl D. M. (1993) Microbial processes in the southern oceans. In: Antarctic microbiology, E. M. Friedmann, editor, Wiley, New York, pp. 163. Kieber D. J., J. McDaniel and K. Mopper (1989) A photochemical source of biological substrates in seawater: implications for carbon cycling. Nature, 241, 637639. Kirchmann D. L., Y. Suzuki, C. Garside and H. W. Ducklow (1991) High turnover rates of dissolved organic carbon during a spring phytoplankton bloom. Nature, 352, 612-614. Lee C. and S. M. Henrichs (1993) How the nature of dissolved organic matter might affect the analysis of dissolved organic carbon. Marine Chemistry, 41, 105-120. Lignell R. (1990) Excretion of organic carbon by phytoplankton: its relation to algal biomass, primary productivity and bacterial secondary productivity in the Baltic Sea. Marine Ecology Progress Series, 68, 8599. Lochte K., P. K. Bjsrnsen, H. Giesenhagen and A. Weber (1997) Bacterial standing stock and production and their relation to phytoplankton in the Southern Ocean. Deep-Sea Research IZ, 44, 321-340. Manuels R. (1994) Dissolved oxygen. Reports on Polar Research, 135, 35-37. Mathot S., B. Queguinier and F. Jochem (1994) Carbon primary production. Reports on Polar Research, 135,6975. Mopper K., X. L. Zhou, R. J. Kieber, D. J. Kieber, R. J. Sikorski and R. D. Jones (1991) Photochemical degradation of dissolved organic carbon and its impact on the oceanic carbon cycle. Nature, 353, 60-62. Moriarty D. J. W. and M. Bianchi (1994) Bacterial production and advection of organic carbon from the Southern Ocean in water at intermediate depths. Eos, Transactions. American Geophysical Union, Supplement, 75, 199. Peltzer E. T. P. and N. A. Hayward (1996) Spatial distribution and temporal variability of total organic carbon along 14O”W in the equatorial Pacific Ocean in 1992. Deep-Sea Research IZ, 43, 1155-l 180. Pomeroy L. R., J. W. Wiebe, D. Deibel, R. J. Thompson, G. T. Rowe and J. D. Paulowski (1991) Bacterial responses to temperature and substrate concentration during the Newfoundland spring bloom. Marine Ecology Progress Series, 75, 143-159. Prego R. (1991) Total organic carbon in the sea-ice zone between Elephant Island and the South Orkney Islands at the start of the austral summer (1988-1989). Marine Chemistry, 35, 189-197. Proctor L. M. and J. A. Fuhrrnan (1991) Roles of viral infection in organic particle flux. Marine Ecology Progress Series, 69, 133-142. Sharp J. (1993) The dissolved organic carbon controversy: An update. Oceanography, 6, 45-49. Sharp J. (1994) The broad community DOC methods comparison. Eos, Transactions, American Geophysical Union, Supplement, 75, 106. Simon M. and F. Azam (1989) Protein content and protein synthesis rates of planktonic marine bacteria. Marine Ecology Progress Series, 51, 201-213. Skoog A. and M. Wedborg (1994) Organic carbon and humic substances in the Weddell Sea. Reports on Polar Research, 135, 168-169. Smetacek V., H. J. W. de Baar, U. V. Bathmann, K. Lochte and M. M. Rutgers van der Loeff (1997) Ecology and biogeochemistry of the Antarctic Circumpolar Current during austral spring: a summary of Southern Ocean JGOFS cruise ANT X/6 of R.V. Polarstern. Deep-Sea Research II, 44, 1-21. Sorokin (1971) Bacterial populations as components of oceanic ecosystems. Marine Biology, 11, 101-105. Sugimura Y. and Y. Suzuki (1988) A high temperature catalytic oxidation method for the determination of nonvolatile dissolved organic carbon in seawater by direct injection of a liquid sample. Marine Chemistry, 24, 105-131. Suzuki Y. (1993) On the measurement of DOC and DON in seawater. Marine Chemistry, 41, 287-288. Veth C., I. Peeken and R. Scharek (1997) Physical anatomy of fronts and surface waters in the ACC near the 6”W meridian during austral spring 1992. Deep-Sea Research II, 44, 2349. Williams P. M. and E. R. Druffel (1987) Radiocarbon in dissolved organic matter in the central North Pacific Ocean. Nature, 330, 246248. Zweifel U. L., B. Norrman and A. Hagstrom (1993) Consumption of dissolved organic carbon by marine bacteria and demand for inorganic nutrients. Marine Ecology Progress Series, 101, 23-32.
Pergamon
II, Vol. 44,No. l-2, pp. 341-353, 1997 ScienceLtd 1997 Published by Elsevier Printedin GreatBritain.All rights reserved 09674645/97 $17.00 + 0.00
Research 0
PII: SO967-0645(96)00071-9
Dissolved organic matter and its utilization by bacteria during spring in the Southern Ocean PAUL
KAHLER,*
PETER
K. BJBRNSEN,f AVAN ANTIAS
(Received 20 November 1995; in revisedform
KARIN
LOCHTE*
and
30 January 1996; accepted 13 June 1996)
AbstractXoncentrations of dissolved organic carbon (DOC) and nitrogen (DON) were measured during early austral Spring 1992 at a number of stations along the 6”W meridian between 47”and 60”s. This included the Polar Front in the north, the zone of melting sea-ice in the south, and waters of the Antarctic Circumpolar Current in between. Concentrations of DOC were low in deep water (34-38 PM) with generally similar or slightly higher values in the surface mixed layer (38-Z pM). DOC:DON ratios are wider in surface water than in deep water, i.e. surface accumulations contain relatively C-rich dissolved organic matter. The highly variable distribution of the surface DOC was not related to hydrographic or biotic features (fronts, plankton development) indicating the lability and transient occurrence of this material. Growth rates of bacteria were determined in subsamples from 5 1 0.8~pm-filtered batches of seawater incubated in the dark at in-situ temperature. Thymidine and leucine uptake and bacterial biomass change as well as changes in dissolved organic carbon in the batches, and oxygen consumption in parallel incubations correlated linearly over 2 weeks of incubation which allowed extrapolation to in-situ conditions. Bacterial growth in these experiments depended strongly on the amount of initial DOC. Growth in water from greater depth (1000 m) containing 38 PM DOC was minimal, as were DOC-decrease and oxygen consumption. Higher rates were observed in surface water slightly enriched with DOC, and highest rates in surface water amended with DOC-rich melted sea ice. Bacterial growth efficiencies (biomass C-increase vs DOC consumed) were about 30%. The experiments showed that at least 4& 60% of the DOC in excess of deep water concentrations was available to bacteria. 0 1997 Published by Elsevier Science Ltd. All rights reserved
INTRODUCTION Little is known about the quantity and quality of dissolved organic matter and its significance for carbon cycling in the ocean. After a period of confusion about the level of dissolved organic carbon (DOC) measured by high temperature catalytic oxidation techniques (Hedges et al., 1993; Suzuki, 1993), this problem has been resolved by establishing a proper blank correction procedure. Recent international intercalibration exercises proved that a number of workers could reproduce DOC-measurements in oceanic samples (Sharp, 1993, 1994). Although recent concentration measurements (with blank correction) are in the lower range of pre-Suzuki values, oceanic DOC is one of the largest carbon reservoirs on earth, and its behaviour is of interest in the context of global carbon
* Institut fiir Ostseeforschung, Seestr. 15, D-181 19 Wamemiinde, Germany. t Marine Biological Laboratory, Strandpromenaden 5, DK-3000, Helsingsr, Denmark. $ Sonderforschungsbereich 313, Universitlt Kiel, Olshausenstr. 40, D-24123 Kiel, Germany. 341
342
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cycling. However, a large pool size is not in itself a reason to assume oceanic DOC to be a relevant component of oceanic, and global, carbon fluxes. Whether there is a pathway of CO2 removal from the atmosphere (and the upper ocean that interacts with it) involving oceanic DOC is largely a question of its reactivity and the timescale of its turnover. A pool of chemically and biologically inert DOC would obviously not interact with other carbon pools or contribute to any carbon fluxes, no matter how much of this material was present. A pool of reactive DOC on the other hand, respired soon after its production, would only transiently affect the carbon budget with no lasting effect on the fluxes in question. Carbon export from the surface via DOC is only possible by accumulation and subsequent deep mixing or downwelling, by eddy diffusion, or by its (efficient) transformation into sinkable particulate material. Any DOC respired in the surface mixed layer can be returned to the atmosphere as CO;?. Kirchmann et al. (1991) reported rapid consumption by bacteria of as much as one-third of a high apparent ambient oceanic DOC-level in the North Atlantic. Their DOCmeasurements, however, were made with an instrumental setup that was later (Suzuki, 1993) shown to produce erroneously high and inconsistent values. Bacterial carbon production amounted to only a few percent of the DOC apparently consumed in their study. In other studies in marine environments, the incorporation of DOC into bacterial biomass was found to be more efficient: 1 l-54% (Baltic Sea, Zweifel et al., 1993) and 40% (Weddell Sea, Bjornsen and Kuparinen, 199 1). Here, we report on the levels of DOC- and DON measured Antarctic waters during Spring 1992 in the Southern Ocean, its availability to bacteria as a food source, the efficiency with which it is transformed into bacterial biomass, and how much is respired.
MATERIAL
AND
METHODS
The results presented are from transects along the 6”W meridian between the Polar Front near 47”s and the ice edge near 6O”S, taken in October and November 1993 during the R.V. Polar-stern expedition ANT X/6. For the positions of the transects and general background, see Smetacek et al. (1997). For the DOC and DON analyses, lo-ml samples were drawn into clean sealed and freshly opened glass ampoules directly from the Niskin bottles, acidified with 200 ~1 42% phosphoric acid, resealed, and stored refrigerated until analysis on board (odd station numbers) or later on land (even numbers). The samples were not filtered to avoid a source of contamination; hence total organic carbon (TOC) and total nitrogen (TN) were measured, DOC and DON were obtained by subtracting particulate, and in the case of nitrogen also dissolved inorganic, C and N, respectively. The magnitude of this correction was 0.5-10 PM POC (see also Fig. 2), 0.1-2 PM PON and 2s-35 PM DIN. The acidified samples were stripped of inorganic carbon by passing a stream of CO*-free gas (oxygen or argon) through them immediately prior to analysis; TOC and TN were then measured by a high temperature catalytic combustion technique with a modified “Dimatoc 100” (Dimatec) total carbon analyser where they were oxidized to CO2 and NO, respectively. Aliquots of 100 ,~l of seawater were injected into a quartz tube containing 50 g 5% platinum-on-alumina catalyst (Degussa) heated to 900°C covered with 0.5 g of platinum wool. The carrier gas was 5% 02 in argon at a flow rate of 100 ml min- ‘. The high temperature and the low oxygen content of the carrier gas were necessary for the complete recovery of nitrogen as NO and did not affect CO2 yield as compared with pure oxygen as
Dissolved organic matter
343
the carrier gas. Glass tubes filled with zinc and bronze, or 10 g of Sulfix (Wake Chemicals) at 500°C served to scrub HCl and SO2 from the combustion gas and ice/water and Mg(ClO& traps to remove moisture. Carbon dioxide was measured in a “Binos 100” (Rosemount) non-dispersive infra-red detector and NO in an “Antek 720” (Antek) chemoluminescence detector in line behind, and the area of the resulting peaks determined with a chromatography program (“Boreal”, Flowtech). At least four injections were made per sample. Calibration was against seawater spiked with glucose and urea (five concentrations) and a standard gas mixture of CO2 and NO in nitrogen. Blanks were determined with “Milli-Q” water twice distilled from persulphate/phosphoric acid according to Benner and Strom (1993). A number of ampoules were filled from a bottle of one of the first casts and the measurement of this water included in each calibration to check the stability of the measurements. This reference water was later measured against water from an international intercalibration (Sharp, 1994) to ensure general comparability of the data. Experience with the combustion unit used had shown that the blank and the response were variable over time with both the DOC and TN measurements. We achieved significant improvements in the reproducibility and long-term stability of calibrations through the use of a catalyst precombusted at 1250°C and the alternate injection of low-carbon deionized water to strip traces of CO* and NO absorbed by sea-salt residues in the cold portion of the combustion tube. Calibrations and measurements of reference water were made before and after each run of 12 samples. The precision of both the TOC and TN measurements was between 0.3 and 3 PM (standard deviation of replicate injections given in Fig. 1). Since this relatively high variability stems from the fact that CO* and NO are deposited and remobilized in the cold portions of the combustion tube, a mean value of four injections is closer to the accurate value than indicated by the variability of the single measurements. The overall error for DOC and DON should include the uncertainties in the determinations of POC, PON, nitrate and ammonia used to calculate them, which are not considered here. For the incubation experiments, water was filtered through 14-cm-diameter/O.8-pm-poresize polycarbonate filters (Costar) with precombusted GF/C (Whatman) prefilters using a peristaltic pump. This was to exclude the bulk of particulate matter and predators of bacteria, but to include bacteria in the filtrate. To avoid clogging of the filters and the buildup of backpressure, the filters were changed frequently during filtration. A third to half of the bacterial count passed into the filtrate. The water used was deep water (from 1000 m depth), surface water (from 20 m depth), and surface water amended with l/3 melted brown sea ice. Two sets of experiments were conducted (at 56”3O’S;6”W, starting 23 October and 13 November). In each, two parallels of each batch were incubated at 1“C in the dark in wellleached 5-l polyethylene bottles, and a number of 100-ml glass bottles for the oxygen determinations. They were subsampled over a period of 2 weeks as indicated in Fig. 3. For the analysis of particulate organic matter in the incubation experiments, 2-l subsamples were filtered on to precombusted GF/F-filters at the beginning and end of the experiments. Although GF/F-filters are not much different in pore size from the polycarbonate filters that we used to filter the water before setting up the experiments (in order to include a seeding population of bacteria in the filtrate), we were able to recover bacteria on these filters. This was because bacteria growing during the incubations were large, and, probably, the small area of the filters (2.5 cm in diameter) caused efficient clogging to trap particles smaller than the nominal pore size. Microscopic inspection of GF/ F-filtrates of the batches showed that only a few percent of the bacteria passed the filters,
344
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DOC pM Fig. 1. Distribution of DOC (solid lines) and DON (broken lines) with depth during spring in water between the Polar Front and the ice edge. Positions are 6”W and 50”3O’S (883); 58”3O’S (917); 59”3O’S (919); 58”s (923), 59”S, ice edge (931); 56”3O’S (942); 55”30’ (944); 52”s (951).
which means that bacterial growth was only slightly underestimated. Particulate organic carbon (POC) and nitrogen (PON) were measured by combustion of the filters in a Carlo Erba Elemental Analyser. Oxygen was measured in triplicate by a Winkler technique with automatic photometric endpoint detection following WOCE recommendations (Manuels, 1994), nutrients by standard procedures (Grasshoff et al., 1983). Growth rates of bacteria were determined by
60
Dissolved
organic
matter
345
the thymidine and leucine uptake method in lo-ml subsamples as described by Lochte et al. (1997). Bacteria were counted by epifluorescence microscopy of subsamples fixed in 2% formaldehyde solution, filtered on to 0.2~pm nucleopore filters and stained with DAPI; bacterial size was determined by comparison with fluorescent beads (Bjornsen and Kuparinen, 1991; Lochte et al., 1997).
RESULTS DOC and DON inventory Figure 1 shows the distribution of DOC and DON with depth for several stations. Note the relative uniformity of the profiles with no or only small increases of DOC, and in some profiles even DON minima, in the surface water. Low values at or slightly below 38 pmol DOC 1-l are found from 300 m down to the greatest depths measured (>4000 m); similarly, DON is constant at 8 PM in deep water, i.e. most variability is near the surface. Figure 2 shows the mean DOC accumulation in the top 100 m (depth-weighted averages) over the background value at 300 m depth along three transects taken between 11 October and 21 November along the 6”W meridian from 47” to 60”s. The surface DOC enrichment is extremely variable at a low level between 0 and 20 PM. There is no trend in the DOC level over the nearly 6-week period between the first and last transects. For comparison, the values of particulate organic carbon are more uniform and show the buildup of phytoplankton stocks in the Polar Frontal region.
Bacterial growth on DOM The experiments were designed to investigate bacterial growth with dissolved organic matter as the sole carbon source excluding mortality by predation which allows the measurement of bacterial production by biomass increase. They were also used as test systems for the calibration of bacterial production measurements (determination of conversion factors; Lochte et al., 1997) by the thymidine and leucine incorporation techniques (cf. Bjornsen and Kuparinen, 1991). Figure 3 shows the time course of the bacterial growth as measured by different parameters and changes in carbon and dissolved oxygen during the incubations. Clearly, the different sources of water support bacterial growth to different degrees. The higher the original content of dissolved organic matter, the higher are growth rates and bacterial biomass yield. The richest substrate is surface water amended with melted sea ice; deep water is the poorest substrate. The two sets of growth experiments (set 2 is a replicate of set 1, set up 3 weeks later at the same location) behaved similarly, with the exception that, in the meltice/surface-water variant of set 2, there was a decline in bacterial numbers and total volume in the last interval of the incubation period, with thymidine and leucine uptake still indicating growth. This odd behaviour may be caused by viral infection and lysis, which also may explain the higher thymidine:leucine uptake ratio. Correlations between the measured parameters are linear, except for the last interval of the second meltice-enriched surface water variant. Correlation coefficients (r’) are generally well above 0.9. Regression coefficients [conversion factors, CF determined following the procedure described by Bjornsen and Kuparinen (1991)] are, however, quite different for each batch. The CF from thymidine incorporation to cell number (1.2 x 1018 cells mol-‘) and from leucine uptake to
346
P. Klhler
et al.
NC
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degr. S Fig. 2. Average contents of DOC in the upper 100 m in excess of that present at 300 m depth (= surface DOC accumulation, closed circles) and POC (lines) between 47% and 60”s along 6”W. Bottom: 11-22 October; middle: 24-31 October; top: l&21 November. The arrow marks the position of the ice edge; the polar front is at 49”s.
bacterial volume production (4.4 x 10” pm3 mol-‘), based on all 12 experimental units are consistent with literature values (Bjornsen and Kuparinen, 1991). The CF from bacterial volume to POC of Table 1 provide a carbon and nitrogen balance of the experiments. The differences between days 1 and 13 (means of two parallels) in DOC, POC, PON, bacterial volume, oxygen, and dissolved inorganic nitrogen are listed. In the deep water, the precision of the TOC and POC measurements was not sufficient to explain the observed low oxygen consumption and bacterial growth in a meaningful way. Deep water DOM was expected to be resistant to bacterial breakdown though (see below), so the small changes observed in the deep water probably give an indication of the maximum
Dissolved
organic
(
matter
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4
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Fig. 3. Bacterial growth determined by 3H-thymidine and 3H-leucine incorporation (cumulative) and change in bacterial numbers and total bacterial volume (two parallels), and oxygen consumption (means of three parallels) in 0.8-pm filtered seawater from 20-m (circles), from 1000 m (squares), and from 20 m enriched with melted brown ice (triangles) filtered through 0.8 pm. Left and right panels represent two sets of experiments conducted (left: growth experiment 1; right: growth experiment 2).
348
P. KLhler et al.
Table 1. Losses and gains of carbon, nitrogen, oxygen and bacterial total volume during the growth of bacteria on natural dissolved organic matter in surface water, deep water and meltice-enriched surface water (growth experiment I) Changes Initial DOC
TOC*
86
-20
52 38
-6 0
Ice-enriched Surface water Surface water Deep water *TOC (total organic
carbon);
particulate
POC (pmol I-‘)
in PON
Oxygen
DIN
10.5
2.45
-26.7
-1.4
493
1.8 1.1
0.55 0.19
-7.5 -2.0
-0.5 -
77 20
organic
carbon
formed during incubation
Bacterial
volume (pm3 I- ‘)
is included
level of organic contamination during incubation and handling of the samples. These data are given only to show the difference in scale regarding the changes observed in surface water. The other two variants (surface water and meltice-enriched surface water) show significant changes for every parameter measured. An increase in mean cell size was observed during the incubations (from 0.06 to 0.09 pm3 in the 20-m batches, and from 0.09 to 0.15 and 0.25 pm3 in the ice-enriched batches) which we interpret as being caused by selection for small bacteria by the filtration at the start and an effect of the absence of predators during the growth phase. This size shift did not affect growth characteristics; the relative proportions of thymidine and leucine incorporation, oxygen consumption and bacterial (volumetric) growth stayed the same. From this, we conclude that they should also apply to the natural bacterial community. DISCUSSION In-situ
DOC and DON concentrations
Deep-water concentrations of DOC are in the range of values encountered elsewhere in the deep ocean; concentrations of 34-38 PM are slightly lower than deep-water concentrations measured in the North Atlantic (42-43 PM, Bodungen and Kshler, 1994; Carlson et al., 1994) and similar to those in the Pacific (35-39 PM, Carlson and Ducklow, 1995; Peltzer and Hayward, 1996), pointing to a very slow breakdown of this material at timescales of ocean mixing along the main routes of deep-water circulation, a process that is almost complete in the region of our study. Hence, this material can be considered extremely recalcitrant under deep-sea conditions. The old age of such material (Williams and Druffel, 1987) is a consequence of this. The occurrence of surface minima of DON in some profiles suggests that a portion of deep-water dissolved organic matter (DOM) has been broken down, i.e. it is not stable under surface conditions. There is evidence that photodestruction and subsequent consumption by bacteria of some of its products is a sink of old DOM (Kieber et al., 1989; Mopper et al., 1991). Photobleaching of gelbstoff (Determann, 1995) also shows that some of this material is reactive under conditions encountered at the sea surface. Figures 1 and 2 show surface maxima though, of DON in some, and of DOC, in most cases. Lower DOC:DON ratios (up to 8) than in deep water (4-5) show that the additional
Dissolved organic matter
349
material is relatively enriched in carbon. Possible sources of dissolved organic matter in surface water include a fraction of primary production (phytoplankton exudates, Bjornsen, 1988) and waste from zooplankton activities (inefficient feeding, leaching and excretion) (Lee and Henrichs, 1993). Addition of viral matter is considered insignificant in terms of carbon (Proctor and Fuhrman, 1991). Under the special conditions of the Southern Ocean, melting sea ice is an additional source of DOM to surface water (see below). Marked differences and patterns, in both space and time, were detected in the occurrence and production of phytoplankton and zooplankton during the cruise. Three zones of different hydrographic conditions (ice edge, Polar Frontal region, and water masses of the Antarctic Circumpolar Current in between, Veth et al., 1997) differed significantly in their biological development. Primary production was enhanced in the Polar Frontal region leading to the buildup of high phytoplankton stocks there, a lesser increase of productivity was observed near the retreating ice edge, and productivity stayed low in the waters in between (Mathot et al., 1994; Jochem et al., 1995). Stocks and productivity of zooplankton closely followed those of phytoplankton (Bathmann et al., 1997). The distribution and development of POC (Fig. 2) give an indication of this overall pattern. The observed differences in DOC concentrations in surface water, however, show no relationship with these biological stocks and activities, which are potential suppliers of DOC. At the ice edge, with ice melt initialising the seasonal cycle of pelagic production, the variability of DOC concentration is already very high. Situations with no enrichment at all, moderate enrichment, and exceptionally high concentrations in surface waters (ice-edge station 931, Fig. 1; not included in Fig. 2) were encountered in this region and under ice cover. This implies that at least part of the additional DOC in surface water is not connected with pelagic production, but is released from sea ice. Except for the extreme value mentioned, variability of the surface DOC enrichment was similar in the other hydrographic zones. Since there was no trend in DOC concentrations over the time between the transects (Fig. 2) we conclude that buildup and breakdown of DOC were generally coupled and in balance. Uncoupling seems to occur only transiently, reflected in higher concentrations at some stations, but the time scale of these accumulations must be much shorter than those of phytoplankton and zooplankton development. Similar results were obtained in a study by Skoog and Wedborg (1994), who measured TOC (total organic carbon) in the Weddell Sea later in the season and found concentrations in the same range as we did. In their study as well, concentrations at stations with high biological activity were not notably different from those at other stations. Bacterial
utilization
of DOC
The magnitude of DOC turnover is not evident from the in-situ concentrations but was determined experimentally. By eliminating larger phytoplankton and zooplankton, and incubating in the dark, the major sources of DOC were removed in the experiments, but bacteria passed the filter. Since heterotrophic bacteria are able to take up dissolved material and to respond quickly to available substrates, they are the most likely agents of DOC breakdown. In Table 2, various parameters derived from the data of Table 1, characterizing bacterial growth and substrate utilization are listed. These parameters show that bacterial growth with natural DOC being the only substrate is not exceptional in any way. The volume:carbon ratios of bacteria grown at 0.26 (surface water) and 0.28 (ice-enriched)
350
P. KLhler et al. Table 2.
Parameters describing bacterial growth and carbon utilization of growth experiment 1 Bacterial
Ice-enriched surface water Surface water
C/vol.
(pg pm-3)
C/N
0.26 0.28
4.3 3.3
Grown
efficiency* 0.30 0.26
RQ 0.75 0.80
Fraction of additional DOC usedt 0.63 0.56
*Delta POC/delta TOC + POC. tAdditiona1 DOC = excess over deep water DOC.
agree well with recently published figures (Fry, 1988; Simon and Azam, 1989); their molar C:N-ratio is about 4. Growth efficiencies, i.e. bacterial carbon gained from DOC consumed are 0.25 and 0.3, respectively, which are within the range observed for many natural substrates (Lignell, 1990 and references therein). These observations are in contrast to those of Pomeroy et al. (199 1) who found that, at low temperatures (1 “C in our case) and at dilute substrate concentrations, bacteria have particularly low conversion efficiencies. The efficiencies that we observed are similar to those observed in temperate marine environments (Bjornsen, 1986; Zweifel et al., 1993). A conversion efficiency of as low as 2%, reported by Kirchmann et al. (1991) for temperate oceanic waters, is considered an artifact. The experiments show that a substantial fraction of the DOM in surface water is biologically labile and can be efficiently transformed into bacterial carbon. Half, or more than half, of the dissolved organic carbon in excess of the deep-water concentration was consumed over the period of 2 weeks. Since growth was still going on when the experiments were terminated, the potentially usable fraction should be even greater. The net consumption of inorganic nitrogen is probably due to the relative C-richness of the surface-accumulated dissolved organic matter. Growth rates of bacteria, which vary between 0.2 and 2 pg C per 1-l day- ’(Lochte et al., 1997), follow the trend of primary productivity, i.e. high rates are connected with the phytoplankton bloom in the Polar Frontal region. Hence, the bacteria can be assumed not to rely on the accumulated DOC, but on a substrate provided directly or indirectly from ongoing primary production. Given a growth efficiency of 0.3, the carbon demand of bacteria can be estimated by multipying their growth rates by 3.3; this results in a C-demand of 0.055-0.55 pmol per 1-l day-‘. If the supply of fresh substrate were cut off and the bacteria were to grow with the accumulated DOC as their sole carbon source, a mean concentration of 10 PM (Fig. 2) could support them for 18 days at the polar frontal zone to a maximum of 180 days near the ice edge, where minimal growth rates were observed. In summary, bacteria can consume enough DOC added from primary production to prevent seasonal accumulation, and they have the potential to cope with the small accumulations observed (whatever their source is) in a matter of months. Implications
for oceanic DOM dynamics
Many findings point to the stability of a substantial fraction of DOC: the old 14C age of DOC (Williams and Druffel, 1987), the absence of seasonal variability of deep-water DOC, and the very low variability of deep-water DOC worldwide (Bodungen and Kahler, 1994; Peltzer and Hayward, 1996; Carlson and Ducklow, 1995). On the other hand, there are
Dissolved organic matter
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reports on the lability of part of the surface water DOC (Amon and Benner, 1994) and of DOC freshly produced by algae (Lignell, 1990; Chen and Wangersky, 1993). The high variability of surface DOC concentrations observed in this study and elsewhere in the ocean (Seltzer and KHhler, in prep.), and the marked seasonality of oceanic surface mixed-layer concentrations of DOC (Bodungen and Kahler, 1994; Carlson et al., 1994) also point to the lability of a large DOC fraction in surface water. From these findings, it can be concluded that dissolved organic matter in the ocean is a mixture of compounds that span a wide range of persistence, from extremely labile to extremely recalcitrant. So far, the magnitude of the recalcitrant fraction and a fraction persistent for less than a year have been discussed. Whether there is a third fraction of intermediate stability is difficult to recognize. Such a component, formed in surface water and stable for years to centuries, should form a permanent gradient below the maximum depth of the mixed layer. In well-stratified waters, this has been observed (Peltzer and Hayward, 1996; equatorial Pacific; Kahler and Peltzer, in prep., North Atlantic). In waters of the Southern Ocean with their high mixing rates in greater water depth, such a gradient, should it exist, would be less steep and probably not detectable with the precision of the DOC measurements. This shows that such a DOM fraction is of minor importance only. It is only such material that would be available for export. Although only a few studies of DOC in the Southern Ocean have been performed, there exists a wide variety of opinions concerning its importance there. The reported levels span a wide range, some of which may reflect analytical difficulties, and some high concentrations are from special marine environments (Dawson et al., 1985). Dafner (1992) found a strong correlation of DOC content and primary productivity and reported DOC concentrations as high as 700 PM in the Polar Frontal Zone, i.e. more than 10 times higher than those that we found during the spring. Moriarty and Bianchi (1994) believe in a source of organic matter (dissolved or suspended) at, or south of, the Polar Frontal Zone, which is advected at intermediate depths to regions far North, in order to explain high bacterial productivity in intermediate waters there. They revive a hypothesis by Sorokin (1971) who had made similar observations: measured bacterial production requires more carbon than supplied from local primary production. This hypothesis has been refuted by Banse (1974), but has since found new attention during the period of confusion regarding the validity of wetchemical DOC determinations following the publication of Sugimura and Suzuki (1988) on the measurement of DOC by high temperature catalytic oxidation (see Karl, 1993 for a recent discussion). Other authors observed particularly low concentrations of dissolved organic matter in oceanic waters around Antarctica (Jackson and Williams, 1985; le Jehan and Treguer, 1985). Prego (199 1) only found substantial DOC accumulations near the pack-ice zone and very low concentrations elsewhere during austral summer in the Weddell Sea. Billen and Becquevort (1991) observed a time lag between primary and secondary production in southern polar waters of Prydz Bay and postulated the temporary accumulation of bioavailable DOC over the period of a month only. It has already been mentioned that the low DOC observed by us during spring and the lack of a relationship between DOC concentrations and biological activity continued later in the year (Skoog and Wedborg, 1994). This is in contrast to equatorial regions where stable accumulations of DOC were observed (Peltzer and Hayward, 1996) and temperate regions where larger transient accumulations occur seasonally (Bodungen and Kahler, 1994; Carlson et al., 1994).
352
P. Kahler
et al.
Hence, the Southern Ocean appears to be a region where DOC is not available for export, either vertically or horizontally, and a region that is poorer in DOC than other oceans. Acknowledgements-We acknowledge the help of Rinus Manuels (dissolved oxygen), Peter Fritsche (nutrients) Laetitia Teissier and Heike Siegmund (POC, PON), and Alexandra Nielsen (bacteria counts). Part of this work (P.K.) was supported by a grant of the German Research Foundation (DFG) and grant No. 03FOlOSF of the German Ministry of Research and Technology.
REFERENCES Amon
R. M. W. and R. Benner (1994) Rapid cycling of high-molecular-weight dissolved organic matter in the ocean. Nature, 369, 5499552. Banse K. (1974) On the role of bacterioplankton in the tropical ocean. Marine Biology, 24, l-5. Bathmann U. V., R. Scharek C. Klaas, C. D. Dubischar and V. Smetacek (1997) Spring development of phytoplankton biomass and composition in major water masses in the Atlantic sector of the Southern Ocean. Deep-Sea Research II, 44, 51-67. Benner R. and M. Strom (1993) A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation. Marine Chemistry, 41, 1533160. Billen G. and S. Becquevort (1991) Phytoplankton-bacteria relationship in the Antarctic marine ecosystem. Polar Research, 10, 245-253. Bjornsen P. K. (1986) Bakterioplankton growth yield in continuous seawater cultures. Marine Ecology Progress Series, 30, 1057-1075. Bjornsen P. K. (1988) Phytoplankton exudation of organic matter: why do healthy cells do it? Limnology and Oceanography, 33, 15 l-l 54. Bjornsen P. K. and J. Kuparinen (1991) Determination of bacterioplankton biomass, net production, and growth efficiency in the Southern Ocean. Marine Ecology Progress Series, 71, 185-194. Bodungen B. and P. Kahler (1994) Dissolved organic carbon in the Atlantic-seasonal and regional patterns. Eos, Transactions, American Geophysical Union, Supplement, 75, 106. Carlson C. A. and H. W. Ducklow (1995) Dissolved organic carbon in the upper ocean of the central equatorial Pacfic Ocean, 1992: Daily and tinescale vertical variations. Deep-Sea Research ZZ,42, 639-656. Carlson C. A., H. W. Ducklow and A. F. Michaels (1994) Annual flux of dissolved organic carbon from the euphotic zone in the northwestern Sargasso Sea. Nufure, 371, 405408. Chen W. and P. J. Wangersky (1993) High-temperature combustion of dissolved organic carbon produced in phytoplankton cultures. Marine Chemistry, 41, 167-172. Dafner E. V. (1992) Dissolved organic carbon in waters of the Polar Frontal Zone of the Atlantic Antarctic in the spring-summer season of 1988-1989. Marine Chemistry, 37, 275-283. Dawson R., W. Schramm and M. Bolter (1985) Factors influencing the production, decomposition and distribution of organic and inorganic matter in Admiralty Bay, King George Island. In: Antarctic nutrient cycles andfood webs, W. R. Siegfried, P. R. Condy and R. M. Laws, editors, Springer, Berlin, pp. 109114. Determann S. (1995) Analyse biologischer und biochemischer Prozesse im Meer mit Fluoreszenzspektroskopie. Ph.D. Thesis, University of Oldenburg, 111 pp. Fry C. (1988) Determination of biomass. In: Methods in aquatic microbiology, B. Austin, editor, Wiley, Chichester, pp. 27-72. Grasshoff K., M. Ehrhardt and K. Kremling (1983) Methods ofseawater analysis. Chemie, Weinheim, 419 pp. Hedges J., B. A. Bergamaschi and R. Benner (1993) Comparative analyses of DOC and DON in natural waters. Marine Chemistry, 41, 121-134. Jackson G. A. and P. M. Williams (1985) Importance of dissolved organic nitrogen and phosphorus to biological nutrient cycling. Deep-Sea Research, 32, 223-235. le Jehan S. and P. Treguer (1985) The distribution of inorganic nitrogen, phosphorus, silicon, and dissolved organic matter in surface and deep waters of the Southern Ocean. In: Antarctic nutrient cycles andfood webs, W. R. Siegfried, P. R. Condy and R. M. Laws, Springer, Berlin, pp. 22-29. Jochem F. J., S. Mathot and B. Queguinier (1995) Size-fractionated primary production in the open Southern Ocean in austral spring. Polar Biology, 15, 381-392. Klhler P. and Peltzer E. (in prep.) Carbon export in the ocean: the role of DOC.
Dissolved
organic
matter
353
Karl D. M. (1993) Microbial processes in the southern oceans. In: Antarctic microbiology, E. M. Friedmann, editor, Wiley, New York, pp. 163. Kieber D. J., J. McDaniel and K. Mopper (1989) A photochemical source of biological substrates in seawater: implications for carbon cycling. Nature, 241, 637639. Kirchmann D. L., Y. Suzuki, C. Garside and H. W. Ducklow (1991) High turnover rates of dissolved organic carbon during a spring phytoplankton bloom. Nature, 352, 612-614. Lee C. and S. M. Henrichs (1993) How the nature of dissolved organic matter might affect the analysis of dissolved organic carbon. Marine Chemistry, 41, 105-120. Lignell R. (1990) Excretion of organic carbon by phytoplankton: its relation to algal biomass, primary productivity and bacterial secondary productivity in the Baltic Sea. Marine Ecology Progress Series, 68, 8599. Lochte K., P. K. Bjsrnsen, H. Giesenhagen and A. Weber (1997) Bacterial standing stock and production and their relation to phytoplankton in the Southern Ocean. Deep-Sea Research IZ, 44, 321-340. Manuels R. (1994) Dissolved oxygen. Reports on Polar Research, 135, 35-37. Mathot S., B. Queguinier and F. Jochem (1994) Carbon primary production. Reports on Polar Research, 135,6975. Mopper K., X. L. Zhou, R. J. Kieber, D. J. Kieber, R. J. Sikorski and R. D. Jones (1991) Photochemical degradation of dissolved organic carbon and its impact on the oceanic carbon cycle. Nature, 353, 60-62. Moriarty D. J. W. and M. Bianchi (1994) Bacterial production and advection of organic carbon from the Southern Ocean in water at intermediate depths. Eos, Transactions. American Geophysical Union, Supplement, 75, 199. Peltzer E. T. P. and N. A. Hayward (1996) Spatial distribution and temporal variability of total organic carbon along 14O”W in the equatorial Pacific Ocean in 1992. Deep-Sea Research IZ, 43, 1155-l 180. Pomeroy L. R., J. W. Wiebe, D. Deibel, R. J. Thompson, G. T. Rowe and J. D. Paulowski (1991) Bacterial responses to temperature and substrate concentration during the Newfoundland spring bloom. Marine Ecology Progress Series, 75, 143-159. Prego R. (1991) Total organic carbon in the sea-ice zone between Elephant Island and the South Orkney Islands at the start of the austral summer (1988-1989). Marine Chemistry, 35, 189-197. Proctor L. M. and J. A. Fuhrrnan (1991) Roles of viral infection in organic particle flux. Marine Ecology Progress Series, 69, 133-142. Sharp J. (1993) The dissolved organic carbon controversy: An update. Oceanography, 6, 45-49. Sharp J. (1994) The broad community DOC methods comparison. Eos, Transactions, American Geophysical Union, Supplement, 75, 106. Simon M. and F. Azam (1989) Protein content and protein synthesis rates of planktonic marine bacteria. Marine Ecology Progress Series, 51, 201-213. Skoog A. and M. Wedborg (1994) Organic carbon and humic substances in the Weddell Sea. Reports on Polar Research, 135, 168-169. Smetacek V., H. J. W. de Baar, U. V. Bathmann, K. Lochte and M. M. Rutgers van der Loeff (1997) Ecology and biogeochemistry of the Antarctic Circumpolar Current during austral spring: a summary of Southern Ocean JGOFS cruise ANT X/6 of R.V. Polarstern. Deep-Sea Research II, 44, 1-21. Sorokin (1971) Bacterial populations as components of oceanic ecosystems. Marine Biology, 11, 101-105. Sugimura Y. and Y. Suzuki (1988) A high temperature catalytic oxidation method for the determination of nonvolatile dissolved organic carbon in seawater by direct injection of a liquid sample. Marine Chemistry, 24, 105-131. Suzuki Y. (1993) On the measurement of DOC and DON in seawater. Marine Chemistry, 41, 287-288. Veth C., I. Peeken and R. Scharek (1997) Physical anatomy of fronts and surface waters in the ACC near the 6”W meridian during austral spring 1992. Deep-Sea Research II, 44, 2349. Williams P. M. and E. R. Druffel (1987) Radiocarbon in dissolved organic matter in the central North Pacific Ocean. Nature, 330, 246248. Zweifel U. L., B. Norrman and A. Hagstrom (1993) Consumption of dissolved organic carbon by marine bacteria and demand for inorganic nutrients. Marine Ecology Progress Series, 101, 23-32.