Production of chromophoric dissolved organic matter by Sargasso Sea microbes

Marine Chemistry 89 (2004) 273 – 287 www.elsevier.com/locate/marchem

Production of chromophoric dissolved organic matter by Sargasso Sea microbes Norman B. Nelson a,*, Craig A. Carlson b, Deborah K. Steinberg c a

Institute for Computational Earth System Science (ICESS), University of California, Santa Barbara, CA 93106, USA b Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA 93106, USA c Department of Biological Sciences, Virginia Institute of Marine Science, Gloucester Point, VA 23062-1346, USA Received 7 February 2003; received in revised form 1 October 2003; accepted 9 February 2004 Available online 1 June 2004

Abstract Time series of chromophoric dissolved organic matter (CDOM) light absorption coefficients indicate a local origin for a large fraction of the CDOM in the upper water column of the Sargasso Sea. In the present study, we demonstrate that CDOM is produced in bacterial culture experiments using Sargasso Sea water and naturally occurring microbial assemblages. Seawater cultures were prepared and grown at in situ temperatures in the dark for periods of weeks. Selected cultures were treated with amendments including inorganic nutrients, glucose, phytoplankton exudates, and zooplankton excretia. In all experiments, when bacterial biomass increased, CDOM increased during the first week of the experiment, followed by a decrease over a longer period of time. Cultures amended with both glucose and inorganic nitrogen and phosphorus produced more CDOM than controls or cultures amended with glucose or inorganic nutrients alone. However, when complex DOM substrates (derived from phytoplankton or zooplankton cultures) were added to seawater cultures, there was a net accumulation of CDOM over the course of the experiments. These data suggest that, in addition to microbial growth, the quality of the substrate plays an important role in net CDOM production. ‘New’ CDOM produced in culture was spectroscopically similar to CDOM appearing below the surface during summer stratification. The results of the present study support a new paradigm for CDOM in the open ocean, which allows for local origin and significant dynamics. Appreciation of CDOM dynamics will, in turn, add to our understanding of microbial productivity, photochemical rate processes, and ultraviolet radiation availability in the global ocean. D 2004 Elsevier B.V. All rights reserved. Keywords: CDOM; Dissolved organic matter; Microbial community; North Atlantic; Sargasso Sea

1. Introduction Seasonal patterns of spectral diffuse attenuation coefficient (a measure of water transparency) in the Sargasso Sea near Bermuda (Siegel et al., 1995) * Corresponding author. Tel.: +1-805-893-3202; fax: +1-805893-2578. E-mail address: [email protected] (N.B. Nelson). 0304-4203/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2004.02.017

reflect the dynamics of a short-wavelength lightabsorbing component that varies seasonally but is not directly correlated to the annual cycle of phytoplankton productivity and biomass (Siegel and Michaels, 1996; Steinberg et al., 2001). Subsequent spectroscopic measurements of light absorption by filtered seawater samples led to the conclusion that the major contributor to the variable light-absorbing component is chromophoric dissolved organic matter

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(CDOM) (Nelson et al., 1998). Traditionally, light absorption by CDOM within oligotrophic oceans far from land was thought to be low (relative to the absorption of particulate matter) and correlated with phytoplankton productivity or terrestrial input of humic material (Kalle, 1966; Bricaud et al., 1981; Højerslev, 1982). These more recent results have demonstrated that the blue light absorption due to

CDOM in the Sargasso Sea (where terrestrial input is negligible on the annual time scale) ranges from ca. 50% to 90% of the total nonwater absorption over the course of a year (Nelson and Siegel, 2002). At this site, an annual seasonal cycle surface temperature and stratification (Fig. 1A) correlates inversely with blue light absorption at the surface, which varies by a factor of 2 over the course of the

Fig. 1. (A) Time – depth contour plot of temperature (jC). (B) Chlorophyll a (mg m
3). (C) Absorption coefficient of CDOM (acdom; m
1) at 325 nm, 1996 – 1999. Also shown are the mixed layer depth computed from the BATS CTD profile (panel A; -o-) and the 1 % of surface irradiance (PAR) depth (panel B; —). The low values of ‘‘background’’ CDOM (e.g., at 150 m) found in 1997 and 1998 were coincident with an anomalous high-salinity water mass present at the surface.

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year (Siegel et al., 1995; Siegel and Michaels, 1996). However, CDOM concentration is only weakly correlated with water column chlorophyll a concentration (Siegel and Michaels, 1996; Nelson et al., 1998; Fig. 1B and C). Since the light absorption by CDOM in the water column relates directly to ocean color, photochemical rate processes, and ultraviolet (UV) transparency, understanding the dynamics of CDOM in the oligotrophic Sargasso Sea is necessary for understanding ocean color remote sensing data (Carder et al., 1989; Garver and Siegel, 1997; Siegel et al., 2002), the distribution of photochemically produced or labile products (such as carbon monoxide, hydrogen peroxide, carbonyl sulfide, or dimethyl sulfide) (Mopper and Kieber, 2002), and UV photobiology (Smith et al., 1992; Kirk, 1994; Arrigo and Brown, 1996). Profiles of CDOM light absorption coefficient constructed from seawater samples collected in the upper 250 m at the U.S. JGOFS Bermuda Atlantic Time series Study (BATS) station near Bermuda exhibited a consistent seasonal cycle over the 1996– 1999 time period (Fig. 1C). CDOM absorption spectra are low in value and are homogenous with depth during the period of convective overturn (December– March; Fig. 1A) and exhibit a surface increase coincident with the spring bloom (Nelson et al., 1998; Fig. 1B). A near-surface minimum and subsurface (60 – 100 m) maximum appear subsequent to stratification, and the subsurface maximum continues to increase throughout the summer months (Nelson et al., 1998; Fig. 1C). In the autumn, with the onset of convective overturn, the profile is homogenized over the course of a few months and reverts back to winter low values. Since the Sargasso Sea is far from terrestrial sources of CDOM input, and the majority of the CDOM light absorption increase occurs subsequent to the spring bloom, Siegel and Michaels (1996) and Nelson et al. (1998) have suggested a local origin for a significant fraction of the CDOM pool. Although some of the annual ‘‘new’’ CDOM appear during the spring bloom season, when phytoplankton growth and biomass are highest (Nelson et al., 1998; Steinberg et al., 2001), the majority of the annual production of CDOM occurs during the summer, when phytoplankton biomass and productivity are at its annual low. This implies a nonphytoplankton origin for the new CDOM. Photochemical production of CDOM is one possible source, although it would

275

appear that the surface minimum in CDOM that appears during the summer is a result of light-mediated destruction of CDOM (Kouassi and Zika, 1992; Nelson et al., 1998; Del Vecchio and Blough, 2002). Furthermore, the majority of CDOM production during the summer occurs at depths where solar irradiance is < 10% of surface levels (Siegel et al., 1995). Deep sources of CDOM upwelling into surface waters also do not appear to be a major factor, as CDOM light absorption in samples collected down to 500 m is lower than those collected in surface waters (unpublished results). Nelson et al. (1998) have demonstrated that summertime CDOM profiles do resemble profiles of microbial abundance and productivity (Carlson and Ducklow, 1996; Carlson et al., 1996; Steinberg et al., 2001), further suggesting that the microbial community mediates CDOM production in some way (Nelson and Siegel, 2002). Nelson et al. (1998) constructed a mathematical formulation for the dynamics of CDOM under conditions of summer stratification in the Sargasso Sea, which related the synthesis of CDOM to the specific growth rate of bacterioplankton, based on concurrent measurements of CDOM concentration, bacterial abundance, and bacterial thymidine incorporation made at the BATS site. Removal of CDOM was related to an exponential decay function simulating the extinction of irradiance with depth. This simple model was found to explain 83% of the variance in the time rate of change of CDOM absorption coefficient during the summer of 1994 and 1995, with an estimated turnover time of 125– 300 days for CDOM production, and 90 days for CDOM photodestruction (Nelson et al., 1998). This result depends upon the assumption that CDOM production is directly related to microbial activity. The purpose of the present study was to test the corresponding hypothesis that Sargasso Sea microbes can produce CDOM under controlled laboratory conditions, and to relate the rates of production in the laboratory to estimates from the field. Seawater culture experiments (Ammerman et al., 1984) conducted using Sargasso Sea water and in situ microbial communities (Carlson and Ducklow, 1996; Carlson et al., 2002) have demonstrated that when bacterial growth is significant in culture, a measurable amount of DOC is removed. We conducted similar seawater culture experiments during different seasons

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and under various conditions of substrate availability in order to test the hypothesis that increase in CDOM light absorption in Sargasso Sea water is due to microbial activity. Additional experiments using organic and inorganic amendments to the seawater (including fresh phytoplankton exudate and zooplankton excretia) were designed to see if CDOM production varied as a function of the quality of the available substrate. As CDOM is a mixture of substances rather than a single compound, it stands to reason that processes that create or destroy CDOM may result in characteristic changes in the absorption spectra, which reflect any selective effect of the production or destruction processes. We examined the absorption spectra of CDOM collected in the field and produced in the laboratory to determine whether the ‘new’ and ‘old’ CDOM pools could be distinguished by absorption spectroscopy.

2. Methods 2.1. Seawater samples We present data generated from seawater culture experiments conducted in the northwestern Sargasso Sea from 1996 to 1999. Sample water was collected from the Bermuda Atlantic Time series Study (BATS; 31j40VN, 64j10VW) site, Hydrostation ‘‘S’’ (32j10VN, 64j30VW) and a coastal station just off the Bermuda Platform located approximately 82, 26, and 10 km southeast of the islands of Bermuda, respectively. Water from 1 to 20 m was collected from the R/V Weatherbird II via Niskin bottles equipped with epoxy-coated springs, on a conductivity, temperature, and depth (CTD) rosette (Knap et al., 1997), or from a small boat via polypropylene bucket. Upon recovery, seawater was transferred to precleaned polycarbonate carboys. All carboys were washed with 5% HCl and rinsed with copious amounts of Millipore Milli-Q water (Milli-Q) and sample prior to each experiment. 2.2. Seawater cultures Seawater culture techniques modified from Ammerman et al. (1984) were employed as described in

Carlson et al. (2002) or Carlson et al. (1999). Briefly, natural assemblages of bacterioplankton collected near the surface were incubated in grazer diluted cultures and allowed to grow on various mixtures of the naturally occurring substrates, or in combination with nutrient and/or organic enrichments (Table 1). Seawater cultures were prepared either by filtering out microflagellate grazers by passing sample water through a 0.8-Am Costar Membra-Fil filter held in a 142-mm all-plastic filtration rig, or by diluting whole (unfiltered) seawater by 70% with a 0.2-Am filtrate. Preparation of 0.8 or 0.2 Am seawater began within a few hours of collection. Seawater was gravity-filtered directly from a Niskin or collection carboy, through an all-plastic filtration rig containing a 142 mm-Costar Membra-Fil filter, into another polycarbonate carboy. The Costar Membra-Fil filters provide a high flow rate and are relatively gentle to cells during filtration, thus reducing cell lysis (Carlson and Ducklow, 1996). Membra-Fil filters initially leach a measurable amount of DOC, so we flushed at least 2 l of Milli-Q water and an additional 0.5 l of sample water prior to collection of filtrate (Carlson and Ducklow, 1996). Each dilution experimental treatment received 7 l of a 0.2-Am filtrate and was inoculated with 3 l of whole seawater. For experiments that used 0.8-Am filtrates as growth media, 10 l of filtrate was generated. All seawater cultures were placed into an environmental chamber and incubated in the dark at in situ temperatures (Table 1). Samples for bacterial cell abundance, 3 H thymidine incorporation (March 1998 only) and CDOM concentrations were drawn at regular intervals for a period of 4– 7 days. In selected experiments, the experimental conditions were maintained and samples were collected at infrequent intervals for up to 50 days. 2.3. Nutrient enrichments A variety of nutrient enrichments were employed to determine if microbial growth would be stimulated and consequently stimulate production of CDOM. Treatments included unamended seawater cultures, seawater cultures grown in the presence of known quantities of inorganic and organic compounds, and seawater cultures grown in the presence of DOM produced by natural assemblages of phytoplankton (phytoplankton exudate) or zooplankton (zooplankton

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Table 1 Experimental parameters for Sargasso Sea seawater cultures from 1996 to 1999 Date of collection

Treatment

Incubation temperature (jC)

Source location

Depth (m)

Culture preparation

February 27, 1996 March 16, 1996 March 25, 1996 January 26, 1998

Unamended Phytoplanktonb Phytoplanktonb Control NH4 + PO4 Glucose Glucose + NH4 + PO4 Control NH4 + PO4 Glucose Glucose + NH4 + PO4 Control NH4 + PO4 Glucose Glucose + NH4 + PO4 Control Glucose + NH4 + PO4 Zooplankton excretia

19.3 19.3 19.9 19.5

HS HS BATS 4 miles SE Spit Buoy

5 5 20 <1

0.8 Am filtratea 0.8 Am filtratea 0.8 Am filtratea 70% dilutionc

20

4 miles SE Spit Buoy

<1

70% dilutionc

20

HS

10

70% dilutionc

22.8

BATS

20

70% dilutionc

March 16, 1998

April 9, 1998

December 4, 1999

Treatments refer to the amendments added to seawater cultures. Culture parameters refer to experimental design used to reduce microflagellate grazing pressure. All incubations were carried out in the dark and at in situ temperature. a Seawater culture was not diluted with 0.2 Am of seawater. Instead, grazers were reduced by passing culture water through a 0.8-Am mixed ester filter. b Microbial seawater culture was initiated after an extended light incubation in which whole water was inoculated with inorganic NO3 (1 AM) and PO4 (0.1 AM), and incubated in the light for several days to produce a phytoplankton bloom in a bottle. After chlorophyll a reached the maximum concentration, the culture medium was passed through a 0.8-Am filter and incubated in the dark. Thus, the culture was in the presence of the remaining inorganic nutrients and any phytoplankton produced DOM during light incubation. c Three liters of whole water was diluted with 7 l of a 0.2-Am filtrate.

excretia). Not all treatments were used in each experiment (Table 1). Amendments for cultures containing known amounts of inorganic and organic compounds included additions of ammonium (NH4Cl), phosphorus (K2HPO4), and labile DOC (glucose). Glucose was chosen as a labile DOC enrichment because it is the most abundant neutral sugar in the open ocean systems, and it is preferentially utilized relative to other monosaccharides (Rich et al., 1996). Enrichments of C, N, or P were added individually, or in combination with final concentrations of 10 AM C, 1 AM N, and 0.1 AM P (Table 1; Carlson and Ducklow, 1996; Carlson et al., 2002). Although the concentrations of inorganic nutrients at the BATS site are maintained at concentrations below colorimetric detection limits for most of the year, annual deep convective mixing (170 to >300 m) and mesoscale eddy pumping can entrain measurable concentrations of N and P into the eupho-

tic zone (Michaels and Knap, 1996; Steinberg et al., 2001). Amendments for cultures containing naturally produced DOM included both DOM derived from phytoplankton cultures and zooplankton excretia. For the phytoplankton exudates experiments, DOM was generated by incubating whole water in a polycarbonate carboy amended with 1 AM NO3, 1 AM SiO4, and 0.1 AM PO4 under natural light exposure for approximately 10 days, when chlorophyll a values indicated that phytoplankton cells were at a stationary growth phase. The whole water was then passed through a 0.8-Am filter and the filtrate was collected and incubated in the dark to assess heterotrophic bacterial growth. The DOM produced during the light phase was likely derived from several processes including direct exudation by phytoplankton, DOM release from grazers, and perhaps hydrolytic breakdown of DOC. The exact mechanism of DOM production was not

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identified in these experiments but was ultimately constrained by the magnitude of primary production. Zooplankton-derived DOM was generated by incubating zooplankton collected at night in short, vertical net tows (0 – 200 m). Animals were immediately sorted from tows and an assortment of copepods, euphausiids, and amphipods was individually pipetted into incubation bottles containing 2 l of 0.2 Am of filtered seawater (generated as described for seawater cultures). The zooplankton were incubated for 9 h at in situ mixed-layer temperature (22.8 jC). The filtrate was generated by first passing the entire bottle contents through a 53-Am Nitex screen to remove zooplankton and their fecal pellets. This filtrate was then passed through a 0.2-Am filter. The final composition of the seawater culture included 3 l of whole water, 2 l of 0.2-Am zooplankton excretia, plus 5 l of a 0.2-Am surface water filtrate. The seawater culture was incubated in the dark at in situ temperatures. 2.4. Spectroscopy Absorption spectroscopy was performed using a Perkin-Elmer Lambda 18 spectrophotometer and 10cm quartz windowed cuvettes. The instrument was zeroed using Milli-Q water in both cuvettes, and all measurements were made with sample and reference at room temperature. Samples for CDOM spectroscopy were filtered using an all-glass filtration unit through 0.2-Am Nuclepore filters that had been pretreated by flushing with 500 ml of Milli-Q water. Filtered samples were stored in acid-washed dark glass bottles (Qorpak) with Teflon (PTFE)-lined lids, in the dark at 4 jC (Mueller and Austin, 1995) until analysis (usually within 3 days). Our previous experience has shown no alteration of samples stored in this manner for up to 30 days (not shown). The spectra were acquired as decadal optical density, corrected for baseline offsets (e.g., Green and Blough, 1994) and were converted to absorption coefficient (m
1) by converting to base ‘‘e’’ (multiplying by 2.303) and dividing by the geometric path length (0.1 m). Absorption spectra were subjected to additional quality control before use. Specifications of the instrument indicate that the limit of detection is approximately 0.06 m
1. However, log-transformed absorption spectra of filtered seawater typically main-

tain linearity from 320 nm down to 0.03 m
1, which typically occurs between 380 and 420 nm in natural samples (Nelson et al., 1998). Log-transformed absorption spectra were tested for linearity vs. wavelength between 320 nm and 0.03 m
1, and were excluded from analysis if the linear correlation coefficient was less then 0.9 or the least squares estimated log slope was less than
0.07 nm
1. CDOM spectra in the UV-A and visible are commonly parameterized using an exponential equation (Bricaud et al., 1981; Green and Blough, 1994), such that: acdom ðkÞ ¼ acdom ðk0 Þe
Sðk
k0 Þ

ð1Þ

where acdom(k) is the absorption coefficient at wavelength k (m
1), acdom (k0) is the absorption coefficient at a reference wavelength k0, and S is the exponential slope parameter. For each spectrum, we calculated the parameter S over the wavelength interval from 320 nm to the limit of detection (estimated as 0.06 m
1) by (1) log-transforming the spectrum and finding the slope by linear regression (hereafter known as LF for linear fit), and (2) fitting an exponential equation to the untransformed data (NLF for nonlinear fit; Blough and Del Vecchio, 2002; Stedmon et al., 2000). There is an inflection point in most spectra at ca. 310 – 315 nm, where the exponential slope changes to a steeper value, making the exponential assumption (Eq. (1)) inappropriate for the complete spectrum. Both LF and NLF methods fit complete (280 nm to limit of detection) spectra poorly because of the change in slope near 310 nm. Slopes computed from LF and NLF fits using the >320-nm data were comparable (n = 874, R2>0.99, rms difference = 5.8 E
4, linear regression slope = 1.0006, intercept =
0.0004), with S values retrieved using NLF being slightly higher. Values of S computed here are therefore smaller and are not strictly comparable to those reported by Nelson et al. (1998) and Blough and Del Vecchio (2002) who used different wavelength intervals (280 – 350 nm, and 290 nm to the detection limit, respectively). Nelson et al. (1998) selected the 280 – 350-nm wavelength band to span the ca. 310-nm inflection point and present a composite S value; analysis of those data and more recent data have suggested that the spectral slopes of the < 310- and >320-nm segments of the spec-

N.B. Nelson et al. / Marine Chemistry 89 (2004) 273–287

trum are independent of each other (not shown). For the purpose of the present study, we elected to use the longer wavelength segment. 2.5. Bacterial cell counts and production Bacterioplankton samples were fixed with particlefree 25% glutaraldehyde (final concentration 1.0%) and stored at 4 jC until slide preparation. All slide preparations were conducted within 48 h of sample collection. Cells were filtered onto Irgalan blackstained 0.2-Am polycarbonate filters, stained with either acridine orange (Hobbie et al., 1977) or 4V– 6V-diamidino-2-phenylidole (DAPI; Porter and Feig, 1980), and enumerated with an Olympus AX-70 epifluorescence microscope (1000  ). Flagellates were enumerated using the same slides used to count bacterioplankton. Bacterial production for the March 1998 experiment was estimated from measurements of high specific activity methyl-3H thymidine (TdR; SA = 90 Ci mmol
1) incorporation into cold 5% trichloroacetic acid extracts following the microcentrifugation method of Smith and Azam (1992). Triplicate 1.7-ml samples were incubated and processed in plastic microcentrifuge tubes. All sample preparation and processing were conducted in subdued light at in situ temperature in a radiation laboratory. Samples were incubated for 1 – 2 h depending on the ambient activity levels, and assayed by liquid scintillation spectroscopy.

3. Results 3.1. Patterns of CDOM distribution in the field Seasonal patterns in CDOM distribution at the BATS site in 1996 –1999 (Fig. 1C) were similar to patterns reported by Nelson et al. (1998) for 1994– 1995. During the spring of each year, CDOM accumulated at the surface, and decreased to below-winter levels at the surface as the summer proceeded. Between 60 and 100 m (starting just below the mixed layer; Fig. 1A) CDOM absorption coefficients continued to increase throughout the summer until convective mixing homogenized the water column. During the summer months, the subsurface peak in

279

CDOM abundance was shallower than the deep chlorophyll maximum layer and the 1% of surface irradiance depth (Fig. 1B). The seasonal pattern of CDOM appears to be superimposed over a ‘background’ of CDOM, which is interannually variable. In 1997 and 1998, an anomalously high-salinity water mass was present, which correlated well with the decrease in ‘background’ CDOM (e.g., 200 m) observed in the field data. The seasonal pattern was still present but of lower amplitude because of the smaller ‘background’ CDOM concentration (Fig. 1C). 3.2. Bacterial culture experiments Three types of bacterial culture experiments were performed (Table 1). The purpose of the experiments was to determine: (a) whether bacteria in culture produce CDOM; and (b) whether CDOM produced in culture was also consumed and (c) delineate conditions under which net CDOM production occurs. The list of experiments discussed is given in Table 1. The first experiment involved unamended seawater cultures. The second experiments used filtered seawater amended with material excreted from phytoplankton in culture. The third experiment type involved unamended cultures and cultures amended with: (1) glucose; (2) nitrate + phosphate; (3) glucose, nitrate, and phosphate; and (4) zooplankton excretia. In most cases where bacterial growth occurred, CDOM appeared in the cultures during the first few days of the experiment. After this, most of the new CDOM disappeared over periods ranging from 5 to 30 days. Rates of microbial growth during log phase, specific rates of CDOM increase during log phase growth, and specific rates of CDOM decrease after the log phase of cell growth has ended are shown in Table 2. A time course of CDOM absorption coefficient and bacterial number within an unamended culture using Sargasso Sea water and microbes is shown in Fig. 2. This experiment was carried out using water collected at Hydrostation S in February 1996, late winter in the Sargasso Sea (Michaels and Knap, 1996). During this cruise, the mixed layer depth was greater than 200 m and it is possible that water collection coincided with elevated primary production and DOC production. Previous experiments conducted in the Sargasso Sea have demonstrated that when bacterial production

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Table 2 Estimated maximum specific growth ratesa (day
1) for microbial cells and CDOM (during log growth), and CDOM specific decrease rates (day
1) in bacterial culture experiments Treatment

l (cells) (day
1)

CDOM production (day
1)

CDOM consumption (day
1)

Unamended; February 1996 (Fig. 2) Phytoplankton culture exudates; March 16, 1996 Phytoplankton culture exudates; March 25, 1996 Glucose, N + P; March 1998 (Fig. 3A) Glucose, N + P; April 1998 (Fig. 3B) Glucose, N + P; December 1999 (Fig. 4) Zooplankton excretia; Dece 1999 (Fig. 4)

0.3

0.40


0.0065

0.4

0.12


0.01

n.a.

0.36


0.01

2.8b

0.35


0.055

0.6

0.18


0.055

0.6

1.2


0.06

1.2

1.1


0.05

The absorption coefficient (m
1) at 325 nm was used to compute specific production and consumption rates. a Specific growth rates were determined from the slope of the natural log of cell abundance (or cell production estimated from 3H TdR incorporation) vs. time during log phase of growth. b Specific growth rate was estimated from 3H TdR incorporation rates (no cell counts were available).

increases significantly in cultures, DOC concentrations are reduced (Carlson and Ducklow, 1996). In the culture, CDOM absorption coefficient increased rapidly over the first 2 days, then declined gradually over the next 30 days to a steady level (Fig. 2). The peak of CDOM absorption was coincident with the transition from logarithmic to stationary growth of bacteria. CDOM light absorption did not decline to the timezero level, even after 43 days of incubation. At this time, the CDOM absorption coefficient was 50% greater than the time-zero value and 50% of the maximum value attained during the first few days of the experiment. In this experiment, replicate samples were collected to qualitatively assess the significance of the observed changes. Standard deviations (Fig. 2) indicated that observed changes in cell concentration and CDOM absorption coefficient were significant.

Specific production rate of CDOM at the outset of the experiment was 0.4 day
1, and the cell division rate was 0.26 day
1. The specific decrease rate of CDOM absorption coefficient after the peak was
0.0065 day
1. A similar culture experiment conducted later in the year, when water column productivity was low, showed no significant microbial growth or new CDOM production (not shown). Growth of bacteria and net CDOM production in amended seawater cultures varied. In two ‘natural DOM amendment’ experiments, phytoplankton cultures in large-volume containers were prepared with filtered seawater (from the BATS site), amended with inorganic nutrients, and allowed to bloom. After the phytoplankton production peaked, the medium was filtered and used as a bacterial growth medium. These experiments had similar results to the February 1996 experiment in that CDOM increased over the first 2– 3 days of the experiment by a factor of ca. 2, then declined over the next 45 –60 days to an intermediate value. CDOM-specific production rates at the outset of the experiments were 0.12 and 0.36 day
1, respectively (Table 2). The beginning of the decline in CDOM concentration coincided with a shift from logarithmic growth to stationary growth of bacteria in each case. The specific rate of decline of CDOM was ca.
0.01 day
1 in both cultures amended with phytoplankton-derived DOM. The third type of bacterial culture experiment involved seawater cultures amended with known quantities of inorganic nutrients and glucose (Figs. 3 and 4), or an undefined mixture of zooplankton excretia (Fig. 4). In an experiment conducted in March 1998, the controls and the amended cultures produced CDOM (Fig. 3A), although the culture containing inorganic and organic amendments produced a slightly larger amount. In all treatments of this experiment, CDOM concentrations returned to the preexperiment values after 6 days. In a similar experiment carried out in April 1998 (Fig. 3B), CDOM light absorption in the control, glucose, and inorganic amendment cultures did not increase significantly in the first 48 h, but the culture amended with both glucose and inorganic nutrients had a significant increase in CDOM absorption (Fig. 3B). In the glucose-amended cultures, the CDOM absorption returned to the background value ( F 10%) within 3 days of the peak (Fig. 3). These results were in

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281

0.8 –1

acdom(325 nm) (m –1), Cells (108ml –1)

: CDOM (m )

0.7

–1

: Cells (ml )

0.6

0.5

0.4

0.3

0.2

0.1 0

2

4

6

8

10

20

40

Days Fig. 2. Time course of a microbial culture experiment using unamended filtered seawater collected at Hydrostation S in February 1996. Diamonds show direct counts of acridine orange-stained bacteria (  108 cells ml
1) using fluorescence microscopy. Dots show absorption coefficient of CDOM at 325 nm (m
1). Vertical bars are standard deviations of replicate samples, which were collected during this experiment.

0.25

A) Mar. 1998

GNP G NP Unamended

B) Apr. 1998

GNP G NP Unamended

acdom (325 nm) (m–1)

0.2

0.15

0.1 0.25

0.2

0.15

0.1

0

1

2

3

4

5

6

7

8

Days Fig. 3. Time course of CDOM absorption coefficient (m
1; 325 nm) in two microbial culture experiments in which filtered seawater was amended with glucose (circles), ammonium and phosphate (inverted triangles), and all three (asterisks). Unamended cultures (dots) are also shown. A) Experiment conducted using water collected in March 1998. B) Experiment conducted using water collected in April 1998.

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Fig. 4. Time course of CDOM absorption coefficient (325 nm) in bacterial cultures, without amendment (dots); with amendment consisting of glucose, ammonium, and phosphate (asterisks); and zooplankton excretia (triangles).

contrast to the experiments that used more complex ‘natural’ sources of DOC for bacterial growth instead of glucose. Estimated specific increase rates of CDOM absorption coefficient were 0.35 day
1 in the March culture and 0.18 day
1 in the April culture experiment. In both experiments, the specific rate of CDOM absorption coefficient decrease was
0.055 day
1 after the peak. In the ‘zooplankton excretia’ experiments, CDOM was produced in both GNP-amended and zooplankton excretia-amended treatments (Fig. 4), but in the GNPamended treatments, almost all the CDOM were removed by the end of 3 days of incubation. In the excretia-amended treatments, some accumulation of CDOM was apparent over and above the initial addition of CDOM from the zooplankton excretia itself. In these experiments, the rate of increase of CDOM at the outset of the experiment was greater than in other culture experiments (Table 2), but the CDOM-specific decrease rate was similar to those reported for previous GNP-amended cultures (Table 2).

3.3. Spectral characteristics of CDOM Differences in the seasonally averaged absorption spectra of CDOM for three depth layers (chosen to reflect the ‘‘summer bleaching’’ layer, the ‘‘summer production’’ layer, and the ‘‘background’’ layer; e.g., Fig. 1) are shown in Fig. 5. Each spectrum is a difference spectrum, where the average spectrum from one season is subtracted from the previous season’s average spectrum, indicating the seasonal change. In the surface (0 –20 m) and mid-depths (40 – 100 m), the winter-to-spring transition is seen as a positive increase in absorption coefficient throughout the spectrum, increasing to shorter wavelengths (Fig. 5A and B). Significant decreases in CDOM absorption spectra occurred at wavelengths shorter than 325 nm in the surface layer during the spring – summer transition (Fig. 5A), indicative of the impact of solar bleaching. Increases of a similar magnitude occurred in the middepth range, indicative of the appearance of new CDOM (Fig. 5B). CDOM increased at the shortest

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283

Fig. 5. Difference spectra (m
1) of multiyear seasonal average absorption coefficient within three depth ranges in the upper ocean in the Sargasso Sea, for: A) surface to 20m depths; B) depths between 40m and 100m; and C) depths between 160m and 200m. The thin solid line shows the difference in absorption spectra between winter (January, February, March) and spring (April, May, and June). The thick solid line is the difference between spring and summer (July, August, and September); the dashed line is the difference between summer and fall (October, November, and December). The spring – summer difference spectra for the surface (A) and mid-depths (B) highlights the effects of bleaching of CDOM at the surface and production in the mid-depths during the summer (Fig. 1).

wavelengths ( < 300 nm) in the surface layer during the summer – autumn transition (Fig. 5A), while a decrease in absorption between 300 and 350 nm occurred in the mid-depths (Fig. 5B). We attribute the summer –autumn transitions to reflect the mixing of bleached surface summer CDOM with ‘‘new’’ subsurface CDOM during the period of mixed layer deepening (Fig. 1A). No significant (above detection limit) changes in the absorption spectra were observed

in the deep (160 –250 m) range throughout the year (Fig. 5C). Mean values of the spectral slope parameter S (nm
1) calculated over the range from 320 nm to the limit of detection (LF) are reported in Table 3. As might be expected from Fig. 5, the largest changes in S occur in the surface and mid-depths during the summer and autumn months. We also computed the spectral slope parameter for ‘newly produced’ CDOM

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Table 3 Value of the exponential slope parameter S (LF) over the wavelength range from 320 nm to the detection limit (nm
1) in absorption spectra from field samples (5-year mean, approximately 140 casts), spring, and summertime culture experiments, and difference spectra of mean absorption spectra (Fig. 5)

Surface (1 – 20 m) Mid (40 – 100 m) Deep (120 – 250 m) Cultures Difference spectra 1 – 20 m 40 – 100 m 120 – 250 m

Winter

Spring

Summer

Autumn

0.0152 0.0159 0.0156

0.0167 0.0159 0.0148 0.0220

0.0181 0.0189 0.0169 0.0250

0.0194 0.0178 0.0165

n.s. n.s. n.s.

0.0224 0.0075 n.s.


0.0204 0.0236 n.s

0.075 n.s. n.s.

For the difference spectra, ‘‘Winter’’ refers to the winter – autumn difference, ‘‘spring’’ refers to the spring – winter difference, ‘‘summer’’ refers to the summer – spring difference, and ‘‘autumn’’ refers to the autumn – summer difference. Difference spectra that had no data with absolute values greater than the detection limit are considered not significant and are denoted ‘‘n.s.’’

in culture, and for the seasonal difference spectra shown in Fig. 5B. Values of S for ‘new’ CDOM in culture were estimated from the difference between the CDOM spectrum recorded at the maximum during a time course and the time-zero CDOM absorption spectrum, assuming that the difference spectrum represents the spectrum of the newly introduced material. The value of S for new CDOM in cultures was found to be between 0.022 and 0.025 nm
1, exceeding the maximum mean values of S from the field (0.0194 nm
1; Table 3). Surface spring minus winter difference spectrum (Fig. 5A) had an S value of 0.022 nm
1, while the mid-depth summer minus spring difference spectrum (Fig. 5B) had an S value of 0.024 nm
1.

4. Discussion Results of the present study clearly indicate a major role for microbes in the production of CDOM in the Sargasso Sea. Bacterial cultures grown under a range of conditions showed increases in CDOM light absorption during the first 48– 60 h of incubation (Figs. 3 and 4), except in cases when there was no significant microbial growth. In all experiments, the majority of this initial CDOM production was then consumed over a similar or slightly longer time

period. Under some conditions, a portion of the newly produced CDOM persisted to the end of the experiment (up to ca. 50 days). Clearly some of the CDOM produced by microbes in these experiments can be categorized as a recalcitrant component of the DOC pool (i.e., material that is unavailable to rapid microbial degradation) (Carlson, 2002; Hedges, 2002). Previous studies have also demonstrated that bacterioplankton can produce recalcitrant DOM in the water column (McCarthy et al., 1998; Brophy and Carlson, 1989; Rochelle-Newall et al., 1999; Ogawa et al., 2001), and that bacterioplankton contain chromophoric materials that can contribute to CDOM in the water column (Determann et al., 1998). Here we demonstrate that under favorable conditions, bacterioplankton can produce recalcitrant DOM with a chromophoric signature. Bacterial cultures grown without amendments to the Sargasso Sea water only occasionally exhibited microbial growth (Carlson et al., 2002) and CDOM production (i.e., Figs. 2 and 3A). Cultures incubated with amendments of both glucose and inorganic nutrients, and zooplankton excretia demonstrated significant bacterial and CDOM production more readily than unamended cultures or cultures amended with just glucose (Fig. 3). Decline of the CDOM concentration after the peak of production and cell growth also differed (Table 2), with the most rapid degradation occurring in the experiments using glucose or zooplankton excretia amendments (Figs. 3 and 4), followed by the experiments using seawater from phytoplankton cultures, and, finally, the experiment where microbial growth and CDOM occurred without amendments (Fig. 2). These results together indicate that the quality of the DOM substrate can control the ultimate lability of the newly produced CDOM. Experiments where more complex ‘‘natural’’ DOM, phytoplankton, or zooplankton-excreted DOM were used as carbon source for the bacteria (Figs. 2 and 4), resulted in more persistent CDOM than experiments where simple addition of glucose was the carbon source (Fig. 3). This result may, in part, explain why little or no correlation between DOC concentration or primary productivity, and CDOM concentration or production rate is found in the Sargasso Sea (Nelson et al., 1998), as a simple measurement of DOC concentration does not reveal the complexity of the available substrate.

N.B. Nelson et al. / Marine Chemistry 89 (2004) 273–287

The specific growth rates of bacteria in the culture experiments (Tables 1 and 2) are ca. three to five times greater than the bacterial growth rates at the BATS site estimated from field data (Carlson and Ducklow, 1996). Nelson et al. (1998) estimated a constant, which related specific CDOM production to specific bacterial production in a simple model of CDOM dynamics for the stratified mixed layer. In this model, the CDOM production rate was estimated at approximately 8% of the specific bacterial production rate, which averaged around 0.07 day
1, indicating a mean CDOM production rate of 0.0056 day
1 (Nelson et al., 1998), which is considerably lower than initial CDOM production rates in culture (Table 2). This result may be due to the ‘batch’ culture procedure introducing an excess of substrate to the culture than is present at one time in situ, or due to the fact that the newly produced CDOM is not diluted faster than it can be consumed or degraded, as it may be in the field. On the other hand, much of the CDOM produced initially in culture appears to be labile and is rapidly degraded after approximately 2 days, regardless of the substrate (Figs. 2 – 5). If we consider the experiments using natural or complex substrate where the initial pulse of CDOM was not completely removed (e.g., Figs. 2 and 4), the apparent production rates of refractory CDOM over the course of the experiment (from time zero to the point where CDOM decrease apparently stopped) were 0.0096 and 0.0071 day
1, respectively. These apparent production rates, while still higher than those estimated by Nelson et al. (1998) for the water column, are definitely comparable. These results provide a clear explanation for the accumulation of CDOM below the surface during the summer (Fig. 1) in the absence of significant phytoplankton production. If suitable DOM is present, the production of CDOM outpaces consumption below the depth of significant solar bleaching, but when bacterial production declines and the mixed layer deepens at the end of the summer, the combined effects of bleaching and other unspecified sinks act to remove most, if not all, of the seasonally produced CDOM (Nelson et al., 1998). We have attempted to independently assess the contribution of the ‘newly produced’ CDOM to the total CDOM pool by examining differences in the spectral characteristics of ‘new’ and ‘background’

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CDOM (Fig. 5, Table 3). If we assume that the log spectral slope (320 nm to the limit of detection) of a mixture of ‘new’ and ‘background’ CDOM is a simple linear combination of the slopes and the fractions, then the fraction of ‘new’ CDOM present in a given sample can be estimated, except in summer surface waters where solar bleaching can also increase the spectral slope parameter (Nelson et al., 1998; Del Vecchio and Blough, 2002). For this purpose, we assume that the minimum S value of the spectra from 120- to 250-m samples (‘‘Deep’’; Table 3) is one endmember, and the spectral slope of the CDOM in culture experiments is the other. Applying this analysis to the mean spectral slopes of the seasonal average spectra, we find that the maximum contribution of ‘‘new’’ CDOM to the total CDOM pool in mid-depth waters during the summer is ca. 45% (Table 4). This is also consistent with the seasonal variation of CDOM light absorption at 325 nm (Fig. 1) where approximately 50% variability occurs over a background concentration (also Nelson et al., 1998). Similar estimates of the contribution of ‘‘new’’ CDOM to the CDOM pool are retrieved if the spectral slope of the mid-depth summer – spring difference spectrum (Fig. 5B, Table 3) or the spectral slope of the surface spring– winter difference spectrum is used (Fig. 5A, Table 3). As net new CDOM production is thought to occur in surface waters in the spring and at the mid-depths in the summer (Nelson et al., 1998), this result is consistent with our supposition that culture-produced new CDOM is similar to field-produced new CDOM. Table 4 Estimated relative contribution of ‘‘new’’ CDOM to the total CDOM pool, based on the exponential slope of CDOM light absorption spectra (Table 3)

Surface (1 – 20 m) Mid (40 – 100 m) Deep (120 – 250 m)

Winter (%)

Spring (%)

Summer (%)

Autumn (%)

4.6 12.6 9.2

21.8 12.6 0.0

37.9 47.1 24.1

52.9 34.5 19.5

This computation assumes the minimum value of spectral slope of ‘‘deep’’ samples is one endmember, and the spectral slope of CDOM from bacterial cultures is the other. The contribution of ‘‘new’’ CDOM in surface waters in the summer is overestimated, as solar bleaching also increases the exponential slope parameter (Green and Blough, 1994; Nelson et al., 1998; Del Vecchio and Blough, 2002).

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The seasonal pattern of the fractional contribution of ‘‘new’’ CDOM to total CDOM (Table 4) provides interesting hints about the interaction between physical and biological processes determining CDOM dynamics. For example, the fractional contribution of ‘new’ CDOM at the deep depth horizon varies throughout the year, reaching a peak in summer and autumn. This change probably reflects the transport of ‘new’ CDOM produced during the summer below the seasonal thermocline in some years by convective mixing. Convective export is an important part of the annual cycle of DOC at the BATS site (Carlson et al., 1994), and this result indicates that CDOM forms part of the DOC that is transported. Convective export, bleaching (Nelson et al., 1998), and microbial consumption (Table 2) may reduce CDOM in the surface layers until spring, when most of the CDOM in the water column appears to be ‘background’ CDOM. In the present study, we have demonstrated that the microbial community, acting on substrates such as natural DOC, zooplankton excretia, or algal exudates, can produce CDOM, which can be consumed by bacteria but that can also persist for over 40 days in some cultures. Evidence from CDOM absorption spectra of laboratory cultures and field samples supports the paradigm of an annual cycle of CDOM superimposed upon a background level of CDOM, which appears to be a water mass characteristic. Our ongoing investigations indicate additional sources of ‘new’ CDOM from some phytoplankton and zooplankton (Steinberg et al., 2004), and we are further investigating the characteristics of ‘new’ and ‘background’ CDOM in the context of solar bleaching and microbial consumption.

Acknowledgements This research was supported by the NSF [Chemical Oceanography program (OCE0196305, OCE9977399, and OCE0241614) and the Biological Oceanography and Molecular and Cellular Bioscience programs] and NASA (SIMBIOS Project, NAS5-00200). We acknowledge the continued support of the BATS program and the captain and crew of the Weatherbird II in making these investigations possible. Lore Ayoub, Matthew Krasowski, Ru Morrison, Greg Earl, Marla

Ranelletti, Stuart Goldberg, and Rachel Parsons provided essential technical assistance. Thanks to Dave Siegel for helpful discussions and three anonymous reviewers for valuable comments.

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