Carbohydrates in phytoplankton and freshly produced dissolved organic matter

Marine Chemistry 63 Ž1998. 131–144

Carbohydrates in phytoplankton and freshly produced dissolved organic matter Andrew Biersmith, Ronald Benner

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Marine Science Institute, UniÕersity of Texas at Austin, 750 ChannelÕiew DriÕe, Port Aransas, TX 78373, USA Received 1 August 1997; revised 12 May 1998; accepted 18 June 1998

Abstract Four taxonomically-diverse, phytoplankton cultures Ž Phaeocystis sp., Emiliania huxleyi, Synechococcus bacillaris, Skeletonema costatum. were grown in batch culture for 14 days, and the particulate and high-molecular-weight dissolved components of the cultures were harvested by tangential-flow ultrafiltration for bulk and molecular-level carbohydrate analyses. Bulk carbohydrates and neutral aldoses accounted for an average of 37% and 20%, respectively, of the particulate organic carbon Ž) 0.1 mm. in the cultures. Glucose was the dominant aldose in phytoplankton cellular material. Ultrafiltered dissolved organic matter ŽUDOM; ) 1000 Da. from the cultures was rich in carbohydrates relative to cellular material. Bulk carbohydrates and neutral aldoses accounted for an average of 66% and 35%, respectively, of UDOM in the cultures. The average CrN value Ž21.6. for UDOM was much higher than the value Ž8.8. for cellular material, reflecting the carbohydrate-rich nature of UDOM. Freshly produced UDOM was characterized by similar contributions of several aldoses, including galactose, glucose, mannose, fucose, xylose, and arabinose. The aldose signatures of phytoplankton UDOM were distinct from the signatures for cellular material and were indicative of heteropolysaccharides. The UDOM produced in phytoplankton cultures was similar in nature to the UDOM isolated from various locations in the surface ocean. Surface ocean UDOM is rich in carbohydrates and is relatively depleted in nitrogen ŽCrN ; 17., and the aldose signature of marine UDOM is similar to that of phytoplankton UDOM. These observations indicate that phytoplankton extracellular releases could be a major source of the dissolved heteropolysaccharides observed throughout the surface ocean. q 1998 Elsevier Science B.V. All rights reserved. Keywords: phytoplankton; carbohydrates; aldoses; dissolved organic matter

1. Introduction Phytoplankton is the primary source of organic matter and carbohydrates in marine systems ŽRomankevich, 1984.. About 5–30% of phytoplankton production is directly released as dissolved or)

Corresponding author. Tel.: q1-512-749-6772; Fax: q1-512749-6777; E-mail: [email protected]

ganic carbon ŽDOC. ŽMague et al., 1980; Fogg, 1983; Lancelot and Billen, 1985; Biddanda and Benner, 1997., which is one of the largest active reservoirs of organic carbon on Earth ŽHedges, 1992.. Predation and viral lysis of plankton are also important processes which contribute to the marine reservoir of DOC ŽJumars et al., 1989; Fuhrman and Suttle, 1993; Strom et al., 1997.. A large fraction Ž; 50%. of photosynthetically produced carbon

0304-4203r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 0 3 Ž 9 8 . 0 0 0 5 7 - 7

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A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

passes through dissolved organic matter ŽDOM. to heterotrophic bacteria ŽAzam et al., 1983; Ducklow et al., 1993.. Carbohydrates represent a significant fraction Ž15–30%. of marine DOC ŽBenner et al., 1992; Pakulski and Benner, 1994., and they are actively released in large amounts by growing phytoplankton ŽAntia et al., 1963; Guillard and Hellebust, 1971; Eberlein et al., 1983, 1985; Biddanda and Benner, 1997. and during grazing ŽDagg, 1974; Lampert, 1978; Strom et al., 1997.. All major classes of carbohydrates occur in marine DOM, including amino sugars ŽKerherve et al., 1995., uronic acids ŽMopper et al., 1995., and aldoses ŽMcCarthy et al., 1996; Borch and Kirchman, 1997; Skoog and Benner, 1997.. Understanding the sources, transformations, and fates of carbohydrates in the marine environment can provide insight into the overall cycling of photosynthetically produced organic carbon. There have been several studies on the production of dissolved carbohydrates by marine phytoplankton Žsee references above. and their consumption by marine heterotrophs ŽBurney et al., 1979, Burney, 1986; Liebezeit et al., 1980; Ittekkot et al., 1981; Eberlein et al., 1983.. However, there are relatively few comparative studies of the molecular composition of carbohydrates in marine and freshly produced phytoplankton DOM. Without this information, our understanding of the sources and diagenesis of dissolved carbohydrates within marine systems is limited. A relatively large fraction Ž; 30%. of DOC can be isolated from seawater for chemical characterization using tangential-flow ultrafiltration with 1000-Da cut-off membranes ŽBenner et al., 1992, 1997.. Samples from different ocean basins indicate that polysaccharides comprise a major fraction of ultrafiltered DOM ŽUDOM. in surface waters ŽBenner et al., 1992; McCarthy et al., 1993, 1996; Aluwihare et al., 1997; Skoog and Benner, 1997.. Polysaccharides rapidly decline in concentration with depth to consistently low concentrations below the oxygen-minimum layer. Molecular-level characterization of aldoses in UDOM indicates little spatial variability in molecular composition ŽMcCarthy et al., 1996; Aluwihare et al., 1997; Skoog and Benner, 1997.. In contrast, the molecular composition and bulk carbohydrate content of phytoplankton has been shown to be highly variable depending on the species ŽParsons

et al., 1961; Myklestad, 1974; Schmidt et al., 1980., stages of growth ŽHanda, 1969; Haug and Myklestad, 1976., nutrient availability ŽAntia et al., 1963; Myklestad and Haug, 1972; Myklestad, 1977., and light conditions ŽHanda, 1969; Varum and Myklestad, 1984.. In order to understand how a complex and variable mixture of phytoplankton-produced carbohydrates is processed and transformed, we must first characterize freshly produced POM and DOM from known sources. In this study, we characterized bulk carbohydrates and neutral aldoses in cultures of four taxonomically-distinct phytoplankton species. Particulate material Ž) 0.1 mm. was separated from dissolved material by tangential-flow ultrafiltration. Structural and cytoplasmic components of cells were separated by high-speed centrifugation. These cultures provided freshly produced POM and DOM which was not exposed to significant microbial degradation. The UDOM gathered from these cultures provided a first look at carbohydrates in freshly produced DOM and enabled us to directly compare the carbohydrate composition of UDOM to its phytoplankton source as well as to UDOM collected from seawater.

2. Materials and methods 2.1. Phytoplankton cultures The following four phytoplankton species were cultured: the prymnesiophyte Phaeocystis sp., the coccolithophore Emiliania huxleyi, the cyanobacterium Synechococcus bacillaris, and the diatom Skeletonema costatum. These phytoplanktons were chosen because of their abundance and wide geographic distribution in the world ocean as well as their taxonomic diversity. Cultures were grown under identical conditions in 235-l polypropylene tanks ŽNalgene biotank. for a period of 14 days. Background concentrations of DOC were kept low by preparing the culture media with deionized, reverse osmosis water and a synthetic basal seawater salt mixture. All cultures exhibited similar growth characteristics and doubling times, with an exponential growth phase between 3–10 days and a stationary phase after 10 days. Dissolved inorganic nitrogen ŽDIN. was almost entirely consumed in the S. bacil-

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

laris and Phaeocystis cultures by the beginning of stationary phase, while substantial concentrations of DIN remained in the S. costatum and E. huxleyi cultures at the end of 14 days. Patterns of DOC release were variable among the four phytoplankton species. Phaeocystis and S. costatum exhibited fairly constant rates of DOC release, while S. bacillaris and E. huxleyi demonstrated maximal DOC release during stationary phase ŽBiddanda and Benner, 1997.. For a more detailed description of the cultures and the culturing conditions and procedures, see Biddanda and Benner Ž1997.. Very low abundances of heterotrophic bacteria were present in the cultures. Biddanda and Benner Ž1997. measured the increase in heterotrophic bacterial abundance throughout the experiments and estimated the bacterial contribution to overall carbon flux. They found that heterotrophic bacterial metabolism accounted for the processing of - 1% of the phytoplankton-produced organic matter during the 14-day growth period. No evidence of viral lysis was observed in the cultures and grazers were not present. Thus, the organic matter produced in each incubation can be considered freshly produced and phytoplankton derived. 2.2. HarÕesting of cultures Following the end of this 14-day period, each culture Ž; 235 l. was divided into POM and DOM fractions by tangential-flow ultrafiltration using a 0.1-mm cut-off, hollow-fiber filter and an Amicon DC10L ultrafiltration system ŽBenner, 1991; Benner et al., 1997.. Tangential-flow ultrafiltration was also used to isolate the high-molecular-weight components of DOM Žincluding colloids., and this material is referred to herein as ultrafiltered DOM or UDOM. Spiral-wound, polysulfone membranes ŽS10N1; 1000 Da cut-off. and Amicon DC 10 and DC 30 ultrafiltration systems were used for the isolation of UDOM. The organic carbon content of each fraction of the cultures was measured to determine the efficiency of carbon recovery by ultrafiltration. The ultrafiltered POM ŽUPOM. and UDOM were concentrated to volumes of 1–2 l, and they were diafiltered with 20 l of Milli-Q water to remove salts. The desalted UDOM concentrates were dried following rotary evaporation using a Savant SpeedVac system ŽBenner et al., 1997..

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The UPOM concentrates were centrifuged at 16266 RCF for 30 min at 48C, and the supernatant was decanted and saved. The pellet was resuspended in 200 ml of Milli-Q water. The resuspension and centrifugation procedures were repeated twice, and the pellet and combined supernatant fractions were dried following rotary evaporation using a Savant SpeedVac system. The pellet fractions were composed of insoluble cellular material and were referred to as cell structural organic matter ŽCSOM.. The water-soluble, supernatant fractions were composed of the cytosol as well as any exopolymer secretions released from the outside of the cells. This fraction was referred to as cell lysate organic matter ŽCLOM.. All ensuing chemical analyses were performed on these two fractions and the UDOM fraction from each phytoplankton culture. Previous studies have fractionated phytoplankton cell material by hot water extraction ŽHanda and Yanagi, 1969., acid and base extraction ŽHaug et al., 1973., and sonication ŽHecky et al., 1973.. We chose the above separation technique because we considered it to be relatively gentle and representative of biological processes, such as predation and viral lysis. The CLOM fraction was produced by osmotic shock, centrifugal rupturing of cells, and centrifugal sheering forces which have been shown to release bacterial and algal exopolymer secretions from cells ŽDecho, 1990.. It also contains any water-soluble extracellular material retained by the 0.1-mm poresize, hollow-fiber filters. 2.3. Measurements Analysis of DOC was conducted on a Shimadzu TOC-5000 analyzer, and all values were blank-corrected ŽBenner and Strom, 1993.. The organic carbon and nitrogen contents of isolated fractions were determined using a Carlo Erba 1108 elemental analyzer following vapor phase acidification to remove traces of inorganic carbon ŽHedges and Stern, 1984.. The concentrations of bulk carbohydrates Žfree q combined. were measured using the MBTH method ŽJohnson and Sieburth, 1977. with the modifications of Pakulski and Benner Ž1992.. All values were within the precision levels found in prior studies Ž"2% standard deviation for C and N determina-

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A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

tions, and "10% standard deviation for MBTH measurements; n s 3.. Molecular-level aldose compositions of the three fractions were determined using the method of Cowie and Hedges Ž1984a.. Samples of 2–4 mg C were pretreated with 12 M sulfuric acid for 2 h at room temperature followed by hydrolysis in dilute Ž1.2 M. acid at 1008C for 3 h. An internal recovery standard Žadonitol. was added after hydrolysis. The sample was neutralized with barium hydroxide and subsequently centrifuged to remove the barium sulfate precipitate. The supernatant was deionized by passage through a mixed-bed of ion exchange resins and dried in a Savant SpeedVac. An absolute recovery standard Žsorbitol. was added to the concentrate followed by an equal volume of 0.4% LiClO4 in pyridine. The aldose anomers were equilibrated at 608C for 48 h and derivatized with Sylon BFT ŽSupelco. for 10 min at 608C. The resulting trimethylsilyl derivatives were analyzed on a Hewlett Packard 5890 gas chromatograph using a non-polar capillary column ŽDB-1, J & W Scientific. and a flame ionization detector. The quantification of individual aldose concentrations was determined by comparison to the adonitol internal standard. Cowie and Hedges Ž1984a. reported sample mean deviations for individual aldoses ranging from 2–10%. For this study, each sample was analyzed twice with average percentage mean deviations for individual aldoses among all samples ranging from 2–9%, with most falling below 5%. The exception was lyxose Ž20%.. This higher variability is thought to be a function of the low levels of lyxose in most samples and the epimerization of a small fraction of xylose. The recoveries of ribose are low following the acid hydrolysis used in this method ŽCowie and Hedges, 1984a..

ŽCLOM. to the initial POC reported by Biddanda and Benner Ž1997.. With the exception of S. bacillaris, an average of 99 " 11% of the initial POC was recovered as CSOM and CLOM. Substantial losses Ž72%. of the S. bacillaris culture during ultrafiltration were likely a result of their sticky mucilaginous sheaths. Earlier investigations with hollow-fiber filters demonstrated that bacterial cells stick to the membrane ŽBenner, 1991; Benner et al., 1997.. An average recovery of 98 " 8.5% of the initial DOC in the cultures was measured in the ultrafiltered and filtrate fractions, indicating minimal carbon contamination or loss. Biddanda and Benner Ž1997. reported that ; 35% of the initial DOC in each of the cultures was recovered in the UDOM fraction. Using the same ultrafiltration membranes and system, UDOM was shown to comprise ; 30% of surface ocean DOC ŽBenner et al., 1992, 1997.. The percentages of carbon found within the various culture fractions ŽCSOM, CLOM, and DOM. are presented in Table 1. About half Ž48 " 9%. of the organic carbon in the cultures was measured in the CSOM fraction. A slightly lower and more variable percentage of carbon Ž35 " 16%. was measured in the CLOM fraction. Due to the previously mentioned filtration losses with the S. bacillaris culture, the Table 1 Distribution of organic carbon ŽOC. among different fractions of phytoplankton cultures Fraction

Culture

mM C

Percentage OC

Phaeocystis E. huxleyi S. bacillaris Ske. costatum

336 257 425 231

48 37 58 48

Phaeocystis E. huxleyi S. bacillaris Ske. costatum

271 380 220 82

39 54 30 17

Phaeocystis E. huxleyi S. bacillaris Ske. costatum

96 65 87 172

14 9 12 35

CSOM

CLOM

3. Results and discussion 3.1. Carbon recoÕeries A carbon mass balance was used for each culture to monitor carbon recovery efficiencies during ultrafiltration. Recovery efficiencies of ultrafiltered particulate organic carbon ŽUPOC. were determined by comparing the organic carbon in cell structural organic matter ŽCSOM. and cell lysate organic matter

DOM

Abbreviations: CSOM, cell structural organic matter; CLOM, cell lysate organic matter; DOM, dissolved organic matter.

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

initial value of POC measured by Biddanda and Benner Ž1997. was used to represent the combined CSOM and CLOM fractions. Thus, the amount of carbon in the CSOM and CLOM fractions of the S. bacillaris culture was estimated by assuming the carbon recovered in these fractions was representative for the POC. S. costatum had the lowest percentage of carbon in the CLOM fraction Ž17%. and the highest percentage Ž36%. of carbon as DOC. All other cultures produced similar percentages Ž12 " 3%. of carbon as DOC. Biddanda and Benner Ž1997., using 0.7-mm pore-size, glass–fiber filters to retain POC, reported similar percentages of total organic carbon as DOC in the S. bacillaris and S. costatum cultures and considerably higher percentages as DOC in the Phaeocystis and E. huxleyi cultures. The 0.1-mm pore-size filter used in the present study retained a greater fraction of carbon as POC in the Phaeocystis and E. huxleyi cultures. Phaeocystis is known to secrete a sticky, carbohydrate-rich mucus which binds cells into colonies ŽGuillard and Helle-

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bust, 1971; Verity et al., 1988; Rousseau et al., 1994. and constitutes a large proportion of the overall colony biomass ŽRousseau et al., 1990.. The lower estimates of DOC in the present study were likely due to greater retention of mucus and colloids by the 0.1-mm pore-size filter. 3.2. Bulk carbohydrates Analyses of bulk combined carbohydrates ŽMBTH method. indicated that carbohydrates comprised a substantial percentage of the organic carbon in all fractions and cultures ŽTable 2.. This analysis of carbohydrates includes basic and acidic carbohydrates as well as the neutral aldoses, which were also measured at the molecular level Žsee below.. Carbohydrate yields are reported here as the percentage of total organic carbon as carbohydrate. Cell structural organic matter ŽCSOM. had the lowest average Ž"SD. carbohydrate yield Ž29 " 11%.. Cell lysate organic matter ŽCLOM. had a higher average

Table 2 Organic carbon ŽOC., nitrogen ŽN., total aldose carbon ŽTAC., and bulk carbohydrate carbon ŽTCHOC. content of different fractions of the phytoplankton cultures Fraction Culture

Dry weight OC ŽPercentage N ŽPercentage CrN TAC TCHOC Percentage Žmg. Žatom. ŽPercentage OC. ŽPercentage OC. TCHOC as aldose dry weight. dry weight.

CSOM Phaeocystis 2103 E. huxleyi 3427 S. bacillaris 793 S. costatum 1770

44.5 20.9 41.6 36.5

8.5 2.9 6.5 6.2

6.1 8.3 7.4 6.9

13.9 24.8 16.8 4.8

27.8 42.5 31.6 16.1

50.0 58.4 53.2 29.8

Phaeocystis 2004 E. huxleyi 2786 S. bacillaris 518 S. costatum 671

37.6 38.0 33.5 34.2

2.8 2.9 4.6 4.2

15.6 15.2 8.5 9.5

27.1 34.3 29.7 19.1

50.9 53.6 54.1 34.5

53.2 64.0 54.9 55.4

Phaeocystis E. huxleyi S. bacillaris S. costatum

4107 6213 1311 2441

41.1 28.6 38.4 35.9

5.7 2.9 5.7 5.7

8.4 11.5 7.8 7.4

19.8 30.5 21.2 8.5

38.1 49.1 39.4 20.9

51.9 62.0 54.0 40.9

Phaeocystis E. huxleyi S. bacillaris S. costatum

551 352 727 812

12.4 12.0 10.6 22.4

0.8 0.6 0.6 1.3

19.3 25.2 21.9 20.1

28.7 30.4 40.9 38.7

56.3 62.2 69.6 74.1

51.0 48.9 58.8 52.2

CLOM

UPOM

UDOM

Abbreviations: CSOM, cell structural organic matter; CLOM, cell lysate organic matter; UPOM, ultrafiltered particulate organic matter Žweighted average of CSOM and CLOM.; UDOM, ultrafiltered dissolved organic matter.

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carbohydrate yield of 48 " 9%. Combining these two fractions, the weighted average carbohydrate yield of phytoplankton cells ŽUPOM in Table 2. was 37 " 12%, with E. huxleyi producing the highest yield Ž49.1%. and S. costatum the lowest yield Ž20.9%.. The UDOM fractions were the most carbohydraterich, with an average carbohydrate yield of 65.5 " 8%. S. costatum UDOM had the highest carbohydrate yield Ž74.1%. and Phaeocystis the lowest Ž56.3%. ŽTable 2.. Carbohydrate yields in UDOM were similar to those measured for total DOM ŽBiddanda and Benner, 1997., with the exception of S. costatum UDOM which was rich in carbohydrates relative to total DOM. The average CrN ratios of these three culture fractions appeared to reflect the variable carbohydrate content of the cultures ŽTable 2.. CSOM had an average CrN value of 7.2 " 1 and the lowest carbohydrate yield Ž29%.. The average CrN of CLOM was 12.2 " 3.7, and this fraction had an average carbohydrate yield of 48%. The average CrN of UDOM was 21.6 " 2.6, and this fraction had the highest carbohydrate yield Ž66%.. This inverse relationship between nitrogen and carbohydrate content indicates that amino sugars are relatively minor components of the carbohydrates in these phytoplankton cultures. 3.3. Neutral aldoses The following neutral aldoses were measured in this study: glucose, galactose, mannose, fucose, xylose, rhamnose, arabinose, ribose, and lyxose. The percentage of total organic carbon in these combined aldoses is referred to herein as the aldose yield. Average Ž"SD. aldose yields of 15 " 8%, 28 " 6%, and 35 " 6% were measured in CSOM, CLOM, and UDOM, respectively ŽTable 2.. This pattern of aldose yields in the various culture fractions was similar to the pattern of bulk carbohydrate yields. The percentage of carbohydrate carbon as aldose was calculated for each fraction of organic matter from the different cultures ŽTable 2.. With the exception of S. costatum CSOM, an average of 54.5 " 4.4% of carbohydrate carbon was measured as aldoses. The combined aldose yields of the two cell fractions ŽCSOM and CLOM. gave a weighted-average aldose yield of 20 " 9% for the various phytoplanktons. E.

huxleyi had the highest Ž30.5%. aldose yield, and S. costatum had the lowest Ž8.5%. yield ŽTable 2.. Yields of individual aldoses for all three fractions and combined particulate material are presented in Table 3. Using similar methods, Hamilton and Hedges Ž1988. measured the aldose content of mixed phytoplankton collected from Saanich Inlet. They reported average aldose yields of 3 " 1.6% during the spring and summer. These values agree with other measurements taken from mixed phytoplankton assemblages in the ocean ŽHanda and Yanagi, 1969; Cowie and Hedges, 1984b; Tanoue and Handa, 1987; Hernes et al., 1996.. In another study, Cowie and Hedges Ž1996. measured the aldose content of three species of cultured diatoms and their cell walls. They found aldose yields of 5–8% for whole cells and 1–4% for cell walls. These findings correspond with the low aldose yields observed in the diatom Ž S. costatum. cells analyzed in this study, but they are much lower than aldose yields in the other phytoplankton that were analyzed ŽTable 2.. The aldose yields Ž; 35%. for phytoplankton culture UDOM were very high, indicating that aldoses were major components of freshly produced UDOM. Aldose yields in UDOM recovered from the surface ocean are considerably lower and are in the range of 10–15% ŽMcCarthy et al., 1996; Skoog and Benner, 1997.. Aldose yields in UDOM from the oxygenminimum layer and deeper are three- to five-fold lower than those in surface water UDOM, indicating that aldoses are produced in surface waters and are reactive components in the upper ocean carbon cycle ŽMcCarthy et al., 1996; Skoog and Benner, 1997.. Molecular-level characterization of aldoses also provides information about the sources, functions, and transformations of carbohydrates. Cell structural organic matter ŽCSOM. aldose compositions were characterized by high mole percentages of glucose Ž28–71%. relative to other aldoses ŽFig. 1a.. Glucose is the most abundant and variable aldose within phytoplankton cellular material due to its function as a storage product in many species ŽHanda, 1969; Cowie and Hedges, 1984b; Tanoue and Handa, 1987; Hernes et al., 1996.. Phytoplankton increase and decrease their levels of storage glucan depending on growth stage, nutrient levels, and light conditions ŽMyklestad, 1989.. Other trends in CSOM include

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

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Table 3 Individual aldose yields of cell structural organic matter ŽCSOM., cell lysate organic matter ŽCLOM., ultrafiltered particulate organic matter ŽUPOM., and ultrafiltered dissolved organic matter ŽUDOM. for the four phytoplankton cultures Culture

Fraction

LYX

ARA

RHA

RIB

XYL

FUC

MAN

GAL

GLU

CSOM CLOM UPOM UDOM

0.04 0.38 0.19 0.81

0.60 2.72 1.55 3.64

0.46 2.22 1.24 1.80

0.26 0.52 0.38 0.46

0.31 4.04 1.98 3.61

0.10 1.21 0.59 1.02

1.03 2.40 1.64 4.41

1.26 7.16 3.89 7.99

9.83 6.29 8.25 4.87

CSOM CLOM UPOM UDOM

0.06 0.77 0.49 1.07

0.43 3.74 2.40 3.52

0.42 0.45 0.44 0.57

0.34 0.28 0.30 0.28

0.78 7.28 4.65 6.20

0.37 3.94 2.50 3.46

3.46 4.54 4.10 4.15

1.12 7.08 4.67 6.90

17.82 6.24 10.91 4.26

CSOM CLOM UPOM UDOM

0.03 0.06 0.04 1.15

0.17 0.30 0.22 2.66

1.76 0.67 1.39 1.02

0.16 0.31 0.21 0.00

0.12 0.36 0.20 3.67

0.14 0.24 0.17 9.33

3.43 2.45 3.09 2.07

1.48 1.86 1.61 11.78

9.47 23.40 14.27 9.15

CSOM CLOM UPOM UDOM

0.08 0.10 0.09 0.39

0.05 0.08 0.06 0.58

0.56 1.60 0.83 1.86

0.18 0.32 0.22 2.61

0.18 1.25 0.46 1.30

0.72 6.31 2.18 4.21

1.09 0.97 1.06 4.97

0.55 2.17 0.98 1.41

1.35 6.32 2.65 21.33

Phaeocystis

E. huxleyi

S. bacillaris

S. costatum

Abbreviations: LYX, lyxose; ARA, arabinose; RHA, rhamnose; RIB, ribose; XYL, xylose; FUC, fucose; MAN, mannose; GAL, galactose; GLU, glucose. Yields are expressed as the percentage of total organic carbon in each aldose.

high mole percentages of mannose Ž5–21%. and galactose Ž5–11%., and low percentages of arabinose, ribose, and xylose. Rhamnose accounted for ; 10% of S. costatum and S. bacillaris CSOM aldoses. S. costatum also had an unusually high mole percentage of fucose Ž16%. relative to the other cultures. Cell lysate organic matter ŽCLOM. aldose compositions were characterized by a much greater degree of variability among cultures than the CSOM fraction ŽFig. 1b.. Glucose was a major aldose, but with the exception of S. bacillaris, glucose was not as dominant in CLOM as it was in CSOM. Arabinose, xylose, and galactose were in similar proportion to glucose in the CLOM of Phaeocystis and E. huxleyi. S. costatum had a much higher mole percentage of fucose than the other phytoplankton species. Unlike cellular components, the UDOM from cultures of Phaeocystis, E. huxleyi, and S. bacillaris contained galactose as the dominant aldose Ž21– 27%., followed closely by glucose Ž13–21%., xylose

Ž10–23%., and arabinose Ž7–14%. ŽFig. 1c.. S. bacillaris UDOM had a much lower percentage of mannose and a much greater percentage of fucose than Phaeocystis and E. huxleyi. S. costatum UDOM aldose composition was quite different from that of the other three cultures. S. costatum UDOM contained a much higher percentage of glucose and a much lower percentages of galactose, xylose, and arabinose ŽFig. 1c.. 3.4. Comparison of UPOM to UDOM in cultures A first step in gaining insight into the possible sources of natural UDOM in the ocean is through the comparison of the aldose composition and yield of freshly produced UDOM and its known source. These comparisons provide the opportunity to determine if UDOM is a unique product of phytoplankton exudation and release or a reflection of the molecular composition of phytoplankton cell components. With these comparisons and a closer examination of the

138

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

Fig. 1. Aldose compositions Žmole% aldose. within different fractions of particulate and dissolved organic matter from four phytoplankton cultures. Abbreviations: CSOM, cell structural organic matter; CLOM, cell lysate organic matter; UDOM, ultrafiltered dissolved organic matter; LYX, lyxose; ARA, arabinose; RHA, rhamnose; RIB, ribose; XYL, xylose; FUC, fucose; MAN, mannose; GAL, galactose; GLU, glucose.

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

growth and physiology of each phytoplankton species, we can deduce more about the origin and function of freshly produced UDOM. Comparison of the aldose composition of Phaeocystis UDOM to that of the CSOM and CLOM particulate fractions indicated that UDOM composition was most similar to that of CLOM ŽFig. 2a.. Both UDOM and CLOM contain galactose as the dominant aldose and glucose, xylose, and arabinose as major aldoses. Similar aldose yields also link these two fractions, which had nearly twice the aldose yield of the CSOM fraction ŽFig. 2a.. It appears that a large fraction of the UDOM produced by Phaeocystis was too large to pass the 0.1-mm pore-size filter used to separate DOM from the cellular material. Any DOM that was retained during filtration would be recovered in the water-soluble CLOM fraction. Janse et al. Ž1996. also observed the retention of Phaeocystis colloidal material by 1-mm pore-size glass–fiber filters. Phaeocystis is known to release an extracellular, polysaccharide-based mucus which serves as a colonial matrix for individual cells ŽGuillard and Hellebust, 1971.. This mucus has been shown to comprise a large fraction of the overall biomass of these colonies ŽRousseau et al., 1990.. The aldose composition of this mucus is variable, but it has been used as a tracer of Phaeocystis DOM in seawater ŽJanse et al., 1996.. As the predominant extracellular product of Phaeocystis cells, this mucus would logically be a major source of UDOM in the cultures. The production of extracellular secretions Žalso referred to as exopolymer secretions or EPS. by marine microbes and algae is thought to function in the creation of microenvironments in which cells are protected from rapidly changing environmental conditions, toxins, grazing, and even digestion. These mucus-like secretions also serve to concentrate nutrients and to link individual cells into mutually beneficial colonies and onto surfaces ŽDecho, 1990.. The major molecular components of EPS are complex heteropolysaccharides containing a wide variety of aldoses in variable compositions. Phytoplankton EPS can be a major source of DOM under bloom conditions ŽGuillard and Hellebust, 1971; Verity et al., 1988; Janse et al., 1996.. The aldose compositions of the UDOM and CLOM fractions of the E. huxleyi culture were also

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similar ŽFig. 2b.. Galactose was again the dominant aldose, with xylose, glucose, mannose, fucose, and arabinose as major components of both fractions. In contrast, the CSOM was glucose-rich, with only minor contributions of galactose, fucose, xylose, and arabinose. The aldose yields of the CLOM and UDOM were similar as well, with the CLOM fraction having a slightly higher aldose yield ŽFig. 2b.. The similarities in aldose yields and compositions of the UDOM and CLOM fractions indicated that water-soluble cytoplasmic material and exopolymer secretions of the CLOM fraction were the probable source of E. huxleyi-produced UDOM. Similar comparisons among the three fractions of the S. bacillaris culture indicated a clear molecular distinction between cell material and its extracellular products ŽFig. 2c.. Both CSOM and CLOM aldose compositions were characterized by high mole percentages of glucose and relatively minor contributions of mannose, galactose, and all other aldoses. In contrast, UDOM contained galactose as its dominant aldose, with substantial contributions of fucose, xylose, and arabinose. S. bacillaris UDOM also contained a considerably higher aldose yield than CSOM and CLOM ŽFig. 2c.. These differences indicated that the extracellular products of S. bacillaris were molecularly distinct from their cell source. Cyanobacteria surround their cell walls with a mucilaginous sheath or matrix which is primarily produced during active growth ŽFogg et al., 1973.. This sheath or matrix is composed of high-molecular-weight heteropolymers predominantly consisting of complex heteropolysaccharides. The composition of these heteropolysaccharides is highly variable among species and is influenced by the physiological state and growth stage of cells ŽDecho, 1990.. Fogg et al. Ž1973. suggested that the primary source of dissolved polysaccharide in cyanobacterial cultures was ‘sloughed-off’ sheath material. Studies of certain species of unicellular cyanobacteria have indicated that during cell division, the sheath surrounding the dividing cells is expanded and eventually ruptured ŽKessel and Eloff, 1975.. These observations provide a possible explanation for the production of a molecularly distinct UDOM fraction in the S. bacillaris culture. The diatom, S. costatum, provided yet another pattern of extracellular aldose release. Unlike the

140 A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144 Fig. 2. Aldose compositions Žmole% aldose. and yields Žpercentage organic carbon ŽOC. as aldose. within different fractions of particulate and dissolved organic matter from individual phytoplankton cultures. Abbreviations: as in Fig. 1; TAC, total aldose carbon Žas percentage OC..

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

other cultures which showed a tendency to excrete UDOM that was depleted in glucose relative to cellular material, S. costatum UDOM was dominated by glucose ŽFig. 2d.. Also distinctive from other cultures is the depletion of galactose in UDOM relative to particulate fractions. Thus, S. costatum UDOM had an aldose composition that was distinct from both its cell source and the aldose compositions of UDOM from the other phytoplankton investigated. Another distinction between S. costatum UDOM and its cell source was the much higher aldose yield in UDOM ŽTable 2.. S. costatum UDOM had over a two-fold higher aldose yield than CLOM and an eight-fold higher yield than CSOM ŽFig. 2d.. Diatoms are known to release storage polysaccharides once the storage capacity of the cells has been reached ŽSmetacek and Pollehne, 1986.. The storage products of diatoms have been identified as primarily b-1,3-D-glucan ŽHanda, 1969; Myklestad and Haug, 1972; Myklestad, 1974, Myklestad, 1989.. Glucose was the dominant aldose in DOM during a diatom bloom in a mesocosm experiment ŽBrockmann et al., 1979., and similar observations have been made during diatom blooms in the surface ocean ŽIttekkot et al., 1981; Ittekkot, 1982. and other mesocosm experiments ŽMopper et al., 1995.. Overall, freshly produced UDOM had an aldose composition which was similar to the complex heteropolysaccharides known to be released by phytoplankton as EPS. This observation is supported by recent results of Aluwihare et al. Ž1997., who found polysaccharide linkages in seawater UDOM that were indicative of a phytoplankton source. Our culture studies generally indicated that freshly produced UDOM has an aldose signature that is unique from phytoplankton cellular material. 3.5. Comparison of culture UDOM and surface ocean UDOM Carbohydrates are the most abundant components of marine DOM, and most carbohydrates in the surface ocean occur in combined form as oligosaccharides and polysaccharides ŽBenner et al., 1992; Pakulski and Benner, 1994.. The depth distribution of combined carbohydrates indicates that they are largely produced and consumed in the upper ocean ŽBenner et al., 1992; Pakulski and Benner, 1994;

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McCarthy et al., 1996.. Phytoplankton is a likely source for the carbohydrates in surface waters, and this study was designed to compare the combined carbohydrates in UDOM produced by marine phytoplankton to those in UDOM collected from the surface ocean. In general, most measurements of phytoplankton UDOM show strong similarities to those of UDOM from the surface ocean. The average CrN ratio of 22 for phytoplankton UDOM is similar to the average CrN ratio of 17 for surface ocean UDOM ŽBenner et al., 1997.. Consistent with the relatively high CrN ratios, phytoplankton UDOM and surface ocean UDOM are very rich in combined carbohydrates. The average bulk carbohydrate yield of 66% for phytoplankton UDOM is slightly higher than the carbohydrate yield of ; 50% for surface ocean UDOM ŽBenner et al., 1992; McCarthy et al., 1993; Aluwihare et al., 1997.. The slightly higher carbohydrate yield in the phytoplankton UDOM is consistent with the slightly higher CrN ratio in this material. Both types of UDOM sample are notable for being depleted in nitrogen and rich in carbohydrates relative to particulate material. Comparison of the average aldose composition of phytoplankton UDOM to that of surface ocean UDOM indicates that the compositions are very similar ŽFig. 3.. Most aldoses in phytoplankton UDOM and surface ocean UDOM occur in similar abundances Ž10–20 mole% ., indicating that heteropolysaccharides are the dominant combined carbohydrates in UDOM ŽFig. 3.. Exopolymer secretions of algae and bacteria have been shown mainly to consist of heteropolysaccharides ŽDecho, 1990., and it appears that exopolymer secretions from phytoplankton could be an important source of the combined carbohydrates that are abundant in surface ocean waters. As noted earlier, these aldose compositions are unique from the glucose-dominated compositions of phytoplankton and marine particulate matter. The only significant difference between phytoplankton and surface ocean UDOM aldose compositions was the enrichment of rhamnose in surface ocean UDOM relative to phytoplankton culture UDOM Žtwo-tailed t-test, p - 0.05.. Aldose yields in surface ocean UDOM were about three-fold lower than those in phytoplankton UDOM ŽFig. 3.. The relatively low aldose yield in surface

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Fig. 3. Average aldose compositions Žmole% aldose. and yields Žpercentage organic carbon ŽOC. as aldose. of ultrafiltered dissolved organic matter ŽUDOM. from four phytoplankton cultures and from surface ocean water in the North Pacific, the Sargasso Sea, and the Gulf of Mexico Ždata from McCarthy et al., 1996.. Error bars represent the standard error of the mean. Abbreviations: as in Figs. 1 and 2.

ocean UDOM could be indicative of diagenetic alteration. Previous studies have shown that decreases in aldose yields are indicative of diagenetic alteration as aldoses are preferentially utilized by microorganisms ŽCowie and Hedges, 1994; Hedges et al., 1994; Hernes et al., 1996; Skoog and Benner, 1997.. The relatively high mole percentage of rhamnose in surface ocean UDOM also could be indicative of diagenetic processes. A diverse array of extracellular enzymes is required to degrade heteropolysaccharides, and it is likely that these enzymes are produced by a diverse bacterial assemblage ŽArnosti et al., 1994.. The aldose composition of UDOM may be preserved during decomposition due to its complexity and resistance to hydrolysis by any single enzyme. Additional studies of the changes in aldose composition and yield during microbial processing of phytoplankton UDOM are needed to verify the utility of these diagenetic indicators for marine DOM. Freshly produced UDOM can be rapidly utilized by marine bacteria ŽAmon and Benner, 1994., and it appears

that combined aldoses in UDOM are important substrates supporting heterotrophic metabolism in the surface ocean. Acknowledgements We thank Bopi Biddanda for assistance with the culturing experiments and Brenda Blank for assistance with ultrafiltration and CHN analyses. We thank Annelie Skoog and the Biogeochemistry group at UTMSI for comments that improved the manuscript. This research was supported by NSF grants OCE-9413843 and OCE-9730223. This is contribution 1069 of the University of Texas Marine Science Institute. References Aluwihare, L.I., Repeta, D.J., Chen, R.F., 1997. A major biopolymeric component to dissolved organic carbon in surface seawater. Nature 387, 166–169.

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144 Amon, R.M.W., Benner, R., 1994. Rapid cycling of high-molecular-weight dissolved organic matter in the ocean. Nature 369, 549–552. Antia, H.N.J., McAllister, C.D., Parsons, T.R., Stephens, K., Strickland, J.D.H., 1963. Further measurements of primary production using a large-volume plastic sphere. Limnol. Oceanogr. 8, 166–183. Arnosti, C., Repeta, D.J., Blough, N.V., 1994. Rapid bacterial degradation of polysaccharides in anoxic marine systems. Geochim. Cosmochim. Acta 58, 2639–2652. Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A., Thingstad, F., 1983. The ecological role of water-column microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257–263. Benner, R., 1991. Ultrafiltration for the concentration of bacteria, viruses, and dissolved organic matter. In: Hurd, D.C., Spencer, D.W. ŽEds.., Marine Particles: Analysis and Characterization, Geophys. Monogr. 63. Am. Geophys. Union, p. 181–185. Benner, R., Strom, M., 1993. A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation. Mar. Chem. 41, 153–160. Benner, R., Biddanda, B., Black, B., McCarthy, M., 1997. Abundance, distribution, and stable carbon and nitrogen isotope compositions of marine organic matter isolated by tangentialflow ultrafiltration. Mar. Chem. 57, 243–263. Benner, R., Pakulski, J.D., McCarthy, M., Hedges, J.I., Hatcher, P.G., 1992. Bulk chemical characteristics of dissolved organic matter in the ocean. Science 255, 1561–1564. Biddanda, B., Benner, R., 1997. Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton. Limnol. Oceanogr. 42, 506–518. Borch, N.H., Kirchman, D.L., 1997. Concentration and composition of dissolved combined neutral sugars Žpolysaccharides. in seawater determined by HPLC-PAD. Mar. Chem. 57, 85–95. Brockmann, U.H., Eberlein, K., Junge, H.D., Maier-Reimer, E., Siebers, D., 1979. The development of a natural plankton population in an outdoor tank with nutrient-poor sea water: II. Changes in dissolved carbohydrates and amino acids. Mar. Ecol. Prog. Ser. 1, 283–293. Burney, C.M., 1986. Bacterial utilization of total in situ dissolved carbohydrate in offshore waters. Limnol. Oceanogr. 31, 427– 431. Burney, C.M., Johnson, K.M., Lavoie, D.M., Sieberth, J.McN., 1979. Dissolved carbohydrate and microbial ATP in the North Atlantic: concentrations and interactions. Deep-Sea Res. A 26, 1267–1290. Cowie, G.L., Hedges, J.I., 1996. Digestion and alteration of the biochemical constituents of a diatom ŽThalassiosira weisflogii . ingested by an herbivorous zooplankton Ž Calanus pacificus .. Limnol. Oceanogr. 41, 581–594. Cowie, G.L., Hedges, J.I., 1994. Biochemical indicators of diagenetic alteration in natural organic matter mixtures. Nature 369, 304–307. Cowie, G.L., Hedges, J.I., 1984a. Determination of neutral sugars in plankton, sediments, and wood by capillary gas chromotography of equilibrated isomeric mixtures. Anal. Chem. 56, 497–504.

143

Cowie, G.L., Hedges, J.I., 1984b. Determination of neutral sugars in plankton, sediments, and wood by capillary gas chromotography of equilibrated isomeric mixtures. Anal. Chem. 56, 497–504. Dagg, M.J., 1974. Loss of prey body contents during feeding by an aquatic predator. Ecology 55, 903–906. Decho, A.W., 1990. Microbial exopolymer secretions in ocean environments: their roleŽs. in food webs and marine processes. Oceanogr. Mar. Biol. Annu. Rev. 28, 73–153. Ducklow, H.W., Kirchman, D.L., Quinby, H.L., Carlson, C.A., Dam, H.G., 1993. Bacterioplankton carbon cycling during the spring bloom in the eastern North Atlantic Ocean. Deep-Sea Res. II 40, 245–263. Eberlein, K., Brockmann, U.H., Hammer, K.D., Kattner, G., Laake, M., 1983. Total dissolved carbohydrates in an enclosure experiment with unialgal Skelotonema costatum culture. Mar. Ecol. Prog. Ser. 14, 45–58. Eberlein, K., Teal, M.T., Hammen, H.K.D., Hickel, W., 1985. Dissolved organic substances during a Phaeocystis pouchetti bloom in the German Bight ŽNorth Sea.. Mar. Biol. 89, 311–316. Fogg, G.E., 1983. The ecological significance of extracellular products of photosynthesis. Botanica Marina 26, 3–14. Fogg, G.E., Stewart, W.D.P., Fay, P., Walsby, A.E., 1973. The Blue-Green Algae. Academic Press, London, pp. 9–17. Fuhrman, J.A., Suttle, C.A., 1993. Viruses in marine planktonic systems. Oceanography 6, 51–63. Guillard, R.R.L., Hellebust, J.A., 1971. Growth and the production of extracellular substances by two strains of P. pouchetti. J. Phycol. 7, 330–338. Hamilton, S.E., Hedges, J.I., 1988. The comparative geochemistries of lignins and carbohydrates in an anoxic fjord. Geochim. Cosmochim. Acta 52, 129–142. Handa, N., 1969. Carbohydrate metabolism in the marine diatom S. costatum. Mar. Biol. 4, 208–214. Handa, N., Yanagi, K., 1969. Studies on water-extractable carbohydrates of the particulate matter from the northwest Pacific Ocean. Mar. Biol. 4, 197–207. Haug, A., Myklestad, S., 1976. Polysaccharides of marine diatoms with special reference to Chaetoceros species. Mar. Biol. 34, 217–222. Haug, A., Myklestad, S., Sakshaug, E., 1973. Studies on the phytoplankton ecology of the Trondheimsfjord: I. The chemical composition of phytoplankton populations. J. Exp. Mar. Biol. Ecol. 11, 15–26. Hecky, R.E., Mopper, K., Kilham, P., Degens, E.T., 1973. The amino acid and sugar composition of diatom cell-walls. Mar. Biol. 19, 323–331. Hedges, J.I., 1992. Global biogeochemical cycles: progress and problems. Mar. Chem. 39, 67–93. Hedges, J.I., Stern, J.H., 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnol. Oceanogr. 29, 657–663. Hedges, J.I., Cowie, G.L., Richey, J.E., Quay, P.D., Benner, R., Strom, M., Forsberg, B.R., 1994. Origins and processing of organic matter in the Amazon River as indicated by carbohydrates and amino acids. Limnol. Oceanogr. 39, 743–761.

144

A. Biersmith, R. Bennerr Marine Chemistry 63 (1998) 131–144

Hernes, P.J., Hedges, J.I., Peterson, M.L., Wakeham, S.G., Lee, C., 1996. Neutral carbohydrate geochemistry of particulate material in the central equatorial Pacific. Deep-Sea Res. 43, 1181–1204. Ittekkot, V., 1982. Variations of dissolved organic matter during a plankton bloom: qualitative aspects based on sugar and amino acid analysis. Mar. Chem. 11, 143–158. Ittekkot, V., Brockmann, U., Michaelis, W., Degens, E.T., 1981. Dissolved free and combined carbohydrates during a phytoplankton bloom in the North Sea. Mar. Ecol. Prog. Ser. 4, 299–305. Janse, I., van Rijssel, M., Gottschal, J.C., Lancelot, C., Gieskes, W.W.C., 1996. Carbohydrates in the North Sea during spring blooms of Phaeocystis: a specific fingerprint. Aquat. Microb. Ecol. 10, 97–103. Johnson, K., Sieburth, J. McN., 1977. Dissolved carbohydrates in seawater: I. A precise spectrophotometric analysis for monosaccharide. Mar. Chem. 5, 1–13. Jumars, P.A., Penry, J.A., Baross, J.A., Perry, M.J., Frost, B.W., 1989. Closing the microbial loop: dissolved carbon pathway to heterotrophic bacteria from incomplete ingestion, digestion, and absorbtion in animals. Deep-Sea Res. 36, 483–495. Kerherve, P., Charriere, B., Gadel, F., 1995. Determination of marine monosaccharides by high-pH anion-exchange chromatography with pulsed amperometric detection. J. Chromatogr. 718, 283–289. Kessel, M., Eloff, J.N., 1975. The ultrastructure and development of the colonial sheath of Microcystis marginata. Arch. Microbiol. 106, 209–214. Lampert, W., 1978. Release of dissolved organic carbon by grazing zooplankton. Limnol. Oceanogr. 23, 831–834. Lancelot, C., Billen, G., 1985. Carbon–nitrogen relationships in nutrient metabolism of coastal marine ecosystems. In: Jannasch, H.W., Williams, P.J. leB ŽEds.., Advances in Aquatic Microbiology 3, 263–321. Liebezeit, G., Bolter, M., Brown, I.F., Dawson, R., 1980. Dissolved free amino acids and carbohydrates at pycnocline boundaries in the Sargasso Sea and related microbial activity. Oceanol. Acta 3, 357–362. Mague, T.H., Friberg, E., Hughes, D.J., Morris, I., 1980. Extracellular release of carbon by marine phytoplankton: a physiological approach. Limnol. Oceanogr. 25, 262–279. McCarthy, M., Hedges, J.I., Benner, R., 1996. Major biochemical composition of dissolved high-molecular weight organic matter in seawater. Mar. Chem. 55, 281–297. McCarthy, M., Hedges, J.I., Benner, R., 1993. The chemical composition of dissolved organic matter in seawater. Chem. Geol. 107, 503–507. Mopper, K., Zhou, J., Ramana, K.S., Passow, U., Dam, H.G., Drapeau, D.T., 1995. The role of surface-active carbohydrates in the flocculation of a diatom bloom in a mesocosm. Deep-Sea Res. 42, 47–73. Myklestad, S., 1989. Production, chemical structure, metabolism, and biological function of the Ž1-3.-linked, b-D-glucans in diatoms. Biol. Oceanogr. 6, 313–326.

Myklestad, S., 1977. Production of carbohydrates by marine planktonic diatoms: II. Influence of the NrP ratio in the growth medium on the assimilation ratio, growth rate, and production of cellular and extracellular carbohydrates by C. affinis var. willei and S. costatum. J. Exp. Mar. Biol. Ecoł. 29, 161–179. Myklestad, S., 1974. Production of carbohydrates by marine planktonic diatoms: I. Composition of nine different species in culture. J. Exp. Mar. Biol. Ecol. 15, 261–274. Myklestad, S., Haug, A., 1972. Production of carbohydrates by the marine diatom C. affinis var. willei: I. Effect of the concentration of nutrients in the culture medium. J. Exp. Mar. Biol. Ecol. 9, 125–136. Pakulski, J.D., Benner, R., 1994. Abundance and distribution of carbohydrate in the ocean. Limnol. Oceanogr. 39, 930–940. Pakulski, J.D., Benner, R., 1992. An improved method for the hydrolysis and MBTH analysis of dissolved and particulate carbohydrates in seawater. Mar. Chem. 40, 143–160. Parsons, T.R., Stephens, K., Strickland, J.D.H., 1961. On the chemical composition of eleven species of marine phytoplankters. J. Fish. Res. Bd. Canada 18, 1001–1016. Romankevich, E.A., 1984. Geochemistry of Organic Matter in the Ocean. Springer Verlag, Berlin, pp. 199–229. Rousseau, V., Voulet, D., Casotti, R., Cariou, V., Lenz, J., Gunkel, J., Baumann, M.E.M., 1994. The life cycle of Phaeocystis ŽPrymensiophyceae.: evidence and hypotheses. J. Mar. Sys. 5, 23–29. Rousseau, V., Mathot, S., Lancelot, C., 1990. Calculating carbon biomass of Phaeocystis sp. from microscopic observations. Mar. Biol. 107, 305–314. Schmidt, W., Drews, G., Weckesser, J., Fromme, I., Borowiak, D., 1980. Characterization of the lipopolysaccharides from eight strains of the cyanobacterium Synechococcus. Arch. Microbiol. 127, 209–215. Skoog, A., Benner, R., 1997. Aldoses in various size fractions of marine organic matter: implications for carbon cycling. Limnol. Oceanogr. 42, 1803–1813. Smetacek, V., Pollehne, F., 1986. Nutrient cycling in pelagic systems: a reappraisal of the conceptual framework. Ophelia 26, 401–428. Strom, S., Benner, R., Ziegler, S., Dagg, M., 1997. Planktonic grazers are a potentially important of marine dissolved organic carbon. Limnol. Oceanogr. 42, 1364–1374. Tanoue, E., Handa, N., 1987. Monosaccharide composition of marine particles and sediments from the Bering Sea and northern North Pacific. Oceanol. Acta 10, 91–99. Varum, K.M., Myklestad, S., 1984. Effects of light, salinity, and nutrient limitation on the production of b-1,3-D-glucan and exo-D-glucanase activity in S. costatum. J. Exp. Mar. Biol. Ecol. 83, 13–25. Verity, P.G., Villareal, T.A., Smayda, T.J., 1988. Ecological investigations of blooms of colonial P. pouchetti: I. Abundance, biochemical composition and metabolic rates. J. Plank. Res. 10, 219–248.