Cycling of some low molecular weight volatile fatty acids in a permanently anoxic estuarine basin

ELSEVIER

Marine Chemistry 47 (1994) 97-113

Cycling of some low molecular weight volatile fatty acids in a permanently anoxic estuarine basin Hanguo Wu, Mary I. Scranton 1 Marine Sciences Research Center, State University of New York at Stony Brook, Stony Brook, N Y 11794-5000, USA

Received December2, 1993; revisionacceptedApril 20, 1994

Abstract

Low molecular weight volatile fatty acids (VFAs) are important products of the anaerobic fermentation of organic matter. However, little is known of the cycling of VFAs in the water column. In this study, the concentrations of acetate, propionate, isobutyrate and butyrate were measured in the water column of a permanently anoxic basin by a static diffusion method. Acetate concentrations varied from 0.8 to 6.1 #M, with the maximum value measured near a peak of anoxyphototrophs. The acetate concentration was relatively uniform except in the peak. The concentration of propionate varied from 44.3 nM to 191.0 nM, with a maximum value found in a layer dominated by the flagellated protozoan, Euglena proxima, which was located just below the depth at which 02 went to zero. The concentration profiles of butyrate and isobutyrate were similar to the concentration profile of propionate. Acetate turnover rate constants varied from 0.1 to 5.1 h -1, A maximum in the acetate uptake rate constant was found during the day in the layer rich in E. proxima, but not at night, and possibly was associated with 02 production from photosynthesis at this depth. Acetate uptake rate constants also were higher during the day as compared with the night in a deeper layer with abundant anoxyphototrophic bacteria, suggesting photoassimilation of acetate by these organisms may be important. In the E. proxima layer, and at the base of the oxycline, most acetate uptake was inhibited by BES in a short term incubation, suggesting that methanogens might be a sink for acetate. Integrated acetate oxidation rates in this system were almost twice as high as the integrated rates of primary production.

1. Introduction

The importance o f low molecular weight volatile fatty acids or VFAs (for example, acetate, propionate, and butyrate) in the terminal steps of anaerobic organic matter decomposition has been recognized for anoxic marine sediments dominated by sulfate reducing or methanogenic activities (Sorensen et al., 1981; Balba and Nedwell, 1982; Sansone, 1986). In particular, acetate plays a key role in the metabolic pathway for decomposition of ICorresponding author.

complex organic compounds (Lovley and Klug, 1982; Sansone and Martens, 1982). Although much work on the cycling of VFAs has been done in marine and freshwater sediments, less is known about the cycling of these compounds in the water column. The goal o f this study (Wu, 1993) was to gain insight into the factors controlling organic matter decomposition at the oxicanoxic interface in the water column by monitoring the uptake rates o f some key intermediates. In particular, we wanted to see how acetate cycling changed at different places in the oxygen profile, and to determine whether light-dependent pro-

0304-4203/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved S S D I 0304-4203(94)00026-A

98

H. Wu, M.I. Scranton~Marine Chemistry 47 : 1994 ~ 97 1t3

Table I Acetate concentrations and uptake rate constants reported for water and sediments Concentration (#M)

Uptake rate constant (h- I )

Environment

Reference

0.0004-0.27 0.0026-0.049 0.003-0.074 0.0046 n/a n/a 0.1- 5.1

Estuary, coastal zone Chemotaxis Dock Long Island Sound Flax Pond Biscayne Bay Black Sea Pettaquamscutt

Billen et al., 1980 Lee, 1992 Wu and Scranton, 1994 Wu, unpubl. Vairavamurthy and Mopper, 1990 Mopper and Kieber, 1991 This study

2.1-3.3 0.78 1.5- 13 0.36-4.2 n/a n/a 0.12-0.24 0.33-1.3 0.97-1.01 n/a n/a 0.05-1.6

Limtjorden, Denmark Colne Point saltmarsh Various Danish coastal waters Cape Lookout Bight Continental shelf Loch Eil, Scotland Loch Eil, Scotland Flax Pond, NY Skan Bay, Alaska Lake Vechten Lowes Cove, Cod Cove, Maine Long Island Sound

Ansbaek and Blackburn, 1980 Balba and Nedwell, 1982 Christensen and Blackburn, 1982 Sansone and Martens, 1982 Sansone and Martens, 1982 Parkes and Taylor, 1983 Parkes et al., 1984 Michelson et al., 1989 Shaw and Mclntosh, 1990 Hordijk et al., 1990 King, 1991 Wu and Scranton, 1994

Water column

0.2 4 0.02 0.085- 0.53 0.35-0.73 0.4 t 5 80 0.8 6.1 Anoxic sediment

0. t--6.0 5.3 2- 70a 54-660 a 10.7 -69.0a 2 15 6.1-6.9 15-70 l 14 4-70 1- 11 1 --41 n/a = data not available.

aMicromoles per liter of sediments.

cesses were important. Since VFAs have been suggested to be key intermediates in the anoxic decomposition of organic matter, we wanted to see if their cycling was as important in the anoxic water column as it appears to be in the sediments. Early studies of acetate uptake (Wright and Hobbie, 1966; Billen et al., 1980) used approaches taken from enzyme kinetics to obtain an upper limit to the acetate concentration and to assess the importance of bacterial heterotrophic activities to cycling of acetate in the oxic water column. Lee (1992) also used enzymatic kinetics to estimate an upper limit of the acetate concentration in the water column and measured uptake rate constants of acetate, along with those of several other organic compounds, to compare the intrinsic rates of organic matter decomposition between oxic and anoxic environments. Vairavamurthy and Mopper (1990), using a gas chromatographic technique, measured acetate concentrations in coastal seawater (from Biscayne Bay, Florida), Mopper and Kieber (1991) measured

acetate concentration in the Black Sea water column and found sharp maxima and minima in the vicinity of the oxic-suboxic interface and suboxic-anoxic interface respectively. Results of these studies are summarized in Table 1. However, no systematic studies on the cycling of VFAs in anoxic water columns have been carried out.

Although the water column has been poorly studied, there are advantages to studying the water column rather than sediments (Lee, 1992). In the water column, both incorporation and respiration can be measured, and processes are likely to occur over a greater depth range than is the case for sediments because diffusion and mixing coefficients are higher. So, it is somewhat easier to sample the oxic/anoxic interface in detail. For example, with the SEABmI~electronic profiling system designed by Donaghay et al. (1992), chemical and biological parameters can be measured simultaneously at a scale of about 5 cm. The anoxic water column is not exactly like the

H. Wu, M.L Scranton~Marine Chemistry 47 (1994) 97-113

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sediment, however. One interesting feature of the anoxic water column is that photosynthetic algae sometimes develop in or just below a pycnocline that is present just above an oxic-anoxic interface (Estep et al., 1986; Steenbergen et al., 1989). They persist there year-round and supply 02 to the anoxic waters. Since the production of 02 occurs only during the day, the organic matter reaching this depth may experience cyclical oxic and anoxic conditions between day and night. This is similar to the oscillation produced by benthic organisms in surface sediment although the time scales are quite different (diel vs. seasonal). In the sediment, such oscillation may promote overall decomposition of organic matter (Kristensen and Blackburn, 1987; Sun et al., 1993). Thus 02 production within the upper portion of the anoxic zone may also play an important role in overall organic matter decomposition. Another major difference between anoxic water columns and sediments is that anoxyphototrophs frequently develop just below the oxic-anoxic interface in the water column since light more often reaches the interface. These bacteria can efficiently use the main fermentative products,

99

such as acetate, in the presence of light and H2S. Acetate is used in these and other organisms for the synthesis of a variety of cellular compounds, such as carbohydrate (Sirev~g, 1975), amino acids (Hoare and Gibson, 1964) and chlorophyll (Bergstein et al., 1981). Perhaps the system most closely analogous to the illuminated anoxic water column is the marine microbial mat. Microbial mats are highly stratified communities and include a variety of microbial types such as cyanobacteria, anoxyphotobacteria, eucaryotic microalgae, and heterotrophic and chemosynthetic bacteria (Stal et al., 1985). As in the illuminated anoxic water column, these populations in mat systems occur in a well defined order, depending on requirements for light, oxygen and other chemical species, but in mats the dimensions are typically on the order of a few millimeters, Compared with several meters in the water column. Therefore in the illuminated water column we have the opportunity to examine processes similar to those going on in mats, but at more convenient spatial scales and in an environment where diffusion is less limiting.

2. Study site: the Pettaquamscutt River Estuary The Pettaquamscutt Estuary (Rhode Island) is a system highly stratified by both salinity and temperature. It includes two brackish basins separated from each other and from the sea by sills less than 1 m deep and from Rhode Island Sound by a shallow channel about 6.5 km long (Gaines and Pilson, 1972; Fig. 1). Salinity at 8-10 m in the lower basin ranged from 21 to 26.5%o during our study, and a sharp density gradient separated the surface mixed layer (,-~ 3 m) from the anoxic bottom layer (Sieburth, 1991). At the surface, a shallow 02 maximum is produced by the photosynthetic activity of cyanobacteria and diatoms (P.L. Donaghay, pers. commun., 1992). Below this maximum is the oxycline which extends between 2 and 3 m. Just below the base of the oxycline, at about 4 m, a secondary 02 maximum is present during the day, in an approximately 10 cm thick layer dominated by the photosynthetic flagellated protist, Euglena proxima. It appears that the E. proxima provide

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tf. Wu, M.I. Scranton~Marine Chemistry 47 (1994) 9 7 113

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are phototrophic although light levels m theH depth strata are usually very low. The light intensity at the surface was around 2000/zE m 2 s while at the anoxyphototroph maximum (~-, 6 m depth) it was only a few tenths of a tzE m 2 s (A.K. Hanson, pers. commun., 1993).

3. Methods

2

3.1 Sampling E

[]

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0

20

40

60

80

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T, % Fig. 2. Typical profiles of O2 (O), H2S ([]), and transmittance ( I ) on August 23, 1993 during the day. During the night, the small oxygen peak at the E. proxima layer disappeared (data not shown).

02 to the water column at the base of the oxycline so that the oxygen concentration in this layer became measurable (15.6 #M) during the day (P.L. Donaghay and J.McN. Sieburth, in prep.). E. proxima are also phagotrophic, and microscopic examination suggests they may be the main consumer of bacteria in this layer (Sieburth, 1991). Biogenic H2S accumulates in bottom waters reaching a concentration of several mM (Hansen, unpubl, data). Below the E. proxima maximum, transmission profiles indicate the presence of a second, smaller particle maximum (Fig. 2). Although no pigment data are available for 1992, in 1991 this feature coincided with a bacteriochlorophyll e maximum (Mason et al., 1993), indicating that the maximum was mainly due to the presence of brown-colored phototrophic Chlorobiaceae such as Chlorobium phaeovibrioides and C. phaeobacteroides, which are the only known species to contain bacteriochlorophyll e (Gloe et al., 1975). These organisms

A SEABIRDelectronic profiling system capable of determining temperature, salinity, density, pH, oxygen, fluorescence, and transmission with a resolution of a few centimeters was deployed from a moored platform (Donaghay et al., 1992). A siphon sampling system for obtaining water from depth for chemical and microbiological studies was integrated with the profiling system. The advantages of the system are that chemical and biological parameters are measured simultaneously at the sampling depth and that the siphon inlet can be dynamically positioned within steep density gradients even in the presence of internal waves. A total of 56 concentration samples were taken, and include a day and a night profile pair, together with samples taken at several depths during a diel cycle. During the diel cycle, a set of samples was taken from 4 depths approximately every 6 h between noon of 23 August and 14:00 of 24 August 1992. Four characteristic layers were studied: the oxic layer where a maximum of diatoms and cyanobacteria was found (~ 2.4 m depth); the base of the oxycline (,-, 3.2 m depth) where the O2 concentration first went to zero as indicated by the oxygen electrode on the CTD: and two transmission minimum layers nucroscopically identified as containing large populations of E. proxima (~ 4.0 m depth) and anoxyphototrophic bacteria (~ 6.0 m depth), respectively.

3.2 Fatty acid concentrations Immediately after collection, seawater samples (0.5 1) were pressure filtered under N 2 through a precombusted G F / F filter followed by a 0.2 #M Gelman Acrocap TM filter. The Acrocap TM filters

H. Wu, M.L Scranton~Marine Chemistry47 (1994) 97-113

were pre-rinsed with 200 ml distilled H20 and with 200 ml of sample. The filtered samples were stored in 500 ml acid washed glass bottles with high density polyethylene amber caps. One ml 10 N KOH, made with MilliQ water redistilled over base, was added to each sample to preserve it and keep the pH value at 11-12. (Residual acetate in the 10 N KOH was apparently the main source of the sample blank.) To desalt and concentrate the sample, a static diffusion method was used as described by Yang et al. (1993). The trapping efficiency was determined for each sample by addition of 14C-acetate tracer before the first diffusion step. The efficiencies for all samples were greater than 80% and were corrected for in calculations of concentrations. After the samples were concentrated, 40 #1 of 0.5 M H3PO 4 were added to 200 #1 of the final concentrate which was vacuum distilled (Christensen and Blackburn, 1982; Michelson et al., 1989). The chromatographic method used an HP 5890A GC equipped with an FFAP column (0.53 mm I.D. × 30 m). The oven was programmed with an initial temperature of 98°C for 3 rain, followed by a temperature program of 15°C min -1 to a final temperature of 180°C for 5 rain. During the field experiment, on three occasions, duplicate samples were taken. Based on these, the overall precision of the method was -4-15%. The acetate blank (for distilled water treated on the platform like a sample) was about 300 nM, depending on the size of sample used. Blanks for the other fatty acids were on the order of a few nM.

101

vial. The following labeled compounds were used: U-14C-acetate, 57.0 mCi mmo1-1 (New England Nuclear, Boston, MA); 2-14C-propionate, 55.7 mCi mmol -I (ICN, Irving, CA). The incubation vials were attached to a rope and suspended at the depth of collection. Spiking the samples and returning them to in-situ depth took between 1 and 3 min. The total radioactivity was determined in three aliquots of 0.5 ml which were taken from the sample treated with 0.5 ml 10 N KOH to evaluate recovery. For most of our samples, the recovery of total activity was greater than 9095%, indicating minimal loss during the experiment. Losses were corrected for in all samples. Duplicate samples were taken for measurement of uptake rate constants during the diel study for all depths and duplicate samples were taken at three depths during the initial day vs night study. Average precision of rate constants was +10%. Incorporation and respiration were measured following a method described by Lee (1992), which was adapted from Wright and Hobbie (1966) and Hobbie and Crawford (1969). The uptake rate constant was determined as the rate constant of incorporation plus respiration. To calculate rate constants, we assumed first-order uptake and used the following equation: k=-

In 1 - ~

(1)

where k is the total uptake rate constant in h -1, t is time in h, a is total activity retained on particles plus that respired to C02, and A is total activity added.

3.3 Rate constants 3.4 Inhibition experiments

At the same time the concentration samples were taken, incubation samples were collected through the siphon tube into 40 ml glass vials which had been prewashed with 10% HC1, rinsed with distilled water and dried. Incubation vials were rinsed with sample and then were allowed to overflow at least two volumes to prevent 02 contamination. A Teflon®-coated silicone disc was added, and then a plastic cap with a hole was screwed on. Care was taken not to trap air bubbles. With a gas tight syringe, 0.18 nanomoles (0.01 mCi) 14C-acetate or propionate was added to each 40 ml incubation

To determine whether methanogens were responsible for the uptake of acetic acid, inhibition experiments were conducted with 2-bromoethanesulfonic acid (BES), an inhibitor of methanogenesis. BES inhibition experiments were conducted at the base of the oxycline during the diel studies. On another day, an incubation with added BES was done at the E. proxima maximum layer. For all BES experiments, 1 ml of a 1 M BES solution (stored under N2 to keep it anoxic) was injected into incubation vials (final concentration of

102

tt, Wu, M.I. Scranton~Marine Chemistry 4/

Acetate, ~M

1994,~ 97 113

Uptake rate, vdVIh

Rate constant, h

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Fig. 3. Verticalprofilesof acetate concentration (A), uptake rate constants (E3),and uptake rates (O) during the day on August 20, 1993 (16.30-19.30: open symbols)and at night on August 21, 1993(03.00-0530: filled symbols).Sunset was at 19.42 on August 20 and sunrise was at 06.09 on August 21. 2 5 m M ) before injecting radiolabeled tracers. Incubations proceeded as described above. The effect of 3-(3,4-dichlorophenyl)- 1,1 dimethylurea (DCMU), an inhibitor of photosystem II, was also tested at the E. p r o x i m a layer. The same treatment was employed for the D C M U stock solution as for the BES to keep it anoxic, but the final concentration in the incubation vial used was 10 mM. This concentration has been proven effective in inhibiting photo-system II (Jorgensen et al., 1979).

4. Results

Profiles of acetate and propionate concentrations, together with the respective uptake rate constants and uptake rates (calculated as concen-

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tration times rate constant), were determined for the afternoon of 20 August (16.30-19.30) and the night of 21 August 1992 (03.00-05.30) (Fig: 3). The acetate concentrations were about 2 # M and did not vary much in the water column, except near the anoxyphototroph layer where a peak value ( ~ 6 # M ) was measured. Propionate concentrations were also relatively constant (at about 80 nM), and showed a peak value of 190 nM at the E. p r o x i m a layer (Fig. 4). The concentrations of butyrate and isobutyrate were constant within a factor of 2, at about 40 nM and 20 nM, respectively, except in the E. p r o x i m a layer, where a much higher concentration was detected (Fig. 5). Uptake rate constants were not measured for the latter two compounds. The rate constants for acetate varied from 0.12 to 2.30 h -l for the daytime and 0.01 to 1.36 h -1 for night and showed a

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Fig. 4. Vertical profilesof propionate concentration (A), uptake rate constants (0), and uptake rates (O) during the day on August 20, 1993 (16.30-19.30: open symbols)and at night on August 21, 1993(03.00-05.30: filled symbols).Sunset was at 19.42on August 20 and sunrise was at 06.09 on August 21.

103

H. Wu, M.I. Scranton~Marine Chemistry 47 (1994) 97-113

Isol~tyrate, nM

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maximum in the E. proxima maximum layer during the day but not at night (Fig. 3). Propionate uptake rate constants were lower, and varied from 0.03 to 0.68 h -1 for the daytime sample and from 0.03 to 0.41 h -1 for the nighttime samples (Fig. 4). Propionate uptake rate constants showed a maximum in the E. proxima layer. In the oxic mixed layer, there was no obvious diel change in concentration, uptake rate constants or uptake rates for either acetate or propionate, although propionate uptake rate constants may have been slightly

higher at night. Below the oxic-anoxic interface (3.2 m), uptake rate constants tended to be higher during the day (average of 1.05 h -1 for acetate and 0.20 h -1 for propionate; Figs. 3A, 4A) than during the night (average of 0.36 h -] for acetate and 0.13 h -] for propionate; Figs. 3B, 4B). During the detailed diel study, concentrations of acetate in the oxic layer were generally lowest at times of high light intensity but varied by only a factor of 2 over a diel cycle (Fig. 6). The concentrations at the base of the oxycline showed no 12

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Fig. 6. Diel variations in acetate concentration (A), uptake rate constant (B) and uptake rate (C) in the oxic layer between August 23 and 24. Sampfing times are indicated on the x-axis. Sunset was 21.39 and sunrise was 06.12.

H. Wu, M.I. Scranton~Marine Chemistry 47 !' 1994) 97-113

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Fig. 7. Diel variations in acetate concentration (A), uptake rate constant (B) and uptake rate (C) at the base of the oxycline between August 23 and 24. Sampling times are indicated on the x-axis. Sunset was 21.39 and sunrise was 06,12.

consistent trend with time of day (Fig. 7) and were constant within a factor of 1.5 except for the first point, which was somewhat higher than the average of the other points. In the E. proxima layer (Fig. 8), acetate concentrations were constant within about 30%. In contrast, in the anoxyphototroph layer (Fig. 9), the concentration varied by a factor of 3, with the highest concentrations at 20:15 and 08:40 (near sunset and sunrise). The concentration observed at 03:40 in the anoxyphototroph layer is lower than concentrations at 20:15 or at 08:40 in the same layer. As such it represents a possibly anomalous value, although we have no specific reason to believe the value is in error. It should be noted that at all times, the concentrations at the anoxyphototroph layer were higher than in other layers (note different concentration scale for Fig. 6A). 10

The rate constants in the oxic layer varied unsystematically and by less than a factor of 2 over a diel cycle (Fig. 6), while at the base of the oxycline, the rate constants seemed to be slightly higher during the day than during the night (factor of 2i3; Fig. 7). In contrast, in the E. proxima layer a larger diel change in the uptake rate constant (factor of 4.1) was observed. The rate constants also were generally higher in this layer than in other layers (compare Fig. 8 with Figs. 6, 7 and 9). The highest value in the E. proximalayer (5.1 ± 0.4 h ~l , which represents turnover of the acetate pool every 12 min) was measured at 08:00. The lowest value, measured at 02:25, was ~ 1.2 h "1. In the E. proxima layer, uptake followed the trend of the rate constants, and was much higher during the day than during the night (Fig. 8). In the anoxyphototroph layer the rate constants varied less but still

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Fig. 8. Did variations in acetate concentration (A), uptake rate constant (B) and uptake rate (C) in the Eugtena proxima maximum between August 23 and 24. Sampling times are indicated on the x-axis. Sunset was 21.39 and sunrise was 06.12.

H. Wu, M.I. Scranton~Marine Chemistry 47 (19941 97-113

105 12

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Fig. 9. Diel variations in acetate concentration (A), uptake rate constant (B) and uptake rate (C) in the anoxyphototroph maximum between August 23 and 24. Sampling times are indicated on the x-axis. Sunset was 21.39 and sunrise was 06.12.

showed a very regular change with light intensity, with the highest value at noon (1.26 and 1.48 h-t; Fig. 9), and lower values at dawn, dusk and at midnight (0.40, 0.43 and 0.37 h-l). Because concentrations increased when rate constants were low, uptake rates in the anoxyphotobacterial layer were almost constant with time (ignoring the point at 03:40). BES inhibition experiments were conducted at the base of the oxycline during our August 23-24 diel studies (Fig. 10). The uptake rate constants in incubation vials varied over the diel cycle as described above. However, uptake rate constants in vials with BES were uniformly low and constant.

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In the E. proxima layer, BES reduced acetate uptake by 70% (Wu, 1993). DCMU inhibition experiments were conducted on two separate days in the E. proxima layer, but no effect of D C M U on acetate uptake was seen in our short term incubations (Wu, 1993).

5. Discussion

Only a few other studies have examined fatty acid concentrations in either the oxic or anoxic water column. Many of these data are summarized in Table 1. The highest concentrations observed in these other studies (Billen et al., 1980; Vairavamurthy and Mopper, 1990) are similar to concentrations we observed in the Pettaquamscutt. In the waters of Flax Pond salt marsh and Long Island Sound, we (Wu and Scranton, 1994; Wu, unpubl. data) have observed acetate concentrations comparable to the lower values seen in oxic coastal waters by Billen et al. (1980) and Vairavamurthy and Mopper (1990), suggesting that at least some of the difference is due to differences in environments. Lee's values (Lee, 1992), which are the lowest reported, might reflect generally lower biological activity in Vineyard Sound as compared to Long Island Sound or might suggest that some part of the chemically determined concentrations include biologically unavailable acetate. Results from the Black Sea suggested that sharp acetate maxima or minima coincided with zones of enhanced microbial activity, especially in the

i(16

tt. 14u, M.t. Scranton~Marine Chemistr~ 47 ;1994j 97 [13

vicinity of the oxic-suboxic interface, the suboxicanoxic interface and near the bottom. Concentrations in the Black Sea were comparable to those we obtained for the Pettaquamscutt (Mopper and Kieber, 1991). In the Pettaquamscutt, the maximum acetate concentrations observed were correlated with high densities of microorganisms, suggesting that production is the result of microbial decomposition of dissolved and/or particulate carbon. DOC concentrations were measured, using the hightemperature combustion method, in the Pettaquamscutt on 20 August 1992 (data not shown). DOC values were almost constant (at about 250 #M) with depth and time of day, suggesting that VFAs do not make up a major portion of the total dissolved organic carbon in this system, although VFAs probably make up a major fraction of the DOC which is rapidly cycled by bacteria. Sharp acetate concentration maxima have been observed in several sediment profiles (Balba and Nedwell, 1982; Sansone, 1986; Michelson et al., 1989; Wu and Scranton, 1994). In modeling the distribution of acetate, Michelson et al. (1989) predicted that the highest production occurred near the sediment surface where organic carbon is highest. In some systems, acetate maxima near the sediment surface have been found to be associated with high sulfate reduction rates (Balba and Nedwell, 1982). Concentrations of acetate (and other VFAs) were at a maximum near the surface in the winter in Cape Lookout Bight, probably due to decomposition of organic matter which accumulates during the fall (Sansone and Martens, 1981). Wu and Scranton (1994) have also shown acetate concentration and uptake rate constant maxima in surficial Long Island Sound sediments following a bloom. Results from some of the many studies of acetate in sediments are presented in Table 1. Concentration maxima do not always correlate with maximum carbon cycling rates. Sansone and Martens (1982) found an acetate concentration maximum below the surface in Cape Lookout Bight, at a depth where sulfate is depleted. In this case the acetate maximum seemed to be caused by the fact that acetate production increased during the summer at the same time that sulfate was

being depleted. Since methanogemc bacteria prefer H2 over acetate as a source of energy (Sansonc and Martens, 1982; Sansone, 1986), acetate accumulated when sulfate depletion began, but methanogenesis from acetate had not yet started. In the Pettaquamscutt water column, sulfate concentrations were high throughout our study period, so this latter mechanism cannot explain the observed maxima. Acetate uptake rate constants for the water column varying from 0.0004 to 0.27 h i have been reported in the literature (Table 1). The rate constants measured for acetate in the Pettaquamscutt were greater than or equal to those measured in the mixed layer in the Scheldt estuary by Bitlen et al. (1980) or by Wu and Scranton (1994) for Long Island Sound or Wu (unpubl. data) for Flax Pond. They were also higher than those determined for the waters of coastal Peru, or Chemotaxis Dock, as reported by Lee (1992). Estuarine (Scheldt) acetate uptake rate constants were much higher than in the Belgian coastal zone of the North Sea and the eastern English Channel. Rate constants reported for sediments in previous studies are comparable to those we measured in the water column of the Pettaquamscutt Estuary (Table 1). The details of acetate cycling in the Pettaquamscutt become much clearer when we focus individually on the processes taking place in the different layers. In particular we found that variations in microbial populations seemed to play an important role in controlling vertical variations in acetate production and consumption. For example, in the oxic layer, uptake rates (calculated as rate constant times concentration) were higher during the periods of low light intensity (average 4.4 #M h-l) than during periods of high light intensity (average 1.9#Mh J). This could suggest either that the heterotrophic activities are intrinsically higher under low light conditions or that there is higher supply, and thus uptake, of acetate at night, perhaps as a result of increased flux of labile organics caused by increased zooplankton grazing. The occurrence of acetate production in the oxic zone is consistent with results obtained by Billen et al. (1980), who suggested that the "high" rate constant (0.0004-0.27 h -j) and "high" concentration (0.2

H. Wu, M.L Scranton/Marine Chemistry 47 (1994) 97-113

4 #M) observed in oxic mixed layers indicated the importance of fermentative catabolism, even in oxic seawater. Our uptake rates are considerably higher than observed by Billen et al. (1980), suggesting that fermentation is even more intense in the Pettaquamscutt. If acetate is predominantly a product of fermentation, our data are consistent with the idea that oxygen production and fermentation occur simultaneously in the oxygenated ocean as has been suggested by Sieburth (1987). It seems most likely that this is the result of anoxic microenvirouments such as those observed in oxic sediment (Jorgensen, 1977) and marine snow (Alldredge and Cohen, 1987). Recently, it was found that even in well-oxygenated ambient water, reducing microniches can persist in marine snow (Shanks and Reeder, 1993). At the base of the oxycline, the acetate uptake rate was still high (1.20-4.80 #M h-l), but no obvious diel change in the uptake rate constant was observed (Fig. 7). In the suboxic zone, both 02 and H2S are very low or absent, but dissolved Fe 2+ and Mn 2+ start to increase (Mason et al., 1993). Iron and manganese reduction have been shown to play an important role in organic matter decomposition in some situations (Lovley, 1991) and manganese-reducing bacteria capable of using fermentation products such as acetate have been isolated from the suboxic zone of the Black Sea (Nealson et al., 1991). It is possible that acetate oxidation is coupled to iron and manganese reduction in the suboxic zone in the Pettaquamscutt Estuary (3.2-5.0 m). However, we have no data which address this point directly. Acetate was cycled most rapidly in the E. proxima layer, a zone where the 02 concentration was measured to be on the order of tens of micromolar during the day but zero during the night (P.L. Donaghay, pers. commun., 1993). 02 production appears to take place in the E. proxima maximum during the day (Sieburth, 1987). The acetate uptake rate constant showed a maximum in the E. proxima peak during the day but not at night (Fig. 3). Paerl et al. (1993) also found that, during the day, light-mediated acetate incorporation rates were higher than acetate uptake rates in microbial mat systems in the dark. In the Pettaquamscutt Estuary, the higher rate constant in the E. proxima

107

layer suggests that 0 2 production may play an important role in acetate metabolism, either directly by limiting heterotrophic uptake or indirectly due to its effect on manganese and/or iron speciation. The lack of an obvious effect of DCMU on the acetate uptake in our short term incubation (Table 1) may suggest acetate uptake at the base of the oxycline is by O2-tolerant anaerobic bacteria or that it occurs in anaerobic microniches, or that DCMU must be added for a much longer time to be effective. Recently, Paerl et al. (1993) noted that DCMU had no effect on lightstimulated DOM uptake rates in microbial mats and suggested that DOM uptake was mediated by bacterial anoxygenic photosynthesis. In the E. proxima maximum, one of the most interesting aspects of the data is that while the acetate uptake rate constant and uptake rate both are very high during the day and decrease at night, concentrations in this layer did not change significantly (Fig. 8). This implies that, during the day, the production is high but is balanced by the acetate consuming population. At night, when lower consumption rates were observed, production must also be lower, since the concentration of acetate did not change. We are not yet able to say whether uptake decreases in response to decreased production or whether some external factor affects both processes simultaneously. Our sampling was about every 6 h, so the bacterial response must be faster than 6 h and is probably much more rapid (based on our uptake rate constants of acetate). In the anoxyphototroph maximum layer, acetate production and uptake occur in the complete absence of 02. A maximum rate constant of 1.48 h -1 (during the day) and a minimum value of 0.37 h -l (during the night) were observed (Fig. 9), perhaps suggesting the importance of light to acetate uptake at this depth. The acetate concentration measured at 03:40 in this layer was comparable to the "day" values and was a factor of 4 lower than values measured for samples collected at 20:15 and 08:40. As indicated in the results we have no specific reason to reject this point. However, since uptake rate constants were about the same at 20:15, 03:40 and 08:40, the large observed fluctuations in concentration would imply a large variation in acetate production rate as well. We do not have

108

H. Wu, M.I. Scranton~Marine Chemistry 47 ,'1994 ) 97 113

adequate data to suggest a cause for such a large production variation. Our understanding of why uptake rate constants might vary is better. Anoxyphototrophs, such as Chlorobium phaeovibrioides and C. phaeobacteroides, are thought to be present in the basin (based on the bacteriochlorophyll e distribution; Mason et al., 1993). These organisms are known to take in acetate and CO2 at rates which increase with increasing light intensity in the range of 0.3 to 20 #E m-Zs -I (Bergstein et al., 1979, 1981). At the phototrophic bacterial layer in the Pettaquamscutt, light intensity (PAR) was only a few tenths of l # E m 2 s-1 (A.K. Hanson, unpubl, data). Growth of Chlorobium species at low light intensity has also been inferred in a study of the Black Sea (Repeta and Simpson, 1991). Our field results are consistent with the observation that available light intensity (or some factor which varies with light intensity) may play an important role in the acetate uptake by anoxyphototrophs in the presence of H2S, Light-independent uptake of acetate, such as by sulfate reducers, methanogens, and/or heterotrophic activities of other microorganisms must also be important at all depths in the Pettaquamscutt as acetate uptake was always observed, even at night. One possibility mentioned earlier is acetate uptake coupled to reduction of iron and manganese oxides. A second possibility is that acetate uptake by sulfate reducers and methanogens is important in the water column. Methane profiles were measured in 1992 and showed pronounced maxima associated with the pycnocline (Scranton et al., in prep.). Since concentrations of methane at the maximum varied by about 40% over a diel cycle (an increase of 0.5 #M 1-~ in 6 h), methanogenesis may be taking place in the water column (Sieburth and Donaghay, 1993; Scranton et al., in prep.). No measurements were made of sulfate reduction rates. A simple mass balance for acetate can be used to estimate production rates from the changes in concentration over time and the uptake rate. Lateral transport from basin wall sediments was not considered because transect studies have found no evidence for lateral variability in T, S or methane along isopycnai surfaces in the basin. The mass

balance of acetate for a particular layer can be written as: dC

--=Pdt

U+S-L

(2i

where P is the acetate production rate, U is uptake rate, S is the rate of supply by vertical advection or diffusion, and L is the rate of loss by vertical advection and diffusion. In the basin, vertical advection is probably small due to the lack of intrusions or upwelling (P.L. Donaghay, pers_ commun., 1991), and is ignored in the subsequent discussion. In order to decide whether vertical diffusion represents an important source or sink for acetate at a particular layer, we will first calculate production for the layer containing the acetate concentration maximum observed at ~ 6 m. Concentration gradients in this layer are particularly large, so vertical fluxes at this depth should be among the highest for the. basin. Since we are considering the budget at a maximum, diffusive supply by physical processes (S) can be ignored. However, loss needs to be considered. The rate of loss, (L), equals Jea where

OC

Jed = Kz x 0--~

(3)

where Kz is the vertical eddy diffusion coefficient. One approach for calculating K~, was proposed by Gargett (1984):

K, = aoN(-l'° to -1.2)

(4)

where a0 is a constant related to energy input to the system and has an upper limit of about 1 x 10-4. and N is the buoyancy frequency:

N = ( - g OP~°'5 where the acceleration due to gravity, g, is 981 cm s -2, P0 is the average density over the interval (here 1.02) and Op/Oz is the density gradient (0.00258 m-l). A value of -1.0 was used for the exponent in Eq. (4). Using these values, Kz in the Pettaquamscutt was found to be 9.5 x 10.4 cm 2 s --~ at 6 m and was almost a factor of 10 lower at 2 m. At the depth of the anoxyphototroph maximum,

H. Wu, M.L Scranton/Marine Chemistry 47 (1994) 97-113

'7, al

10"

a

6

rate of 4.2 #M h -1 at 6 m (see Fig. 3). Even if our calculation underestimates Jcd by an order of magnitude, the loss by eddy diffusion is still very small relative to uptake. Similar calculations were made in the E. proxima layer, with similar results. Since the anoxyphototroph layer had the steepest concentration gradients and a relatively high value of Kz, it appears that diffusion of acetate to or from any layer considered in the Pettaquamscutt Estuary will be small. Thus Eq. (2) can be simplified to: P = U+

0

Diel #

.~

g

~

"'

~-~

(6)

or

1

10

b

6

Diel #

109

1-2

2-3

3-4

4-5

Fig. 11. Estimated average acetate production rates derived from Eq. (6). Numbers on the x-axis indicate the two diel study profiles used in calculating the average acetate production rates. (A) Data from the oxic layer are designated D, from the base of the oxycline O, and from the E. proxima maximum II. (B) Data from the anoxyphototroph layer are designated (3. An alternative calculation for the anoxyphototroph layer was made by replacing the measured value for diel 3 with the average of the concentrations measured during diel 2 and 4. These production rates are shown using A. Approximate time intervals are as follows: 1-2=14.00-19.30; 2-3=19.30-02.00; 3-4=02.0008.00; 4 - 5 = 08.00-14.00.

OC/Oz was 7.1 x 10-5 #mole cm -4 so the vertical diffusive flux was only 2.4 x 10 - 4 #mole cm -2 h -l. If the thickness of the peak is taken to be 10 cm, the loss due to diffusion is 2.4 x 10-2 #M h -1, which can be compared with the measured uptake

In other words, the production rate is roughly equal to the uptake rate plus any change in concentration with time. By measuring the uptake rate constant and the concentration of a substrate as a function of time at a certain depth, the production rate can be estimated from Eq. (7). For acetate uptake rates, we used an average of the uptake rates for the intervals considered [(1/2) (kt C1 -+-k2C2)],where C1, kl and 6"2, k2 refer to the concentration and uptake rate constant at times 1 and 2, respectively. Production changes over time in all four layers calculated using this approach are shown in Fig. 11. In general, AC/At was small, so production primarily mirrors uptake, While no obvious diel variations in acetate production rates were observed in either the oxic layer or the base of oxycline, larger did changes occurred in the E. proxima layer and the anoxyphototroph layer. In the E. proxima layer, the high daytime production rate may be due to enhanced production, caused by enhanced production of precursors during photosynthesis. The variations calculated for the anoxyphototroph layer are strongly influenced by the low concentration measured at that depth measured during middle of the night. If we assume the concentration for 03.40 at the anoxyphototroph layer should be the average of the concentrations of 20.15 and 08.40, the production rate in this layer seems to be relatively constant (Fig. 11).

~ll

tt. Wu, M.l. Scranton~Marine Chemisttiv 47 ,'1994) 97 113

Sieburth and Donaghay (1993) and Scranton et al. (in prep.) have suggested that methane production may be an important process in the water column of the Pettaquamscutt Estuary. One way of determining whether methanogenesis is an important sink for acetate is to use BES (2-bromoethanesulfonic acid), a specific inhibitor of methanogenesis (Oremland and Capone, 1988). In the Pettaquamscutt Estuary we measured acetate uptake with and without BES at the depth of the E. proxima maximum, where Scranton et al. (in prep.) observed diel variations in methane concentrations, and at the base of the oxycline. BES reduced acetate uptake to < 30% of the control rate. If about 70% of the acetate was being converted to CH 4 and CO 2, total amounts of CH4 produced would be about 4 #M h ~. However, estimates of methane production rates from methane concentration changes predict a value closer to 0.1 #M h l (Scranton et al., in prep.). Even if part of the acetate were "unavailable" to methanogens, and thus the calculated acetate uptake rate were overestimated, the predicted methane production seems to be much higher than observed. For the two to agree, 97.5% of acetate would have to be unavailable to methanogens. Since CH4 oxidation rates were not measured, it is not possible to resolve this apparent inconsistency. It is also possible that BES may be affecting something other than methanogens. Total integrated uptake rates of acetate from the surface to 7 m were about 600 mmol C m 2 d-~. This is almost twice as high as the primary production integrated to the same depth (~ 336 mmol C m -~2 d-I; A.K. Hanson and J. Prentice, unpubl. data). Direct comparisons between acetate uptake rates and primary production rates at specific depths also showed that in both the suboxic and anoxic zones, acetate uptake rates were much higher than primary production. There are several possible reasons for the high integrated acetate uptake. Acetate oxidation rates which are higher than sulfate reduction rates have been observed in sulfate-reduction dominated sediments on a number of occasions, and there is some evidence that biologically less-available pools may be present, either in complexed form or adsorbed to sediment or mineral surfaces and to particulate

organic matter (Parkes et al., 1984; Shaw et ai., 1984; Gibson et al., 1989; Wang and Lee, 1993). Shaw and McIntosh (1990) have suggested thai the discrepancy between acetate uptake rates and sulfate reduction rates may be due to release of acetate during porewater extraction. Since water column samples were gently filtered under nitrogen at low pressures rather than being subjected to centrifugation, this source of' error should not be a problem in our Pettaquamscutt data. However, complexation of acetate might be important in the water column as well as the sediments (Parkes et al., 1984: Thompson and Nedwell, 1985; Michelson et al., 1989), and the extent ot nature of this speciation might also vary across the oxic- anoxic interface. Unfortunately, we have no data which directly bear upon the issue of speciation of acetate in the water column, Acetate uptake rates could also appear higher than primary production if allochthonous compounds exist which can be decomposed into acetate precursors. Findlay et al. (1992) has recently shown that the Hudson River is a heterotrophic system in which bacterial production exceeds algal productivity by a factor of two or more. Since the Pettaquamscutt is a narrow estuary with residential, marshy and forested areas along its banks, input of terrestrial organic matter to this system is likely to be important. Still another possibility is that acetate uptake is primarily by pathways other than acetate oxidation to CO2. Acetyl-coenzyme A (acetyl-CoA) is involved in many enzymatic reactions, and acetate in the form of acetyl-CoA is the precursor of many cellular compounds (Lehninger, 1978). Since most of the acetate uptake we observed in our very short time course incubations was in the lbrm of assimilation rather than respiration, it is possible that acetate could be released from grazed cells and then reassimilated as DOC. Whatever the case, we are unable to quantify the relative importance of these processes to the acetate cycle at this time. The fact that the integrated acetate uptake rate was twice as high as primary production suggests that acetate plays an important role in organic matter decomposition in this system. In addition to acetate, we measured propionate. butyrate and isobutyrate concentrations in the

H. Wu, M.L Scranton~MarineChemistry47 (1994) 97-113

Pettaquamscutt River. The pool sizes of propionate, isobutyrate and butyrate were much smaller than that of acetate. The uptake rate constants for propionate were also lower than for acetate. (Uptake rate constants were not determined for butyrate and isobutyrate.) Few previous studies have determined natural abundances of propionate, although concentrations of 1 to 24 #M were measured in whole sediment extractions of the organic rich Cape Lookout Bight sediment (Sansone and Martens, 1982), and Ansbaek and Blackburn (1980) found propionate concentrations of 100-600 nM for Limtjorden sediments. In the Pettaquamscutt Estuary, highest propionate concentrations (187 nM) and propionate uptake rate constants (0.67 h -1) were found at the E. proxima maximum layer. Like acetate, propionate is a common product of many fermentations (Mclnerney et al., 1979), and propionate-oxidizing, sulfate-reducing bacteria (such as Desulfococcus spp., Desulfobulbus spp., and Desulfosarcina spp. (Widdel and Pfennig, 1984) have been isolated from both freshwater and marine sediments. At high sulfate concentrations, some sulfate reducing bacteria may directly metabolize both propionate and butyrate completely to CO2 (Laanbroek and Pfennig, 1981). Other sulfate reducers (for example, Desulfobulbus spp.) oxidize propionate incompletely (Widdel and Pfennig, 1984). Since significant rates of CO2 production were observed in incubations with 2-14Cpropionate, sulfate reducers which completely oxidize propionate to CO2 are probably active in the Pettaquamscutt. However, propionate also can be assimilated by some methanogens (Eikmanns et al., 1983) and can be metabolized by methanogenic co-cultures (Koch et al., 1983; Houwen et al., 1987). We did not perform any BES inhibition experiments with propionate.

6. Summary and conclusions Low molecular weight fatty acids are present at nanomolar to micromolar concentrations in the Pettaquamscutt River Estuary. Acetate and propionate uptake rate constants in the suboxic zone are highest where both 02 and H2S concentrations

111

are low or undetectable and also in an anoxyphototroph maximum found at the top of the H2S concentration gradient. Acetate uptake increased during the day in the E. proxima layer, coincident with photosynthetic oxygen production in this layer. Oxidation and fermentation appear to occur simultaneously in the oxygenated zone and the E. proxima maximum layer. The high uptake of acetate in the anoxyphototroph layer during the day is also consistent with the importance of light intensity to the acetate cycle in this layer. Inhibition experiments using BES show that acetate might be one of the precursors of methane production in the E. proxima maximum layer. The high integrated acetate oxidation rates for the basin imply that acetate plays a very important role in organic matter decomposition in the anoxic water column as it does in anoxic sediments. The fast turnover of acetate also indicates the importance of fermentative processes in the pycnocline of the anoxic water column.

Acknowledgments Our studies in the Pettaquamscutt have been highly labor intensive and could not have been made without the help of Percy Donaghay, A1 Hanson, Wayne Warren, Adam Cantu, Jennifer Prentice, Kathy Hardy, Dan Sullivan, and Tim Finnegan of the Graduate School of Oceanography, University of Rhode Island, and Elizabeth Lamoureux and Heloisa Borges of the Marine Sciences Research Center. Percy Donaghay and AI Hanson generously provided unpublished hydrographic and productivity data. John Sieburth first called our attention to the Pettaquamscutt and encouraged our participation in an earlier study. Radioisotope studies were carded out in the MERL Radiation Facilities at URI where Ken Hinga greatly facilitated our use of radioisotopes. Drs. Cindy Lee and Gordon Taylor of MSRC provided many useful comments on the data and its interpretation. Support for this research was provided by NSF grant 90-17737 (M.I.S.). This is contribution 953 from the Marine Sciences Research Center.

112

tl. Wu. M.t. Scranton~Marine Chemistry 47 ~1994) 9 7 11.3

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