Methane Production During Zooplankton Grazing on Marine Phytoplankton

Methane Production During Zooplankton Grazing on Marine PhytoplanktonAuthor(s): Marie A. de Angelis and Cindy LeeSource: Limnology and Oceanography, Vol. 39, No. 6 (Sep., 1994), pp. 1298-1308Published by: American Society of Limnology and OceanographyStable URL: http://www.jstor.org/stable/2838133 .

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Limnol. Oceanogr., 39(6), 1994, 1298-1308 ? 1994, by the American Society of Limnology and Oceanography, Inc.

Methane production during zooplankton grazing on marine phytoplankton

Marie A. de Angelis' and Cindy Lee Marine Sciences Research Center, State University of New York, Stony Brook 11794-5000

Abstract Zooplankton produced CH4 while grazing on marine phytoplankton during laboratory experiments.

We consider anaerobic microniches within zooplankton digestive tracts to be the most likely site for such methanogenesis. Methane production appeared to be zooplankton species-specific rather than dependent on the phytoplankton species being grazed. The amount of CH4 produced (4-20 nmol copepod-I d-1) was sufficient under experimental conditions to make a significant contribution to the formation and maintenance of oceanic subsurface CH4 maxima.

The methanogenic substrates monomethylamine and trimethylamine were constituents of the diatom, dinoflagellate, and flagellate phytoplankton species used in the CH4 production feeding experiments. Dimethylamine was present in two of the three algal species. Trimethylamine was the most abundant methylamine in all cases and was present in sufficient quantity to account for the CH4 production observed during copepod feeding experiments. Methanogens in these experiments would need to convert only 4- 12% of the phytoplankton methylamines available to copepods during grazing to produce CH4 concentrations observed in the experimental flasks.

Dissolved CH4 profiles in the upper oceanic water column are frequently characterized by a subsurface maximum with levels 2-3 times more supersaturated with CH4 than are surface waters (e.g. Scranton and Brewer 1977; Brooks et al. 1981; Conrad and Seiler 1988). Advec- tion of CH4 from nearshore point sources such as hydrocarbon seeps (Cynar and Yayanos 1991) and anoxic slope sediments (Brooks et al. 1981) undoubtedly contributes to the subsurface CH4 maximum in coastal regions. However, Scranton and Brewer (1977) and Conrad and Seiler (1988) have shown that ad- vection alone is insufficient to maintain such a well-developed feature against large diffusional gradients over the time and distance

1 Current address: Department of Oceanography, Hum- boldt State University, Arcata, California 95521. Acknowledgments

We thank Gordon Wolfe for supplying [14C]DMS and Xu-Chen Wang for help with the methylamine analysis. Sigruin J6nasd6ttir maintained the phytoplankton and copepod cultures throughout the study and helped with the feeding experiments. We thank Mary Scranton and Amy Michelson for help and discussions during the initial stages of this work. We thank Mary Landsteiner and the crew of the RV Clifford Barnes for help obtaining zooplankton from Puget Sound and John Hedges for shiptime. Gayle Heron helped identify Calanus pacificus.

This research was supported by the National Science Foundation (OCE 91-15681) and by the Coastal Marine Scholar Program at the Marine Sciences Research Center.

that the CH4 maximum persists; rather, in situ production of CH4 must occur within the well- oxygenated upper water column.

The only known mechanism for production of CH4 under normal seawater conditions is biosynthesis by methanogens-a strictly anaerobic process. To explain methanogenesis in the well-oxygenated upper water column, various researchers have postulated the existence of reducing anoxic microzones in the guts of zooplankton and higher organisms (Oremland 1979; Traganza et al. 1979) and in particles such as fecal pellets, detrital material, algal cells, and marine snow (Scranton and Brewer 1977; Brooks et al. 1981; Burke et al. 1983).

Traganza et al. (1979) suggested anoxic mi- croenvironments in the digestive tracts of herbivorous zooplankton as the sites of CH4 production and cited strong correlations between the depths of maximum zooplankton biomass and subsurface CH4. Evidence that viable methanogens are present in the oxygenated upper water column and associated with zooplankton was provided by Oremland (1979), who observed evolution of CH4 from concen- trated subsurface plankton samples incubated under anoxic conditions. Cynar and Yayanos (1991) successfully isolated methanogens from a mixed plankton sample obtained from a tow through the mixed layer off the coast of Cali- fornia. In both cases, the plankton assemblage was dominated by zooplankton, specifically

1298



Methane production from zooplankton 1299

copepods, although phytoplankton and particles were also present. Recently, Marty (1993) reported the presence of viable methanogens in copepods, in copepod fecal pellets, and in sediment trap particles, as indicated by the production of CH4 from these freshly collected materials under anoxic conditions.

The precursor for CH4 production in the upper water column has not been conclusively identified. Methanogens that use H2 and CO2 or simple organic acids such as acetate require strict anoxic conditions to produce CH4 (Zin- der 1993). In addition, sulfate reducers are able to outcompete methanogens for these substrates (Schonheit et al. 1982; Zinder 1993). Given these requirements and the large supply of sulfate in seawater, it is unlikely that anoxic conditions would be extensive enough in upper water-column microniches for the develop- ment of sulfate depletion necessary for ace- toclastic or hydrogenotrophic methanogenesis to occur.

In recent years, several methylated compounds that do not require sulfate depletion to produce CH4 have been identified as substrates for marine methanogens. These include methylamines, dimethylsulfide, and methanol (Oremland and Polcin 1982; King et al. 1983; Kiene et al. 1986). All of these methylated compounds are direct or indirect products of phytoplankton metabolism and are possible precursors for CH4 production in the upper water column. In particular, Cynar and Yaya- nos (1991) and Marty (1993) isolated or identified methanogens capable of using methylamines in mixed zooplankton samples obtained from the upper water column. If such compounds are indeed the precursors of CH4 in this environment, methanogenesis may not need the sulfate depletion required to reduce CO2 or cleavage of acetate to CH4. Instead, simple anoxic conditions may suffice. Such anoxic conditions have been reported in particles (Alldredge and Cohen 1987) and may occur in invertebrate guts.

This paper reports the results of a laboratory study designed to test the hypothesis that methanogens in anaerobic microenviron- ments associated with the digestive tracts or fecal pellets of herbivorous zooplankton can produce significant quantities of CH4 under typical upper water-column conditions. Ma- rine copepods were fed 14C-labeled phyto-

plankton, and the 14CH4 produced over time was monitored. We describe a sensitive method which involves the separation of 14CH4 from other volatile carbon compounds and its subsequent conversion to 14C02, allowing us to detect the production of small quantities of CH4. We also determined cell contents of mono-, di-, and trimethylamines for all phytoplankton species used in the feeding experiments so we could determine whether the amount of methylamines released by zooplankton grazing is sufficient to support the observed levels of CH4 production.

Methods Zooplankton sampling and culturing-Co-

pepods were chosen as the experimental zooplankton because they are abundant grazers of phytoplankton in the upper water column (Frost 1980). Copepods were collected on an incoming tide in Stony Brook Harbor, New York, with a 210-,um-mesh zooplankton net. The contents of the cod-end were diluted into a bucket filled with ambient water and immediately returned to the laboratory. Adult and C5 stages of Temora longicornis and Acar- tia tonsa were sorted and - 50 animals placed in each of several 2- or 3-liter beakers filled with glass-fiber-filtered (Whatman GF/C) ambient water or seawater obtained from a depth of 15 m in Long Island Sound. Copepods were also obtained from vertical tows with a 330- ,um-mesh plankton net to a depth of 50 m in Puget Sound, Washington. Adult and late-stage copepodites of the species Calanus pacificus were sorted, placed in 2.5-liter plastic insulated bottles filled with GF/C-filtered Puget Sound surface water, and fed Thalassiosira weissflo- gil. The bottles were packed in insulated con- tainers lined with Blue Ice and flown back to Stony Brook. All animals were kept in culture in a O0?C (1992) or 16?C (1991) cold room and fed a mixture of the phytoplankton T. weissflogii, Rhodomonas lens, and Prorocentrum minimum twice a week. The water was lightly aerated and was changed about every 10 d.

Phytoplankton culturing-The diatom T. weissflogii, the cryptophyte R. lens, and the dinoflagellate P. minimum were used as the experimental food. For each experiment, a phytoplankton culture was started in a 2-liter batch culture using f/2 media (Guillard 1975) and grown under fluorescent illumination on



1300 de Angelis and Lee

Table 1. Summary of CH4 production feeding experiments.

[Initial algal] Algal sp act Exp. Copepod species Phytoplankton species (cells ml-') (mCi mmol-1)

91-1 Temora longicornis Thalassiosira weissflogii 12,500 5.2 91-2 Acartia tonsa Rhodomonas lens 28,000 13 91-3 A. tonsa Prorocentrum minimum 6,100 8.7 92-1* T. longicornis T. weissflogii 12,100 9.9 92-2* T. longicornis P. minimum 5,200 7.2 92-3* T. longicornis R. lens 17,500 8.9 92-4 Calanus pacificus T. weissflogii 18,000 14.5 92-5 C. pacificus P. minimum 7,700 6.3

* Concurrent CH4 oxidation rate experiments were carried out in separate flasks under identical conditions but with nonlabeled phytoplankton, as described in the text.

a 14: 10 L/D cycle. The growth of the culture was monitored daily by cell counts. When the culture was at an early exponential growth stage, it was split into two 1-liter sterile screwcap Erlenmeyer flasks. One flask received 0.5- 1 mCi NaH14CO3. The other flask was not radiolabeled and was used in parallel CH4-oxidation experiments, as described below. The cultures were incubated an additional 48-72 h to allow uptake of the radiolabel and to assure uniform labeling of the algae (Welschmeyer and Lorenzen 1984).

Experimental design-A series of feeding experiments was carried out in which copepods were fed 14C-labeled phytoplankton, and the production of 14CH4 was monitored over time. Each feeding experiment involved a single copepod and phytoplankton species (Table 1). Algal concentrations were chosen so that the copepods were not initially food limited (excess food conditions) and were feeding at maximum rates for the first 3-6 d of the experiment. Feeding rates for each copepod species were determined prior to radiolabeling experiments by incubating a given number of copepods with excess T. weissflogii and measuring grazing rates over time. For R. lens and P. minimum, copepods were fed similar concentrations of food, on a cell volume basis, as for experiments with T. weissflogii.

For each separate feeding experiment, 250- ml Erlenmeyer flasks were autoclaved and filled with a mixture of filtered (Whatman GF/C) 15-m Long Island Sound water and radiolabeled algal culture to a final volume of 200 ml. The volumes of seawater and algae varied for each experiment and were dependent on algal culture cell density and desired algal concentration in the experimental flask. A headspace

of - 100 ml was present in all flasks. Three C. pacificus or five T. longicornis or A. tonsa adult copepods were added to half the flasks. The other flasks served as controls and contained labeled algae at the same initial concentration as the zooplankton flasks but no copepods.

Flasks were sealed with autoclaved two-hole silicone stoppers with 3.2-mm (1/8") nylon tub- ing serving as inlet and outlet tubes. Each tube was equipped with two-way nylon stopcocks (Pharmaseal) that were closed immediately after flasks were stoppered. The inlet tube was long enough to reach the bottom of the flask, while the outlet tube remained above water level. All materials had been previously tested in the presence of copepods and found to be nontoxic. This arrangement allowed water subsamples to be drawn from the flasks for algal counts without loss of gases from the headspace and allowed flasks to be connected to the stripping-oxidation line without loss of headspace, as described below. Separate stor- age experiments in which 14CH4 was added to flasks containing sterile seawater indicated that there was no detectable loss of CH4 from the flasks for at least 1 week. After this time, slow loss of CH4, presumably through the silicone stoppers, was observed over time.

Flasks were incubated in the dark at 100 (1992 experiments) or 16?C (1991 experiments) for up to 21 d. Temperatures matched those of Long Island Sound from which T. longicornis and A. tonsa were originally collected and sub- sequently cultured. Flasks with and without copepods were tested for algal cell concentration and 14CH4 production every 2-3 d for as long as copepods were actively swimming. Flasks were gently swirled daily for the dura- tion of the experiment.




For each sampling day, flasks with and without copepods (sometimes in duplicate) were swirled, and 3-ml subsamples were withdrawn through a 5-ml disposable syringe attached to the stopcock of the outlet tube. The flask was inverted so that the outlet tube was in the seawater. The stopcock was opened and the syringe flushed several times. One 3-ml subsample was placed in a screwcap test tube with three drops of Lugol's solution and stored in the dark for algal cell counts. Algal cell density was determined by counting a minimum of 300 cells with a model S50 Sedgwick-Rafter counting cell (Electron Microscopy Science). Presence of fecal pellets, if any, was noted, as well as their state of preservation (intact, frag- mented).

A separate 3-ml subsample was passed through a 0.22-gum filter (Millipore GSWP) and allowed to dry. The filter was then counted in Permafluor E+ on a Packard model 1600CA liquid scintillation counter (LSC) to determine the amount of radiolabeled phytoplankton still available for ingestion and conversion to CH4. After each 3-ml subsample was withdrawn, an equivalent amount of air was added by syringe to the flask to maintain atmospheric pressure. Samples were then shaken vigorously for -2- 3 min. Because CH4 is extremely insoluble in water (McAuliffe 1971; Wiesenburg and Gui- nasso 1979), shaking resulted in the removal of >90% of the CH4 to the headspace. Samples were then attached to the oxidation line described below to measure 14CH4 production.

CH4 production measurements - Produc- tion of 14CH4 was measured with an oxidation line similar to that of Crill and Martens (1986), but with several modifications. A Pd catalyst was used instead of CuO to convert CO2 to CH4 because Pd has been demonstrated to be a more efficient catalyst for oxidizing CH4 (An- derson et al. 1961). In addition, a chromato- graphic column was added to separate CH4 from other volatile carbon compounds.

The '4CH4 was stripped from the sample by a stream of prepurified N2 bubbled through the incubation flask at a rate of 40 ml min- 1. The stripped gas was passed through two indicating Drierite (10/20 mesh) columns (0.95 x 15.2-cm Pyrex) to remove water vapor and an Ascarite (30 mesh) trap to remove any 14CO2 originally present in the sample. Addition of 14CO2 standards injected through a septum port

directly into the oxidation line verified that Ascarite quantitatively removed CO2 at the highest levels expected from the measured radiolabeled algal concentrations (-0.2 mg CO2). The gas then passed through a 60/80 mesh HayeSep Q column (0.64-cm x 1 -m stainless steel, preconditioned overnight at 175?C with a flow of prepurified N2), which separates CH4 from other volatile carbon-containing compounds.

After the CH4 passed through the column, a four-way valve was switched to vent the re- mainder of the stripped gases to the hood. Im- mediately after the four-way valve, 02 was added to the N2 stream at a flow rate of 40 ml min -I (total flow, 80 ml min- 1). The gas stream then passed through a Pd on alumina catalyst in a 1.28- x 30.5-cm stainless steel column at 650?C where CH4 was converted to CO2 at a known conversion efficiency. The catalyst was a mix (- 2: 1) of 3.2-mm Pd on alumina pellets (Aldrich) and Pd powder. Tests with injection of known concentrations of 14CH4 over 3 or- ders of magnitude gave highly reproducible conversion efficiencies of 80% under the spec- ified temperature and flow conditions. Higher catalyst temperatures and lower flow rates yielded higher conversion efficiencies but resulted in shorter column life and longer analysis time, respectively. Oxidation-column conditions were chosen to maximize conversion at a reasonable analysis time.

After oxidation, the gas stream passed through a series of three scintillation vials, each containing 0. 5 ml of Carbosorb E (Packard) to trap any 14CO4 produced. Tests with 14CH4 standards indicated that CH4 was stripped, oxidized, and collected in 60 min under the above conditions. Samples were run for a total of 70 min (20 min in valve position A and 50 min in valve position B) to be certain that all labeled CO2 was collected. The scintillation vials were then removed, 6 ml of Permafluor V or E+ (Packard) scintillation cocktail was added to each, and the vials were counted in an LSC. Standards consisting of diluted stock 14CH4 were run at the end of each day to check conversion efficiencies.

The 14CH4 used in each experiment was as- sayed by a modification of the method of Zehnder et al. (1979). A small hole was drilled in a scintillation cap of a 20-ml glass scintillation vial and the liner removed and replaced




with a 18-mm Teflon-faced silicone septum (Pierce). The same volume of 14CH4 standard as had been introduced to the oxidation line was injected through the septum into 20 ml of Permafluor V. The vial was allowed to sit in the dark for 1 h to permit the 14CH4 to equil- ibrate with the Permafluor and was then counted in an LSC. The counts were corrected for the solubility of CH4 in Permafluor V; solubility was determined by adding a fixed volume of pure CH4 into varying volumes of Permafluor V in septum-capped 20-ml scintillation vials and analyzing the headspace for CH4.

Radiolabeled dimethylsulfide (DMS) and trimethylamine (TMA) were injected into the oxidation line under the same conditions under which samples had been run to test for possible interferences. No "4CO2 was collected in either case. Blanks were run periodically to check for sample-to-sample contamination.

CH4 oxidation measurements-If the rate of CH4 oxidation is sufficiently high in the incubation flasks, CH4 production in the flasks may be underestimated, as methanotrophic bacteria may oxidize some fraction ofthe 14CH4 produced during a feeding experiment. For this reason, we determined CH4 oxidation rates on replicate samples for selected 1992 feeding experiments (92-1, 92-2, 92-3).

For determination of CH4 oxidation rates, the experimental design was identical to that described for CH4 production experiments (i.e. for a given algal and copepod species combi- nation, the same seawater volume, number of copepods, and algal concentration were used). However, algal cultures were not radiolabeled; instead, 14CH4 to a final concentration of 60 nM was added to all flasks. As in the CH4 production experiments, two sets of flasks were run-one set with copepods and phytoplankton and one set with phytoplankton alone. Methane oxidation was measured as production of 14C02 using a modification of the method of de Angelis et al. (1993).

We recovered the 14C02 produced by oxidation of the added 14CH4 by acidifying the sample with 10 N H2SO4 to a final pH of 2 and stripping the sample with a stream of He at 75 ml min-I for 30 min. The stripped 14C02 was collected in a series of two scintillation vials containing 0.5 ml of Carbosorb E. After stripping, the vials received 6 ml of Permafluor E+ and were counted in an LSC. NaH14CO3

standards were run at the beginning and end of each day to test for trapping efficiency. In all cases, >98% of the added NaH14CO3 was recovered. Blanks were run at frequent inter- vals to check for contamination.

Methylamine content of phytoplankton- Phytoplankton culture volumes of 250-500 ml were passed through glass-fiber filters (What- man GF/F), placed in 40-ml acid-washed vials containing 20 ml of 6 N trace-metal grade HCI, capped, sonicated, and allowed to sit overnight. Vials were vortex mixed and allowed to stand for 45 min so particles could settle. The acid was then pipetted into 50-ml pear-shaped flasks and rotoevaporated to dryness. Samples were redissolved in 200 ,gl of deionized water and analyzed with the gas chromatography (GC) method described by Yang et al. (1993). This acid extraction also removes some qua- ternary amines that would be converted to methylamines during the evaporation and GC steps.

Specific activity of labels used-The specific activity (mCi mmol-1) of the radiolabeled phytoplankton for each separate feeding experiment was determined by passing 3 ml of the well-mixed labeled stock algal culture through a 0.47-,gm filter (Millipore HAWP) and rinsing well with distilled water. The filter was dried and counted in Permafluor E+ by LSC to determine dpm ml-l of stock culture. This value was multiplied by the total volume (- 1,000 ml) of the stock culture to yield total algal dpm. Total algal dpm was multiplied by total mCi of bicarbonate added divided by 2.2 x 109 dpm mCi-1 to obtain the fraction of original label incorporated into algal cells. This fraction was multiplied by the original specific activity of the NaH14CO3 to yield the specific activity of the experimental phytoplankton cultures in units of mCi mmol-1. This algal specific activity was used to convert units of dpm 14CH4 to units of total pmol CH4.

The labeled CH4 used in methane oxidation experiments was made from NaH14CO3 by a modification of the method of Daniels and Zei- kus (1982). Specific activities were determined for all synthesized 14CH4 and exceeded 50 mCi mmol-1 for all batches used.

Results Grazing of radiolabeled phytoplankton-

Copepods actively grazed the radiolabeled phytoplankton, as indicated by decreasing al-




5-9/-I 50 > 2.0-9- 0

CL E 2 'o 01 V3 .... N

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0 2 4 6 8 10 120 4 8 12 6 Time (days) Time (days)

4t 80'> 3o 92-t 13 80>

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Fig. 1. Radiolabeled algal cell (dashed lines) and '4CH4 (solid lines) vs. incubation time for feeding experiments with Temora longicornis. Each point represents sampling from a separate flask incubated for the time shown.

gal cell counts in flasks containing copepods. In most cases, grazing began immediately and continued for 4-9 d, until algal cell counts were < 10% of initial concentrations. Cell counts remained constant thereafter; this presumably represents a condition of food limitation under which copepods no longer graze efficiently. The only exception was in experiment 91-3 in which A. tonsa reduced R. lens levels by about half.

In flasks containing phytoplankton only, algal cell counts remained at initial concentrations for most experiments. For experiments 91-1 and 92-3, a slight decrease in cell counts in the control flasks was observed near the end of the incubation period. In both cases, the cells appeared to be senescent and suffering from light deprivation.

Methane production -Production of 14CH4 was observed in flasks containing copepods and radiolabeled phytoplankton in 50% of the feeding experiments (Fig. 1). CH4 production was observed in all experiments in which T. longicornis was fed either T. weissflogii, P. minimum, or R. lens. No 14CH4 production was observed with A. tonsa feeding on R. lens

or P. minimum or C. pacificus feeding on T. weissflogii or P. minimum.

Methane production by T. longicornis feeding on T. weissflogii was observed in separate experiments in 1991 and 1992 under similar conditions, except for temperature of incubation. A similar pattern of algal grazing and CH4 production was observed in both years. Meth- ane was produced for -6-7 d, at which time CH4 levels reached a maximum (Fig. 1). CH4 was produced as long as food was plentiful and feeding occurred. The shapes of the CH4 production curves were similar in 1991 and 1992, but production was higher from similar levels of labeled phytoplankton in the 1991 experiments. The higher CH4 production may be partially attributable to higher incubation temperatures in 1991 (16?C) than in 1992 (10?C).

A slightly different pattern of 14CH4 production was observed for T. longicornis feeding on P. minimum and R. lens (Fig. 1). In these experiments, CH4 was produced within the first 2 d of feeding. Methane levels then remained constant, indicating no additional production of CH4. As was the case for T.




Table 2. Methane production rates (pmol d-l copepod- 1) and ratios of CH4 production to methylamine available to copepods grazing on phytoplankton for feeding experiments with Temora longicornis.

Exp. Phytoplankton species CH4 production Mean CH4: TMA Mean CH4 MMA

91-1 Thalassiosira weissflogii 7.6 0.09 0.4 92-1 T. weissflogii 3.9 0.04 0.2 92-2 Prorocentrum minimum 20.4 0.09 0.3 92-3 Rhodomonas lens 15.1 0.12 1.3

longicornis feeding on T. weissflogii, CH4 was produced only during active feeding when food was not limiting.

No 14CH4 production was observed in control flasks containing labeled T. weissflogii and no copepods in either the 1991 or 1992 experiments. Slight 14CH4 production (< 300 dpm), however, was observed for control flasks containing R. lens or P. minimum, consistent with a previous report of small amounts of CH4 produced from algal cultures (Scranton 1977).

Rates of CH4 production were determined over the time interval of the experiment during which 14CH4 increased (i.e. before '4CH4 lev- eled off or decreased). Conversion of dpm to total moles of CH4 yielded production rates of CH4 by T. longicornis ranging from 3.9 to 20 pmol CH4 d-1 copepod-& (Table 2).

Methane oxidation-In all three experiments for which separate determinations of CH4 oxidation rates were made, 14CH4 was taken up in flasks containing copepods and phytoplankton as well as in control flasks containing only phytoplankton. However, CH4 oxidation rates in the copepod flasks were con- sistently higher than in flasks without copepods (Fig. 2). Oxidation of CH4 increased with time. Uptake of labeled CH4 in T. weissflogii flasks was more erratic over time than in either R. lens or P. minimum flasks. Separate CH4 oxidation experiments with replicate flasks also followed this trend, with much higher variability in oxidation rates in flasks containing T. longicornis and T. weissflogii than in those containing T. longicornis and P. minimum (data not shown). Rate constants for CH4 oxidation for flasks with copepods ranged from 0.0002 to 0.0 14 d-1 (0.02-1.4% available CH4 oxidized d-l).

Methylamine contents of phytoplankton cells-Trimethylamine (TMA) and monomethylamine (MMA) were present in all phytoplankton species used in the feeding experi-

ment (Table 3). Dimethylamine (DMA) was present in small quantities in two of the three species tested but was undetectable in the T. weissflogii culture. TMA was the dominant methylamine in all cases.

Discussion CH4 production associated with grazing by

herbivorous zooplankton-The production of CH4 associated with copepods grazing on phytoplankton in seawater was clearly demonstrated. CH4 production was significant only when copepods were present and actively feeding on phytoplankton. In cases in which only phytoplankton were present, CH4 production was extremely low or undetected. Previous studies carried out by Oremland (1979), Cynar and Yayanos (1991), and Marty (1993) have shown the existence of viable methanogens and the production of CH4 in zooplankton and par- ticle samples incubated under anoxic conditions. However, our study demonstrates CH4 production associated with living zooplankton under the oxygenated conditions typical of the upper water column. We assume that CH4 production must occur in anoxic microniches within the copepod guts or in particles. Meth- ane production associated with zooplankton from the upper water column appears to be species-specific and not a general feature of all mixed-layer zooplankton. This may well result from physiological differences among zooplankton that affect the formation or maintenance of suitable anoxic microniches in the digestive tract. This specificity may account for much of the variability that has been reported in the occurrence and magnitude of midwater oceanic CH4 maxima (Burke et al. 1983).

Methane oxidation experiments supplied additional indirect evidence that CH4 production in the upper water column is associated with zooplankton activity. In all CH4 uptake experiments, CH4 oxidation rates were signif-




...45- E - 92-1 0.

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0

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Time (days) 6

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Fig. 2. Methane oxidation vs. time in flasks containing copepods and phytoplankton (solid lines) and in flasks containing phytoplankton only (dashed lines) for 1992 Temora longicornis feeding experiments. Each point represents sampling from a separate flask incubated for the time shown.

icantly higher in flasks containing both copepods and phytoplankton than in flasks containing only phytoplankton. This stimulation of CH4 oxidation activity would be expected if the CH4 produced in copepod guts (or other anoxic microniches) was released to the sur- rounding water column where it would be available for consumption by CH4-oxidizing bacteria.

It is not reasonable to extrapolate the results of limited laboratory experiments of CH4 pro-

Table 3. Methylamine content of phytoplankton cultures. MMA-Monomethylamine; DMA-dimethylamine; TMA-trimethylamine.

Methyl- [Methylamine] amine (fmol (Mmol g

Phytoplankton species species cell-') dry wt-1)

Prorocentrum minimum MMA 1.2 0.7 DMA 0.4 0.3 TMA 4.3 2.5

Rhodomonas lens MMA 0.3 2.5 DMA 0.01 0.1 TMA 1.7 16.5

Thalassiosira weissflogii MMA 0.4 1.1 DMA nd nd TMA 1.6 4.8

duction by a single species of copepod to the oceanic water column. However, the rates we obtained can be used to estimate whether the CH4 production rates associated with the zooplankton we measured are of sufficient magnitude to make a significant contribution to observed upper water-column CH4 maxima. The observed range of CH4 production rates by T. longicornis in the laboratory under the temperature and food availability conditions in Long Island Sound in spring was 3.9-20.4 pmol CH4 copepod-I d-I (Table 2). The water- column density of T. longicornis in Long Is- land Sound during the spring bloom has been reported to be -50 copepods liter-' (Peterson 1985). This value is averaged over the water column, hence densities are higher at given depths. This number includes adult copepods, nauplii, and copepodite stages. Using these values of CH4 production and copepod densities, we obtain potential rates of CH4 production by T. longicornis of 0.2-1.0 nmol liter-' d-1. Upper water-column CH4 maxima are frequently on the order of 1-2 nmol liter-I above ambient levels. Diffusion of CH4 against such gradients is relatively slow-on the order of 0. 1-0. 3 nmol cm- I d- I (Scranton and Brew- er 1977; Ward 1992). Additional studies of more zooplankton species are required to determine more accurately the contribution of this mechanism to upper water-column dissolved CH4 levels. Other microniches for methanogenesis, including fish guts (Oremland 1979) and particles (including fecal pellets, Marty 1993), may also exist in the upper water column. However, the results of this study in-




dicate CH4 production associated with zooplankton grazing of phytoplankton can occur at rates high enough to make significant con- tributions to the formation and maintenance of oceanic subsurface CH4 maxima.

We postulate three possible sites for upper water-column methanogenesis associated with zooplankton based on the results of our feeding experiments. The first site for CH4 production is within algal cells, with subsequent CH4 re- lease when the cells are grazed by zooplankton. This mechanism is similar to that proposed for dimethylsulfoniopropionate (DMSP) re- lease from phytoplankton cells (Dacey and Wakeham 1986). Some algal cultures have been observed to produce small amounts of CH4 (Scranton 1977). This mechanism is not likely to be operating in this case, however, since CH4 production in the feeding experiments should have been independent of the grazing copepod species (because any grazing or mechanism breaking up the algal cells should have released any CH4 present). In fact, CH4 production was found to be zooplankton species- specific.

The second site for CH4 production is within copepod fecal pellets. Fecal pellets are the right size and composition to promote formation of anoxic microniches within which methanogens might be active. Marty (1993) presented evidence that methanogens, most likely orig- inating from the gut of the copepod, are associated with freshly produced copepod fecal pellets. Some CH4 production may have arisen from fecal pellets, but for two reasons we do not believe this mechanism is the main source of the CH4 produced in our feeding experiments. First, anoxic conditions are most likely to arise in larger fecal pellets, and we observed CH4 production with the copepod which produced pellets of intermediate size (T. longicornis) and not with the copepod which produced the largest pellets (C. pacificus). Second, we observed no correlation between CH4 production and fecal pellet formation in the experimental flasks. In all cases where CH4 production occurred, fecal pellet formation was maximum after CH4 production had peaked or stopped altogether.

The third site for methane production is within the digestive tracts of herbivorous zooplankton. We favor this explanation for the CH4 production we observed. Reducing con-

ditions, gut passage times, and other physiological variables are some of the factors that are likely to determine whether zooplankton guts can support active methanogens. These factors are likely to differ among zooplankton species. Therefore, zooplankton species-dependent CH4 production might well be expected, as we observed.

Methylamines as substrates for CH4 production -Another goal of our investigation was to examine methylamines (TMA, DMA, MMA) as possible precursors to CH4 produced during herbivorous zooplankton grazing. Methyl- amines are breakdown products of glycine be- taine and other quatemary amines that func- tion as osmoregulators in a variety of marine phytoplankton and plants (Kneifel 1979). TMA is also reported to be common in marine algae (Steiner and Hartmann 1968) and has been widely reported as a substrate for methanogenesis in various marine environments (Or- emland and Polcin 1982). Initial studies (C. Lee and T. Cowles unpubl. data) identified DMA as the major methylamine excreted by zooplankton. Because DMA is not found in large amounts in algae (Table 3), this methylamine may arise from the demethylation of TMA. This demethylation process could also result in production of CH4.

Methylamines have been specifically identified as substrates for methanogens isolated from samples of upper water-column zooplankton (Cynar and Yayanos 1991; Marty 1993). Indirect evidence for the presence of sufficient algal methylamines to support growth of methanogens was provided by Sieburth and Keller (1991); they found MMA-oxidizing bacteria to be associated with specific algal cultures. However, it is not known whether ingested phytoplankton can supply adequate methylamines to support CH4 production associated with zooplankton grazing. No com- prehensive quantitative assessment of the methylamine content of marine algal species has been carried out similar to the detailed analysis of algal DMS by Keller et al. (1989).

From the methylamine contents of the phytoplankton (Table 3) and the number of cells grazed, it is possible to estimate the maximum amount of methylamines available for conversion to CH4 in each of the feeding experiments. The available methylamines can then be compared to the amount of CH4 produced




in each experiment. The molar ratio of CH4 produced to maximum available TMA or MMA from ingested algal cells is presented in Table 2. The CH4 production ranged from 4 to 12% of available TMA and from 20 to 130% of available MMA. The ratio of CH4 to TMA in feeding experiments involving three different species of phytoplankton was similar even though the phytoplankton had different TMA contents. Thus, CH4 production associated with copepods was correlated with TMA availability in these experiments. Although these experiments do not prove that methylamines were being used as precursors for CH4 produced in association with zooplankton feeding on phytoplankton, they clearly demonstrate that sufficient TMA was present to account for the observed CH4 production in the experimental flasks.

The ratio of CH4 produced to MMA available was much higher than for TMA and exceeded unity in the experiment with R. lens. That is, more CH4 was produced than could be accounted for by conversion of MMA. It is unlikely that 100% conversion of methylamines to CH4 would occur, and because of diffusional loss to the water column, it is unlikely that all phytoplankton methylamine ingested by the copepods in the feeding experiments would be released or made available in the gut or in fecal pellets. It is also probable that heterotrophic bacteria consumed some of the methylamines. Methylamines are rapidly converted to CO2 by heterotrophs (Budd and Spencer 1968; Bicknell and Owens 1980). Therefore, it appears that MMA was not the sole methanogenic precursor, although it may have made some contribution to CH4 production during copepod feeding.

DMS has also been identified as a substrate for methanogenesis in the presence of sulfate (Kiene et al. 1986) and is produced by a wide variety of phytoplankton species (Keller et al. 1989). The production of DMS is highly species-specific for phytoplankton. For example, most dinoflagellates and coccolithophorids are major DMS producers, while in general, dia- toms and cryptomonads produce little or no DMS (Keller et al. 1989). In our experiments, CH4 production by T. longicornis was lowest when feeding on T. weissflogii, which has a relatively low DMS content. Methane production was highest for T. longicornis feeding on

P. minimum (EXUV), which produces large amounts of DMS. However, copepods feeding on R. lens, which does not produce DMS, also yielded relatively large amounts of CH4. Therefore, it appears that although DMS can- not be ruled out as a methanogenic substrate in the feeding experiments, it was not the sole precursor of CH4.

The results of our experiments and methylamine analyses of phytoplankton support the work of Cynar and Yayanos (1991) who isolated a methylamine-utilizing methanogen from zooplankton samples collected from well- oxygenated coastal waters. These results are also consistent with the report by Marty (1993) of methylotrophic methanogens associated with copepods and their fecal pellets. Although TMA and other methylamines have been identified by these workers as substrates for upper water-column methanogens in zooplankton samples, our study demonstrates that sufficient TMA is available from zooplankton grazing on phytoplankton to account for the levels of CH4 produced by this mechanism in seawater.

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Submitted: 14 July 1993 Accepted: 24 January 1994

Amended: 23 February 1994



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Methane Production During Zooplankton Grazing on Marine Phytoplankton