Appl Environ Microbiol 65(7): 3148-3157

PMC: PMC91469

PMID: 10388716

Nitrogen, Carbon, and Sulfur Metabolism in Natural <em>Thioploca</em> Samples

MATERIALS AND METHODS

Samples were collected in January and February 1997 on the continental shelf within the Bay of Concepción, central Chile, onboard the research vessel Kay Kay, and the laboratory work was performed at the Marine Biological Station of Dichato, both of the University of Concepción. Sampling was performed at 34 m of water depth at station 7 (32), at 36°36′5"S, 73°00′6"W. At this station, at the time of sampling, the percentage of organisms in the upper 2 cm of the sediment found to be T. araucae was 39 to 67%. The ratio of the biovolumes of T. araucae and T. chileae is approximately 70:30, and therefore, the majority of the community consisted, in biovolume, of T. araucae.

Collection.

Sediment samples were obtained by a Rumohr corer with Plexiglas cores (inside diameter, 9.5 cm), stored at 4°C, and processed within 8 days of sampling. The top 1 to 2 cm was removed and placed on ice in an N₂-filled glove bag (Sigma-Aldrich, Zwÿndrecht, The Netherlands). Thioploca sheaths with bundles of trichomes (1 to 2 cm in length) were collected with forceps and transferred to synthetic medium (containing [per liter] 25 g of NaCl, 6 g of MgSO₄ · 7H₂O, 1 g of CaCl₂, 0.5 g of K₂HPO₄, 0.1 g of KH₂PO₄, and 0.5 g of NaHCO₃ [pH 7]) or to synthetic medium without NaHCO₃, supplemented with 0.05% (wt/vol) thioglycolate and 1 mg of catalase per liter (Sigma-Aldrich). The latter medium (hereafter referred to as medium, unless stated otherwise) was found to give the best results. Media were sparged with N₂ for 30 min for anoxic conditions and subsequently stored at 4°C. Shortly before use, media were sparged again with N₂ for 10 min, unless stated otherwise, while kept cold. Under an N₂ atmosphere in a glove bag, the sheaths with trichome bundles were washed twice by transfer into fresh medium and were incubated in airtight 6-ml vials equipped with rubber septa (Exetainer; Labco, High Wycombe, United Kingdom). The medium in the vial was changed twice by decanting under an N₂ atmosphere, with minimal disturbance of the sheaths and trichome bundles. The incubation volume was adapted to the conditions of the experiment performed. Under anoxic conditions, substrates were injected through the rubber septum. Residual sediment, left after washing Thioploca trichome bundles, was used as a control. In these controls, the amount of sediment used was 5 to 10 times higher than the estimated contamination of sediment attached to washed sheaths with trichome bundles. Another control consisted of disrupted Thioploca sheaths with trichome bundles (equal to the amount used in the experiment) which had been disintegrated mechanically by a Potter-Elvehjem homogenizer (Fisher Scientific, Zoetermeer, The Netherlands). This control was necessary because observations under the fluorescence microscope had shown that the sheaths were covered with epibiontic bacteria, including sulfate-reducing filamentous bacteria of the genus Desulfonema (36, 40). After the Potter-Elvehjem treatment, it was observed under the fluorescent microscope that Thioploca trichomes were mechanically disrupted, while the majority of the epibiontic bacteria remained intact. Bundles consisting of sheaths with bundles of trichomes will hereafter in this paper be referred to as “trichome bundles,” while sheaths with Potter-Elvehjem-treated bundles will hereafter be referred to as “disrupted trichome bundles.”

The method of handpicking Thioploca sheaths with trichome bundles had a bias towards T. araucae, which led to a majority (80 to 90% in biovolume) of this species in the samples.

Incubation.

For each experiment, approximately 100 Thioploca trichome bundles were collected in a final volume of 3.5 ml under an N₂ atmosphere in gas-tight vials, unless stated otherwise. Vials were incubated with substrates in a water bath at approximately 12°C. At specific time intervals, samples were taken with a syringe previously flushed with dinitrogen and analyzed for ammonium, nitrite, sulfide, thiosulfate, and sulfate.

Analytical procedures.

Nitrite and ammonium in the supernatant were determined colorimetrically (as described in references 14 and 5, respectively). Intracellular nitrate concentrations were measured with a miniaturized version of the standard colorimetric method of Grasshoff et al. (13). Nitrate was measured in 100-μl extracts of rinsed and dried Thioploca trichomes. Trichomes 5 to 40 mm in length were dissected under the microscope with the help of forceps and needles. Length and width of these trichomes were measured and, after washing and drying, the filaments were resuspended in 50 μl of distilled water to measure the nitrate concentration. Biovolume was calculated from trichome length and width. An average nitrate concentration (n = 27) of 160 ± 150 mM was found. Protein was determined by the microbiuret method of Goa (11). The observed protein concentrations were in agreement with calculations for expected protein content. Where no protein measurements were available (in experiments where labeled compounds were used), a protein content of 315 ± 95 μg was assumed for 100 Thioploca sheaths with trichomes (which is based on an average of 34 protein measurements of Thioploca samples). Thiosulfate was derivatized with monobromobimane (4) and analyzed by reversed-phase high-performance liquid chromatography (28). Sulfide was determined either colorimetrically according to the method of Cline (2) or by the method described for thiosulfate determination. Standards for sulfide and thiosulfate were prepared in degassed sulfate-free medium (MgCl₂ instead of MgSO₄ and without thioglycolate and catalase). Sulfate was determined by nonsuppressed ion chromatography as described by Ferdelman et al. (6). Since high concentrations of chloride interfere with sulfate analysis by ion chromatography, chloride was removed from the samples by adding 40 mg of Ag^{-loaded cation exchange (Ag 50W-X8; Bio-Rad) per 150-μl sample and incubating for 2 h at room temperature. After centrifugation and filtering, the sample was analyzed. Standards were treated in the same way.}

^{N experiments and mass spectrometry.}

Under anoxic conditions, a concentrated solution of Na^NO₃^{was added to gas-tight vials, each with 120}Thioploca bundles in 4.5 ml of medium. No direct protein measurements could be performed and, therefore, a total protein content of 378 ± 114 μg was assumed on the basis of the average protein content of 100 Thioploca bundles (see above). The headspace was changed with He, and at certain time intervals samples were taken for analysis. Total nitrite and ammonium concentrations were measured immediately after centrifugation. To determine the concentration of ^NO₃^,^NO₂^{, and}^NH₄^{, samples were removed with a syringe, centrifuged, sterilized by passing the supernatant through a 0.2-μm-pore-size filter (Dynagard; Microgon Inc., Laguna Hills, Calif.), acidified to pH 4 to 5, and stored at −20°C until the time of analysis. At the end of the experiment, 1 to 2% (final concentration) formaldehyde was added to the vial and pressure was equilibrated with He. Vials were stored at 4°C until the headspace could be analyzed for}^N₂. Analysis and mass spectrometry were performed at the Institute of Biological Sciences, University of Aarhus, Aarhus, Denmark. For determining concentrations and isotopic compositions of ^NO₃^and^NO₂^{, samples were neutralized and incubated with denitrifying bacteria to convert these compounds to N}₂ for mass spectrometry analysis (29). The labeling pattern of the obtained N₂ gives an indication of the ratios of labeled and unlabeled nitrate and nitrite present in the samples. However, the denitrifiers used can reduce both nitrate and nitrite. Therefore, the recovered N₂ is the product of the reduction of nitrate as well as nitrite present in the medium. Standards with known concentrations (120 μM) of ^NO₃^and^NO₂^{were included in the assay and confirmed that the conversion efficiency was consistent (standard deviation = 4%) and that residual nitrate and nitrite concentrations were insignificant.}

To analyze the isotopic composition of the NH₄^{formed, hypobromite was added for specific oxidation of ammonium to N}₂ (30). The mass spectrometer measured singly and doubly labeled dinitrogen (^N₂ and ^N₂) in excess of the natural background. From this, the recovery of added ^{N in the sampled N}₂, NO₃^{, NO}₂^{, and NH}₄^{was calculated. Dinitrogen is formed by random isotope pairing and, therefore, the ratio of labeled nitrogen recovered as}^N₂ versus the recovery as ^N₂ was used as a minimum estimate of the ^N:^{N ratio in the source (}26). If the source is isotopically uniform and constant, the estimate is correct. If several pools are involved, i.e., discrete intracellular nitrate pools in the incubation vial or nitrate and nitrite in the water samples, the true representation of unlabeled nitrogen cannot be much (at the most, 0.5 nmol) less but can be higher, depending on the pool sizes and isotopic variations.

NaH^CO₃, [2-^{C]acetate, and [}^{H]acetate incorporation experiments.}

Several vials were incubated with approximately 30 Thioploca bundles in 1.3 ml of medium, in the dark. On the basis of the average protein content of 100 Thioploca bundles (see above), the total protein for 30 bundles was assumed to be 94.5 ± 28.5 μg. One hundred micromolar NaNO₃^{, 25 μl of CO}₂ gas (headspace was approximately 5 ml), and, for all experiments, 1 mM HCO₃^{were added to the medium. Under anaerobiosis, 0.005 nmol of [}^{H]acetate (∼500 μCi) or 0.034 μmol of a [}^{C]acetate solution (∼5 μCi) was added for the labeled-acetate experiments. For the labeled-bicarbonate experiments, 0.139 μmol (∼10 μCi) or 1.85 μmol of a neutralized NaH}^CO₃ solution (∼100 μCi for microautoradiography experiments) was added. Experiments with [^{C]bicarbonate were performed in the absence as well as in the presence of approximately 70 μM sulfide. At certain time intervals, a vial was opened and the supernatant was analyzed for NO}₂^{and NH}₄^{. The pellet of}Thioploca bundles or debris from disrupted bundles was washed four times (by vigorous mixing and subsequent centrifugation) in medium containing 10 mM acetate (when incubated with labeled acetate) or containing 10% trichloroacetic acid (when incubated with NaH^CO₃) and then added to 2.4 ml of H₂O and 7.5 ml of scintillation liquid (EcoLite [+]; ICN Biomedicals). This suspension was subsequently analyzed in a scintillation counter (Packard liquid scintillation analyzer model 1600 TR). When necessary, CO₂ was trapped by suspending a small cup filled with 100 μl of 2 M NaOH in the gas-tight vial. After the vial was opened, this solution was neutralized and added to scintillation liquid and counted. Experiments with [^{C]acetate took 3 h, experiments with NaH}^CO₃ required 4 h of incubation (to test the influence of oxygen, trichomes were incubated for 22 h), and trichomes used for microautoradiography were incubated for 4 h or 20 to 22 h.

Microautoradiography.

Microautoradiography was performed on the experiments described above. Following incubation with [^{H]acetate or with NaH}^CO₃, bundles were washed six times with medium containing 1 mM acetate or 1 mM HCO₃^{, respectively. Individual sheaths were then sorted onto 25-mm hydroxyapatite Millipore filters (Millipore Corp., Bedford, Mass.) or left in 2 ml of medium containing 2% formaldehyde. Filters were subsequently washed in filter-sterilized (first through 0.45-μm-pore-size then through 0.2-μm-pore-size Gelman filters [Millipore]) medium with 1 mM phosphate buffer. After drying, the filters were stored at 4°C. At the end of the cruise, the filters and samples, stored in 2% formaldehyde, were analyzed. Some filters were stained with 2% (wt/vol) erythrosin-B (Sigma-Aldrich) and were subsequently destained by placing them face up on deionized H}₂O-saturated pieces of gauze. Filters were air dried, attached to microscope slides, and optically cleared by fuming acetone (27). Cleared filters were prepared for microautoradiography by dipping in Kodak NTB-2 nuclear track emulsion. After exposure (1 to 3 weeks), autoradiographs were developed (Kodak D-19 developer), fixed, rinsed, and air dried prior to microscopic examination with a Nikon Labophot 2 phase-contrast microscope at ×200 to ×400 magnification. Photographs were recorded on either Ilford Pan-F fine-grain black-and-white or Kodachrome 200 color slide 35 mm film.

Calculations. (i) ^{N experiments: the ratio between added [}^{N]nitrate and intravacuolar [}^N]nitrate.

Addition of 100 μM [^{N]nitrate in 4.5 ml of medium yields 0.45 μmol of [}^{N]nitrate. One hundred twenty}Thioploca bundles have a protein content of approximately 0.38 ± 0.11 mg (see analytical procedures). Assuming that 50% of the dry weight is protein, and 24% of the wet weight is dry weight and knowing that 90% of the cell is vacuole (as measured in this study and by Maier et al. [20]), then the total wet weight of 120 bundles is 0.38 × 2 × [100/24] × [100/10] = 31.7 ± 9.17 mg, of which 28.5 ± 8.25 mg is vacuolar liquid. Assuming that 1 mg is equal to 1 μl of liquid in the vacuole, then the volume of all vacuoles in the bundles used for the experiments will be approximately 28.5 ± 8.25 μl. If all the added [^{N]nitrate is transported into the vacuoles, then this would lead to a concentration of the label of 15.8 ± 4.57 mM (0.45 μmol in 28.5 μl). Since the vacuoles are filled with an average of 160 mM (see Materials and Methods) unlabeled nitrate, the labeled nitrate will be diluted to 9.9% ± 2.85%. If}Thioploca trichome bundles were damaged and all internal nitrate were released, then approximately 5.6 μmol (160 mM in 28.5 μl) would be released into 4.5 ml of medium. This would lead to an increase in nitrate concentration of 1.2 mM, i.e., a 12-fold increase, which would be visible during measurement of the ^N:^{N ratio of the external nitrate pool.}

(ii) Sulfide oxidation: the ratio between observed sulfide reoxidation rates and specific activity of Thioploca.

Sulfate reduction rates measured in sediments at station 7 at the time of sampling were approximately 30 mmol m^day^{(26 to 37 mmol m}^day^[34]). If all sulfide produced from this reduction was subsequently oxidized by the Thioploca mats, then the mats should be able to oxidize sulfide at the same rate, which is equal to 20.8 μmol m^min^{. Schulz et al. (}32) estimated the wet biomass of trichomes without sheaths to be 50 to 120 g m^{. Assuming an average of 85 g (wet weight) m}^{and knowing that 90% of the biovolume is taken up by the central vacuole, the active cytoplasm weighs approximately 8.5 g (wet weight) m}^{. This active cytoplasm is then responsible for the sulfide oxidation rate as stated above, which would give a specific rate of 20.8/8.5 = 2.4 μmol min}^{g of wet weight}^{. Assuming that 24% of the wet weight is dry weight and that 50% of the dry weight is protein, the sulfide oxidation rate in vivo should be 2.4 × (100/24) × 2 = 20.4 nmol min}^{mg of protein}^{. In analogy, Ferdelman et al. (}6) found an in vivo sulfate reduction rate of approximately 17.5 mmol m^day^{for station 7. This reduction rate corresponds to a sulfide oxidation rate by}Thioploca of 12 μmol m^min^{. Making the same assumptions as above, this oxidation rate is equal to 11.8 nmol min}^{mg of protein}^.

Collection.

The method of handpicking Thioploca sheaths with trichome bundles had a bias towards T. araucae, which led to a majority (80 to 90% in biovolume) of this species in the samples.

Incubation.

Analytical procedures.

^{N experiments and mass spectrometry.}

NaH^CO₃, [2-^{C]acetate, and [}^{H]acetate incorporation experiments.}

Microautoradiography.

Calculations. (i) ^{N experiments: the ratio between added [}^{N]nitrate and intravacuolar [}^N]nitrate.

(ii) Sulfide oxidation: the ratio between observed sulfide reoxidation rates and specific activity of Thioploca.

RESULTS

Cultivation and survival.

Thioploca bundles, consisting of 10 to 20 trichomes in a sheath, were collected from the top 2 cm of the sediment with forceps and cleaned by several transfers through medium. In developing the method, there were three parameters to be considered. Firstly, motility of Thioploca trichomes under a microscope was a measure of viability (19, 31). Secondly, it was observed during the initial experiments that high nitrite concentrations (50 to 100 μM), in addition to high nitrate concentrations, were obtained within 1 h of anoxic incubation of the bundles, suggesting lysis of the cells. Thirdly, in previous studies of Thioploca and large Beggiatoa species, it was observed that these organisms are highly sensitive to oxygen (16, 22) and that catalase is required in the growth medium of Beggiatoa species (1). These considerations led to several improvements of the final cleaning procedure. To avoid contact with oxygen, all steps of the method (collection, washing, and incubation) were performed under a dinitrogen atmosphere. Sparging of the medium during incubation for anoxic conditions was avoided, because this mechanically affected the trichomes. To keep the growth medium anoxic, thioglycolate was added as a reducing agent and catalase was also included in the synthetic medium. The endogenous ammonium production rate did not increase after thioglycolate was included in the medium, suggesting that this compound was not used as a carbon source. Survival experiments, as monitored under the microscope (31), showed that the medium highly improved survival and that trichomes did not show a decrease in motility over 2 days of incubation. From similar survival experiments, it was further concluded that Thioploca cells could get damaged when transferred through a liquid-gas interface. To avoid this damage, washing was performed twice by draining off approximately 80% of the medium, such that the trichomes were still in the liquid, and then fresh medium was added.

Further improvement of the method was obtained by keeping the sediment on ice during collection and by washing the trichomes and omitting bicarbonate in the medium, since removal of CO₂ during sparging of the medium caused an increase in pH. In all subsequent experiments these cleaned Thioploca trichomes were used.

N metabolism.

Inside Thioploca cells, intravacuolar nitrate concentrations measured up to 500 mM, with an average of 160 ± 150 mM (n = 27). Thioploca trichome bundles, incubated in medium without addition of NO₃^{, produced NO}₂^{and NH}₄^{. The average NH}₄^{production (with internal sulfur available but no added external electron donor) of eight independent measurements was 1.0 ± 0.3 nmol min}^{mg of protein}^{, whereas NH}₄^{production by the controls (disrupted trichome bundles or NO}₃^{-supplemented sediment) was 0.07 ± 0.03 nmol min}^{mg of protein}^{and 0.03 ± 0.01 nmol min}^{mg of protein}^{, respectively. Nitrite production was negligible (<0.1 nmol min}^{mg of protein}^{) in most experiments but was sometimes observed at a maximum production rate of 1 nmol min}^{mg of protein}^.

^{N-labeling experiments.}

To determine whether Thioploca reduces NO₃^{to NH}₄^{or to N}₂, experiments were performed by using ^NO₃^{. After addition of}^NO₃^{, total NO}₂^{and total NH}₄^{, as well as}^NO₃^/^NO₂^and^NH₄^{, were monitored over time (the}^{N-labeling method does not differentiate between labeled NO}₃^{and NO}₂^{; see Materials and Methods). At the end of the experiment, total}^N₂ and the ratio between unlabeled and (singly or doubly) labeled N₂ were determined. Figure Figure1A1A shows that 95% of the externally available NO₃^{or NO}₂^{originated from the supplied}^NO₃^{. During the course of the experiment, the specific labeling of the extracellular nitrate pool remained 95%, indicating that the trichomes were not damaged and did not release}^NO₃^{(see calculations in Materials and Methods). Figure}Figure1A1A shows that nearly all of the nitrate was taken up linearly during the course of the experiment in approximately 3.5 h. As calculated in Materials and Methods, if all of the ^NO₃^{were taken up by}Thioploca trichomes, the label would be diluted inside the vacuoles to 9.9% ± 2.85% (see calculations in Materials and Methods). The NH₄^{produced during the experiment (1.8 ± 0.4 nmol min}^{mg of protein}^{) was 44% (at 85 min) and 48% (at 145 and 215 min) labeled (Fig.}(Fig.1B).1B). This difference in specific labeling between the externally available nitrate pool and the NH₄^{produced indicates that the internal nitrate of}Thioploca trichomes contributes substantially to the total NH₄^{production. However, the amount of label is not diluted as much as would be expected if all the labeled nitrate were first taken up in the vacuole and subsequently reduced. Therefore, it seems that in the cytoplasm the [}^{N]nitrate is readily reduced before it reaches the vacuole. Figure}Figure1C1C shows that at the end of the experiment N₂ had also been produced, but the amount was only 15% (nanomoles of nitrogen per nanomole of nitrogen) of the total amount of nitrogen compounds produced. The specific labeling of the N₂ was substantially higher than that of NH₄^{, suggesting that epibionts might be responsible for this production, although the amount of unlabeled N}₂ is a minimum estimate (see Materials and Methods). This implies that, although N₂ appeared to not be a major product of the washed Thioploca sample, the present data cannot completely rule out that Thioploca can reduce nitrate to N₂, i.e., denitrify, in addition to the observed full reduction of nitrate to ammonium.

An external file that holds a picture, illustration, etc.
Object name is am0791832001.jpg

FIG. 1

Distribution of label during ammonium and dinitrogen production by Thioploca trichome bundles after incubation in medium with [^{N]nitrate under a helium headspace. (A)}^{N- and}^{N-labeled nitrate and nitrite in the growth medium; (B)}^{N- and}^{N-labeled ammonium in the growth medium; (C) total amount of}^{N and}^{N derived from dinitrogen species in the headspace (}^N₂, ^N₂, ^N₂, ^N₂). Symbols: ▴, total ammonium measured colorimetrically; ■, nitrite measured colorimetrically.

Sulfur metabolism.

To measure sulfide oxidation rates, Thioploca trichomes were incubated in medium. After addition of approximately 50 μM sulfide to the vials, sulfide, nitrite, and ammonium concentrations were observed over time. An ammonium production rate by the Thioploca suspension of approximately 1.9 nmol min^{mg of protein}^{was observed, whereas the controls (sediment samples and samples treated in a Potter-Elvehjem homogenizer) showed activities of 0.02 and 0.04 nmol of NH}₄^min^{mg of protein}^{, respectively (Table}(Table1).1). The sulfide consumption rate decreased with decreasing sulfide concentrations, but the average maximum rate was approximately 4.2 nmol min^{mg of protein}^{, while the controls showed a 10- to 200-fold lower consumption rate. Since the control, which contained sediment, represented an overestimate of the amount of sediment attached to the trichomes, the contribution of the sediment to the total activity in live}Thioploca experiments was negligible. Trichomes, which had been collected and incubated 2 days before the experiment was performed, showed continued motility under the microscope, an ammonium production rate of 3.2 nmol min^{mg of protein}^{, and a sulfide consumption rate of 5.5 nmol min}^{mg of protein}^{, after addition of sulfide to the incubation medium. The cells appear to reduce their metabolic activity when no external substrate is present (NH}₄^{production rate is approximately 1 nmol min}^{mg of protein}^{) but are able to respond quickly when substrate is encountered again (NH}₄^{production increases to 3.2 nmol min}^{mg of protein}^{). An internal nitrate concentration of 160 mM would be sufficient for approximately 200 h (given our estimate that 90% of the cell is vacuole and that 1 mg of vacuolar liquid is equal to 1 μl), with an NH}₄^{production rate of ± 1 nmol min}^{mg of protein}^{. This experiment indicates that trichomes are still active and motile after 2 days and that the internal NO}₃^{is sufficient for at least 2 days of normal metabolism without external supply of fresh substrate. In an experiment where two different concentrations of sulfide were added to}Thioploca suspensions (Fig. (Fig.2),2), a small accumulation of thiosulfate, which was higher when the sulfide concentration was higher, was observed. This thiosulfate accumulation suggested that this compound may be a by-product or an intermediate in sulfide oxidation, and therefore, Thioploca might be able to oxidize thiosulfate to sulfate. To investigate this possibility, approximately 100 μM thiosulfate was added to Thioploca trichome bundles incubated in sulfate-free medium with MgCl₂ instead of MgSO₄ and without thioglycolate and catalase, to avoid interference with analytical measurements. In these experiments (results not shown) only a slight thiosulfate consumption was observed.

TABLE 1

Specific rates of ammonium production and sulfide consumption by Thioploca trichrome bundles incubated in medium

Sample	NH₄^{production rate (nmol min}^{mg of protein}⁾	HS^{consumption rate (nmol min}^{mg of protein}⁾
Thioploca sheaths with trichomes	1.9	4.2
Thioploca sheaths with trichomes (2-day-old culture)	3.2	5.5
Disrupted Thioploca sheaths with trichomes	0.04	<0.02
Sediment (with 100 μM NO₃⁾	0.02	0.5

An external file that holds a picture, illustration, etc.
Object name is am0791832002.jpg

FIG. 2

Thiosulfate production by Thioploca trichome bundles incubated anoxically in medium with two different initial sulfide concentrations. Open symbols, 100 μM initial sulfide concentration; closed symbols, 400 μM initial sulfide concentration. Symbols: triangle, ammonium; circle, thiosulfate; diamond, sulfide.

Freshly harvested cells contain high concentrations of elemental sulfur (200 nmol mm^{) and nitrate (160 mM), which could influence the observed oxidation rates. In line with the calculations made above for consumption of internal nitrate during starvation, it can be calculated that the internal sulfur would be sufficient for approximately 170 h. Thus, it was possible that partially starved cells would show higher oxidation rates. Therefore, suspensions of 50}Thioploca trichome bundles were sulfur starved for 45 h by incubation in 4.5 ml of sulfate-free medium in the presence of 50 μM nitrate. After 45 h, approximately 135 μM sulfide or 70 μM thiosulfate was added to the suspensions and ammonium, sulfide, thiosulfate, and sulfate concentrations were monitored over time (results not shown). During anoxic sulfur starvation, productions of NH₄^{and SO}₄^byThioploca trichome bundles were approximately 1 and 2 to 3 nmol min^{mg of protein}^{, respectively. In the control with disrupted trichome bundles, NH}₄^{and SO}₄^{production levels were 0.1 and 0.45 nmol min}^{mg of protein}^{, respectively. This activity was ten times lower than the activity of the trichome bundles, indicating that epibiontic bacteria are not responsible for the observed sulfate production. Subsequent sulfide addition (135 μM) to these sulfur-starved trichome bundles led to an initial sulfide consumption rate of 10.7 nmol min}^{mg of protein}^{, which was the largst oxidation rate observed, while ammonium and sulfate production did not increase significantly. There was no production of sulfite during these experiments; however, accumulation of thiosulfate was observed in the disrupted control (up to 21 μM) during starvation. This control contained sulfur compounds released from the ruptured}Thioploca trichome bundles, suggesting that epibiontic bacteria may be responsible for thiosulfate accumulation. After addition of sulfide to intact, starved trichome bundles, thiosulfate accumulation also occurred (up to 10 μM). After addition of thiosulfate to sulfur-starved Thioploca trichome bundles, only a low thiosulfate consumption rate was measured, which was equal to the rate observed previously. Measurements of this consumption rate also showed high variability. Addition of thiosulfate had no effect on ammonium or sulfate production.

Carbon metabolism.

In order to gain some insight into the carbon source used by Thioploca for its cell material, experiments were performed with radioactively labeled acetate and bicarbonate additions (Table (Table2)2) in combination with microautoradiography.

TABLE 2

Specific rates of NH₄^{production and}^{C incorporation by}Thioploca trichome bundles incubated in medium after addition of [^{C]acetate or NaH}^CO₃

Sample	NH₄^{production rate (nmol min}^{mg of protein}⁾	^{C incorporation rate (nmol min}^{mg of protein}⁾
Thioploca trichomes + ∼5 μCi of [^C]acetate	4.0	0.37
Thioploca trichomes + ∼10 μCi of NaH^CO₃	1.3	0.5–0.8
Thioploca trichomes + ∼10 μCi of NaH^CO₃ + 70 μM Na₂S	1.7	0.3–0.5
Thioploca trichomes + ∼100 μCi of NaH^CO₃	1.2	0.25–0.35
Thioploca trichomes + ∼100 μCi of NaH^CO₃ + 2% O₂	0.65	0.36–0.48
Disrupted trichomes + ∼3 μCi of [^C]acetate	<0.01	<0.01
Disrupted trichomes + ∼100 μCi of NaH^CO₃	<0.01	0.01

(i) NaH^CO₃.

Addition of NaH^CO₃ to a Thioploca suspension resulted in a linear incorporation rate of 0.4 to 0.8 nmol min^{mg of protein}^{and an NH}₄^{production rate of approximately 1.3 to 1.7 nmol min}^{mg of protein}^{. Addition of sulfide (ca. 70 μM) did not have a significant effect on the incorporation rate. The presence of low concentrations of oxygen (ca. 10% air saturation) also did not have a significant effect on the rates of NaH}^CO₃ fixation. Control experiments with disrupted trichome bundles showed a ^{C fixation rate of only 0.01 nmol min}^{mg of protein}^{, which is 1 to 3% of the rates observed in intact}Thioploca suspensions. Intact bundles obtained from these experiments were used for microautoradiography. Uptake of ^{C appeared to be largely dominated by}Thioploca trichome bundles, since there was no significant uptake of label in epibiontic microbial cells associated with the sheaths. Microautoradiography and control experiments with disrupted trichome bundles both show that the measured uptake rate of ^{C by the}Thioploca suspension primarily represents the activity of the trichome bundles and not of epibionts. Differences in intensity of labeling among trichomes were observed, but no differences were observed that could be due to possible differences in the physiology of the two major species in the sample (T. araucae and T. chileae). Among individual trichomes, labeling was homogeneously distributed along their entire length, and labeling was concentrated along the transverse walls (Fig. (Fig.3),3), suggesting the presence of a vacuole.

An external file that holds a picture, illustration, etc.
Object name is am0791832003.jpg

FIG. 3

High-magnification microautoradiogram of a single Thioploca trichome incubated with NaH^CO₃.

(ii) [^{C]- and [}^H]acetate.

After addition of [^{C]acetate to}Thioploca trichome bundles, an acetate uptake rate of approximately 0.4 nmol min^{mg of protein}^{and an NH}₄^{production rate of 4 nmol min}^{mg of protein}^{were observed. Unaccounted loss of label was less than 10%.}^CO₂ production was not significant (less than 2% of acetate incorporation), indicating that under these conditions (i.e., in the presence of internal sulfur) acetate was not used as a significant energy source. Control experiments with disrupted trichome bundles showed an incorporation rate of less than 0.01 nmol min^{mg of protein}^{, which was less than 2% of the activity of intact}Thioploca trichome bundles.

Bundles incubated with [^{H]acetate and showing similar activities, as described above, were examined by microautoradiography. Results indicate acetate uptake by trichomes as well as by bacteria associated with the sheath (Fig.}(Fig.4).4). However, taking into account the volume ratio between trichomes and attached bacteria, uptake of label was largely dominated by Thioploca trichomes. This indicated that measured uptake rates of label were primarily due to Thioploca trichomes. No differences were observed between the two species of Thioploca present. Labeling with the soft β emitter ^{H gives a higher resolution than labeling with}^{C and, therefore, more clearly shows the difference between uptake of label by epibionts and by}Thioploca trichomes. Figure Figure55 shows, more clearly than with ^{C label, that the}^{H label is situated along the transverse cell walls. This reflects the presence of a large central vacuole, leaving the cytoplasm concentrated along the cell walls. The results with}^{H labeling showed uniformity in cell to cell labeling along the entire length of a trichome, as described above for}^{C labeling, as well as differences in cellular labeling between individual trichomes. Uniform trichome labeling was observed immediately after addition of the labeled acetate and increased in intensity with time, indicating accumulation of label, reflecting measured uptake rates.}

An external file that holds a picture, illustration, etc.
Object name is am0791832004.jpg

FIG. 4

Low-magnification microautoradiogram of a Thioploca sheath with trichomes incubated with [^{H]acetate, showing}^{H uptake by trichomes and associated bacteria. The heavily labeled trichomes are out of focus to show uptake by bacteria situated on the sheath. This image has been selected for its high concentration of epibionts and is not representative of the overall results from the microautoradiography.}

An external file that holds a picture, illustration, etc.
Object name is am0791832005.jpg

FIG. 5

High-magnification microautoradiogram of Thioploca trichome incubated with [^H]acetate.

Cultivation and survival.

N metabolism.

^{N-labeling experiments.}

FIG. 1

Sulfur metabolism.

TABLE 1

Specific rates of ammonium production and sulfide consumption by Thioploca trichrome bundles incubated in medium

Sample	NH₄^{production rate (nmol min}^{mg of protein}⁾	HS^{consumption rate (nmol min}^{mg of protein}⁾
Thioploca sheaths with trichomes	1.9	4.2
Thioploca sheaths with trichomes (2-day-old culture)	3.2	5.5
Disrupted Thioploca sheaths with trichomes	0.04	<0.02
Sediment (with 100 μM NO₃⁾	0.02	0.5

FIG. 2

Carbon metabolism.

TABLE 2

Specific rates of NH₄^{production and}^{C incorporation by}Thioploca trichome bundles incubated in medium after addition of [^{C]acetate or NaH}^CO₃

Sample	NH₄^{production rate (nmol min}^{mg of protein}⁾	^{C incorporation rate (nmol min}^{mg of protein}⁾
Thioploca trichomes + ∼5 μCi of [^C]acetate	4.0	0.37
Thioploca trichomes + ∼10 μCi of NaH^CO₃	1.3	0.5–0.8
Thioploca trichomes + ∼10 μCi of NaH^CO₃ + 70 μM Na₂S	1.7	0.3–0.5
Thioploca trichomes + ∼100 μCi of NaH^CO₃	1.2	0.25–0.35
Thioploca trichomes + ∼100 μCi of NaH^CO₃ + 2% O₂	0.65	0.36–0.48
Disrupted trichomes + ∼3 μCi of [^C]acetate	<0.01	<0.01
Disrupted trichomes + ∼100 μCi of NaH^CO₃	<0.01	0.01

(i) NaH^CO₃.

FIG. 3

High-magnification microautoradiogram of a single Thioploca trichome incubated with NaH^CO₃.

(ii) [^{C]- and [}^H]acetate.

FIG. 4

FIG. 5

High-magnification microautoradiogram of Thioploca trichome incubated with [^H]acetate.

DISCUSSION

Filamentous sulfur bacteria of the genus Thioploca occur along the continental shelf off the coast of Chile and Peru. High sulfate reduction rates in Thioploca mats have been reported (6). Thioploca species are able to store internally high concentrations of sulfur globules and nitrate. It is assumed that these vacuolated Thioploca species use their internally stored nitrate as a terminal electron acceptor for sulfide and sulfur oxidation (7). The product of nitrate reduction, however, was still unknown. Also, Thioploca trichome bundles have been shown to take up both CO₂ and acetate, but quantitative data were lacking (19). Therefore, this study was undertaken to investigate carbon, nitrogen, and sulfur metabolism in Thioploca species.

A method was developed to collect and clean individual sheaths with bundles of trichome bundles from the top 2 cm of the sediment. After being collected and washed under an N₂ atmosphere, trichomes were still motile and could be used for physiological experiments. In the early stages of method development, high cellular or extracellular nitrite and nitrate concentrations were observed, possibly as a result of lysis of the cells. However, after adjustments (anoxic conditions, medium supplemented with thioglycolate and catalase, low temperature, and avoiding transfer through the gas-liquid interface) these nitrite and nitrate accumulations were no longer observed. Thioploca trichome bundles incubated for 2 days still showed activity comparable to activities measured immediately after incubation (Table (Table1),1), indicating that trichome bundles were able to survive and remain physiologically intact in the synthetic medium.

Nitrogen metabolism.

During experiments performed with intact Thioploca trichome bundles, without addition of external substrate, an ammonium production rate of approximately 1 nmol min^{mg of protein}^{was observed. Since in these experiments the only available substrates were internally stored sulfur and nitrate in}Thioploca trichome bundles, it is highly unlikely that epibiontic bacteria were responsible for this NH₄^{production. Experiments using [}^{N]nitrate resulted in uptake of all the labeled nitrate in approximately 3.5 h. The specific label of the external NO}₃^{pool remained 95% and was, therefore, not diluted during the course of the experiment, indicating that trichome bundles were not damaged and leaking NO}₃^{. Analysis showed an increase in NH}₄^{production (1.8 ± 0.4 nmol min}^{mg of protein}^{) immediately after addition of labeled NO}₃^{. The specific label of NH}₄^{produced was maximally 48%. This indicates that NH}₄^{is produced from a different NO}₃^{pool than the external pool, since the external pool was more heavily labeled. The only other source of NO}₃^{is the internal NO}₃^ofThioploca trichome bundles, which is not available to epibiontic bacteria. This indicates that Thioploca species reduce NO₃^{to NH}₄^{. Another argument is that there was no electron donor for NO}₃^{reduction available in these experiments, except the internally stored sulfur.}

If all the NO₃^{were taken up by}Thioploca trichome bundles, this would lead to an increase in NO₃^{of 15.8 ± 4.57 mM within the vacuole (see calculations in Materials and Methods). Since the average NO}₃^{concentration in the vacuoles was found to be 160 mM, this would correspond to a dilution of the}^NO₃^{to a specific labeling (}^NO₃^:^NO₃^{) of 9.9% ± 2.85% (see calculations in Materials and Methods). If this NO}₃^{pool were subsequently reduced, then labeling of the NH}₄^{would be much lower than 48%. The fact that the produced NH}₄^{is more heavily labeled suggests that during transport of the labeled NO}₃^{across the membrane into the thin layer of the cytoplasm, it is readily reduced. If the transport rate of nitrate from the vacuole into the cytoplasm is in the same order of magnitude, then this would explain why the actual specific labeling of the cytoplasm is near 48%.}

N₂ was also detected in the headspace and was more heavily labeled than the NH₄^{produced. However, since the amount of unlabeled N}₂ was a minimum estimate (see Materials and Methods), the specific labeling of the produced N₂ can actually be lower than shown in Fig. Fig.1C,1C, suggesting that the produced N₂ may also have a different specific labeling than the external pool of NO₃^{. Therefore, on the basis of these data, one cannot completely exclude the possibility that}Thioploca can also reduce NO₃^{to N}₂. The amount of N₂ produced, however, was approximately 15% of the amount of NH₄^{produced, emphasizing that under the conditions tested, reduction of NO}₃^{to NH}₄^{is the preferred pathway in}Thioploca, and this pathway is probably used for energy conservation. Conservation of energy from NO₃^{reduction to NH}₄^{has also been found in}Sulforospirillum deleyianum, which uses sulfide as an electron donor (3), and in Campylobacter species (33), where H₂ was used as an electron donor. The ecological implications of the finding that Thioploca prefers to produce NH₄^{are significant, since this means that nitrate reduction by}Thioploca does not lead to nitrogen loss in this vast ecosystem along the entire coast of Chile and Peru.

Sulfur metabolism.

After addition of sulfide to Thioploca trichome bundles in a particular experiment, a sulfide oxidation rate of approximately 4.2 nmol min^{mg of protein}^{was observed in the absence of external nitrate. The NH}₄^{production was 1.9 nmol min}^{mg of protein}^{, resulting in a ratio of 2.2 between sulfide oxidized and NH}₄^{produced. If the sulfide were oxidized to elemental sulfur and NO}₃^{were reduced to NH}₄^{, then the expected ratio of sulfide to ammonium would be 4. If sulfide were oxidized to sulfate then a ratio of 1 would be expected. The observed ratio suggests that the sulfide is oxidized to both sulfur and sulfate, since there was no significant accumulation of other (intermediate) sulfur species (i.e., sulfite and thiosulfate). Analogous to observations in marine}Beggiatoa (23), it is likely that the immediate product of sulfide oxidation is elemental sulfur, which is stored in Thioploca as globules. The elemental sulfur is then oxidized to SO₄^{in a second, independent step, as suggested by Fossing et al. (}7). In experiments without addition of sulfide, sulfate production was observed at a rate of 2 to 3 nmol min^{mg of protein}^{, which must have originated from internal elemental sulfur. In the presence of sulfide, the SO}₄^{production rate did not increase significantly, suggesting that sulfide is oxidized to sulfur and that further oxidation of sulfur to SO}₄^{occurs independently of the presence of sulfide. In these two experiments, the ratio of SO}₄^{to NH}₄^{produced was approximately 1.5 in the absence and approximately 1.7 in the presence of sulfide. If NO}₃^{is reduced to NH}₄^{and sulfur is oxidized to SO}₄^{, then a ratio of 1.3 is expected. This is in agreement with the observed ratio in the absence of sulfide, indicating, again, that}Thioploca trichome bundles reduce most NO₃^{to NH}₄^{under the conditions tested. It was also observed that addition of different concentrations of sulfide (100 μM and 400 μM) did not result in a significant increase in NH}₄^{production (Fig.}(Fig.2).2). This reconfirms that oxidation of sulfide, and subsequently sulfur, occurs independently. The observed ratios indicate that net sulfur accumulation will occur when external sulfide is present. Addition of sulfide led to a small accumulation of thiosulfate (S₂O₃^{) in the medium, suggesting that S}₂O₃^{may be an intermediate in sulfur oxidation to sulfate. However, addition of S}₂O₃^{to trichome bundles showed only a very low consumption of S}₂O₃^{. Starvation of the trichome bundles for 45 h in the presence of NO}₃^{did not enhance this consumption rate. Accumulation of S}₂O₃^{during starvation of disrupted trichome bundles indicates that}Thioploca cells may not be responsible for the observed accumulation in previous experiments. At present, due to variations in the measurements, it cannot be determined whether or not Thioploca produces S₂O₃^{as an intermediate.}

Sulfate reduction rates measured in sediments from station 7 at the time of sampling were approximately 30 mmol m^day⁽34). If all sulfide produced from this reduction were subsequently oxidized by the Thioploca mats then Thioploca cells should be able to oxidize sulfide with a rate of 20.4 nmol min^{mg of protein}^{(see Materials and Methods). In comparison, Ferdelman et al. (}6) measured an average SO₄^{reduction rate of 17.5 mmol m}^day^{, indicating that}Thioploca should be able to oxidize sulfide with a rate of 11.8 nmol min^{mg of protein}^{(see Materials and Methods). The average sulfide oxidation rate observed during our experiments was 5 nmol min}^{mg of protein}^{, which increased to 10.7 nmol min}^{mg of protein}^{after starvation. Compared to the above-mentioned reduction rates, this oxidation rate observed in}Thioploca could be responsible for 25 to 91% of the observed SO₄^{reduction rates measured in the sediments. This indicates that}Thioploca species may be able to oxidize the majority of the sulfide produced in the sediment of the continental shelf. These data are in agreement with observations by Ferdelman et al. (6), who found an oxidation capacity for Thioploca of 35% of the sulfide production in the sediment.

Carbon metabolism.

Addition of [^{C]bicarbonate resulted in an incorporation rate of 0.4 to 0.8 nmol min}^{mg of protein}^{. The presence of sulfide did not increase the incorporation rate significantly. The measured SO}₄^{production rate (generated from internal sulfur) was 2 to 3 nmol min}^{mg of protein}^{, which is equivalent to an average of 1.3 nmol min}^{mg of dry weight}^{, assuming that 50% of dry weight is protein. From these data we can predict the CO}₂ fixation rate, assuming that 12.5% of the electrons produced go to CO₂ fixation (assuming a yield of 8 g (dry weight) · mol of sulfide^[23, 38]). The oxidation of sulfur to SO₄^{produces six electron equivalents. Given the fact that CO}₂ reduction to biomass (dry weight) requires four electron equivalents, the predicted rate of CO₂ fixation would be 0.125 × (6/4) × 1.3 = 0.24 nmol min^mg^{(dry weight). This rate is equivalent to 0.49 nmol min}^{mg of protein}^{(assuming that 50% of the dry weight is protein), which is the rate observed, suggesting that}Thioploca species can grow autotrophically by using internally stored sulfur and NO₃^{for energy generation. Results obtained with microautoradiography confirm earlier qualitative experiments by Maier and Gallardo (}19) and indicate that the CO₂ fixation measured can be attributed to Thioploca trichome bundles and not to epibiontic bacteria. Ferdelman et al. (6) measured a CO₂ fixation rate in cleaned Thioploca suspensions of 2,400 ± 700 nmol day^g^{(wet weight). Assuming that the wet weight of trichomes is 10% of the wet weight of sheaths and trichomes (}32), that 10% of the wet weight of trichomes is cytoplasm, that 24% of the wet weight of the cytoplasm is dry weight, and that 50% of the dry weight is protein (see calculations in Materials and Methods), then the fixation rate was estimated to be 1.4 ± 0.4 nmol min^{mg of protein}^{. This rate is approximately three times as high as the rate observed in our study.}

Experiments performed with [^{C]acetate in the absence of sulfide resulted in an uptake rate of approximately 0.4 nmol min}^{mg of protein}^{. Microautoradiography showed that epibiontic bacteria also incorporated acetate, but the majority of the label (>50%) was taken up by trichomes. Labeling experiments performed with}Thiobacillus neapolitanus showed that obligate autotrophs are able to incorporate acetate via an incomplete trichloroacetic acid cycle, lacking the enzyme α-ketoglutarate dehydrogenase (18), resulting in an acetate incorporation rate of 20 to 30% of the CO₂ fixation rate. However, for the Thioploca trichome bundles the acetate uptake rate was approximately equal to the CO₂ fixation rate, which strongly suggests that Thioploca species are facultative chemolithoautotrophs, as previously shown for a marine Beggiatoa strain (15) and as has also been suggested for the large vacuolated Beggiatoa spp. from the Guaymas Basin (25). Production of ^CO₂ was not observed after the addition of [2-^{C]acetate, suggesting that acetate, under these conditions, was used only as a source for cell carbon, since total oxidation of acetate for energy would release}^CO₂. Since Thioploca has internally stored sulfur, which is available as an energy source, it would be most beneficial, strategically, to use acetate as the primary carbon source. This economic use of energy and carbon sources is typical for mixotrophic growth (12).

Labeling experiments with bicarbonate and acetate followed by microautoradiography showed localization of the label along the transverse walls, indicating the presence of the central vacuole.

The ecophysiological experiments presented here indicate that Thioploca is a facultative chemolithoautothroph, capable of fixing CO₂ and assimilating available acetate when sulfur or sulfide is present as an energy source. This use of acetate as a carbon source when other substrates are present as an energy source is typical behavior for organisms capable of mixotrophic growth. In spite of its ability to rapidly respond to fluctuations in both NO₃^{and sulfide, its metabolic strategy seems to be geared toward continuous, but extremely slow, growth which is apparently unaffected by such fluctuations. Indeed, the large reservoir of both NO}₃^{(average 160 mM) and sulfur (200 nmol mm}^{) indicates a turnover time for NO}₃^{and sulfur of 8 to 10 days. Based on the observed rate of autotrophic CO}₂ fixation, Thioploca would grow with a doubling time of 69 to 139 days under the laboratory conditions tested (0.4 to 0.8 nmol of CO₂ min^{mg of protein}^{is equal to 0.4 to 0.8 nmol of carbon min}^{mg (dry weight) of carbon}^{, assuming that 50% of the dry weight is carbon. One milligram of carbon is equal to 0.08 mmol of carbon, and thus, it would take 69 to 139 days to incorporate this amount. Assuming that}Thioploca can grow mixotrophically on acetate, this doubling time could be increased to 26 to 52 days. Although this may be an underestimate, such a rate coincides with the observed increase in biomass (wet weight) of 1 g m^day^{as has been observed for station 6 by H. N. Schulz (}31). This increase would lead to a doubling time of approximately 70 days, assuming an average of 85 g (wet weight) m^{for trichomes without sheaths (see Materials and Methods). In general, however, we should remember that samples used in this study were mixed populations and, therefore, differences in activity between the two species used may occur.}

In spite of its low growth rate, the evidence presented here shows that Thioploca is one of the major players in sulfur and nitrogen cycling of the sediment along the west coast of South America.

Nitrogen metabolism.

Sulfur metabolism.

Carbon metabolism.

Labeling experiments with bicarbonate and acetate followed by microautoradiography showed localization of the label along the transverse walls, indicating the presence of the central vacuole.

In spite of its low growth rate, the evidence presented here shows that Thioploca is one of the major players in sulfur and nitrogen cycling of the sediment along the west coast of South America.

Department of Biotechnology, Kluyver Laboratory for Biotechnology, Delft University of Technology, Delft, The Netherlands^{; Institute of Biological Sciences, University of Aarhus, Aarhus, Denmark}^{; Max Planck Institute for Marine Microbiology, Bremen, Germany}^{; Institute of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina}^{; Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts}^{; and Departamento de Oceanografia, Universidad de Concepción, Concepción, Chile}⁶

^{Corresponding author. Mailing address: Delft University of Technology, Kluyver Laboratory for Biotechnology, Julianalaan 67, 2628 BC, The Netherlands. Phone: 31 15 2782416. Fax: 31 15 2782355. E-mail:}ln.tfleDUT.MTS@neneuK.G.J.

Received 1998 Dec 8; Accepted 1999 May 7.

Abstract

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with ^{N compounds and mass spectrometry and found that these}Thioploca samples produce ammonium at a rate of 1 nmol min^{mg of protein}^{. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min}^{mg of protein}^{. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min}^{mg of protein}^{and could be increased to 10.7 nmol min}^{mg of protein}^{after the trichomes were starved for 45 h. Incorporation of}^CO₂ was at a rate of 0.4 to 0.8 nmol min^{mg of protein}^{, which is half the rate calculated from sulfide oxidation. [2-}^{C]acetate incorporation was 0.4 nmol min}^{mg of protein}^{, which is equal to the CO}₂ fixation rate, and no ^CO₂ production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-^{C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.}

Abstract

Massive communities of Thioploca species occur as dense mats in the top sediment underlying the oxygen minimum zone of the continental shelf off the coast of Chile and Peru (17). Extending down to 5 to 10 cm into the sediment, the total biomass (including sheaths) of these colorless sulfur bacteria may be as high as 800 g (wet weight) m⁽32), potentially covering several thousands of square kilometers along a 3,000-km stretch of coast.

Thioploca chileae and Thioploca araucae are the two dominant species in the mat, measuring 12 to 22 and 28 to 42 μm in diameter, respectively (32, 36). Both species produce 2- to 7-cm-long trichomes (filaments), each of which consists of a uniseriate row of many vacuolated cells. Morphologically and phylogenetically, they are similar to vacuolated Beggiatoa species (24, 37), and it has been suggested that their physiology might be similar as well. A chief difference between the genera is, however, that Thioploca produces characteristic bundles of usually 10 to 20 trichomes, surrounded by 10- to 15-cm-long sheaths up to 1.5 mm in diameter. Individual trichomes can glide independently within the sheaths and extend up to 3 cm into the water phase above the sediment (16). In general, Thioploca species and Beggiatoa species appear to occupy different niches, the former living in vertical and horizontal sheaths down to 10 cm in sediments that contain relatively little sulfide. In contrast, Beggiatoa species live in the top layer of sediments that have relatively high sulfide concentrations. Since their discovery by V. A. Gallardo (8, 9), it has been assumed that the Thioploca mats play a crucial role in balancing the sulfur cycle of their marine habitat by reoxidizing all, or at least a substantial portion, of the sulfide produced in the sediment. The sulfide results from high rates of bacterial sulfate reduction, up to 2.4 g of sulfur m^day⁽6), driven by extremely high primary productivity (up to 9.6 g of carbon m^day^{) over the continental shelf (}7). Recently, Thioploca spp. have been identified off the coast of Namibia (10), where similar oceanographic conditions exist, i.e., upwelling, high primary productivity, and oxygen-depleted bottom water.

Given that the high remineralization rates of organic compounds result in the often-observed depletion of oxygen in the bottom water overlying the sea floor (7, 9, 32), the question arose as to which electron acceptor might be used for the reoxidation of all the sulfide produced in these sediments. When it was discovered that the vacuolated Thioploca species living in the mat were capable of accumulating up to 500 mM nitrate from the overlying water (containing ∼25 μM [7]), it was hypothesized that Thioploca species would be able to use the nitrate as a terminal electron acceptor for sulfide oxidation (7). It was assumed that nitrate would be reduced to dinitrogen, although no experimental data was available to support this (7). The question was, therefore, still open as to whether dinitrogen gas or ammonium would be the product. This was particularly interesting in view of the finding by McHatton et al. (21) that vacuolated Beggiatoa species are also capable of accumulating and reducing nitrate and in view of conflicting observations by others with respect to the final product of nitrate reduction by the nonvacuolated Beggiatoa alba, i.e., ammonium or dinitrogen gas (35, 39).

So far, it has not been possible to cultivate Thioploca species in pure culture. The same is true for the vacuolated (nitrate accumulating) Beggiatoa species. Hence, little is known about their (eco)physiology, specifically, their sulfide and sulfur oxidation rates, abilities to respirate oxygen and/or nitrate, growth rates, or capabilities to grow autotrophically, heterotrophically, or mixotrophically. Clearly, this knowledge is essential for understanding the role of Thioploca species in their habitat.

McHatton et al. (21) studied partially purified cultures of naturally occurring populations of large vacuolated Beggiatoa species and showed that these organisms contain substantial activities of membrane-bound nitrate reductase, indicating that they may indeed be capable of using nitrate as the terminal electron acceptor. Significant activities of ribulose-1,5-bisphosphate carboxylase were also detected, evidencing that the vacuolated marine Beggiatoa species are capable of autotrophic growth. Using rinsed samples of Thioploca material from a mat, Ferdelman et al. (6) were able to demonstrate CO₂-fixing capacity in these preparations, indicating that Thioploca has an autotrophic potential.

Since Thioploca species live at high densities in the mats of the Chilean marine sediments and could be seen with the naked eye, we decided that it was possible to obtain samples of these organisms sufficiently pure to allow the performance of physiological experiments. We developed a simple method by which we handpicked individual sheaths with trichome bundles with forceps from the top 2 cm of sediments incubated under an N₂ atmosphere. After they had been collected and washed, cells were used for various experiments. By using radiolabeled and unlabeled substrates, a study was made of carbon, nitrogen, and sulfur metabolism. The observed activities were compared with data obtained from field measurements. The results indicate that Thioploca species are (metabolically) highly active under anoxic conditions and that they can play a significant role in the total oxidation of sulfide in the mat under anoxic conditions in the presence of nitrate. They appear to be facultative chemolithoautotrophs with a mixotrophic potential, meaning they can use sulfide or sulfur as an energy source for growth and CO₂ fixation and can use acetate under these conditions as an additional carbon source. Evidence presented in this study points to ammonium as the end product of nitrate reduction, although conversion to dinitrogen gas cannot be ruled out. Oxygen at approximately 10% air saturation did not inhibit the observed CO₂ fixation.

ACKNOWLEDGMENTS

This research was part of a joint project between the Max Planck Institute of Marine Microbiology, Bremen, Germany, and the University of Concepción, Concepción, Chile. We greatly appreciate all the enthusiasm, help, and support that we received from the people at the Estación de Biologia marina in Dichato, the crew of the Kay Kay, the staff from the University of Concepción who helped us with the ^{C scintillation counter, and all the members of the scientific party present. Furthermore, we thank the reviewers for their many helpful suggestions to improve the manuscript.}

This study was supported by the Max Planck Society, the University of Concepción, the Delft University of Technology, the Netherlands Organization for Scientific Research (NWO project R83-151), the Woods Hole Oceanographic Institution (contribution no. 9730), the FONDAP-HUMBOLDT Program, and the National Science Foundation (OCE 94-15985).

ACKNOWLEDGMENTS

REFERENCES

References

1. Burton S D, Morita R YEffect of catalase and cultural conditions on growth of Beggiatoa. J Bacteriol. 1964;88:1755–1761.[Google Scholar]
2. Cline J DSpectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr. 1969;14:454–458.[PubMed][Google Scholar]
3. Eisenmann E, Beuerle J, Sulger K, Kroneck P M H, Schumacher WLithotrophic growth of Sulforospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia. Arch Microbiol. 1995;164:180–185.[PubMed][Google Scholar]
4. Fahey R C, Newton G LDetermination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high performance liquid chromatography. Methods Enzymol. 1987;143:85–96.[PubMed][Google Scholar]
5. Fawcett J K, Scott J EA rapid and precise method for the determination of urea. J Clin Pathol. 1960;13:156–159.[Google Scholar]
6. Ferdelman T G, Lee C, Pantoja S, Harder J, Bebout B M, Fossing H ASulfate reduction and methanogenesis in a Thioploca-dominated sediment off the coast of Chile. Geochim Cosmochim Acta. 1997;61:3065–3079.[PubMed][Google Scholar]
7. Fossing H A, Gallardo V A, Jørgensen B B, Hüttel M, Nielsen L P, Schulz H N, Canfield D E, Forster S, Glud R N, Gundersen J K, Küver J, Ramsing N B, Teske A, Thamdrup B, Ulloa OConcentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca. Nature. 1995;374:713–715.[PubMed][Google Scholar]
8. Gallardo V ANotas sobre la densidad de la fauna sublitoral del norte de Chile. Gayana Zool. 1963;10:3–15.[PubMed][Google Scholar]
9. Gallardo V ALarge benthic microbial communities in sulphide biota under Peru-Chile subsurface counter current. Nature. 1977;286:331–332.[PubMed][Google Scholar]
10. Gallardo V A, Klingelhoeffer E, Arntz W, Graco MFirst report of the bacterium Thioploca in the Benguela ecosystem of Namibia. J Mar Biol Assoc UK. 1998;78:1007–1010.[PubMed][Google Scholar]
11. Goa JA micro-biuret method for protein determination: determination of total protein in cerebrospinal fluid. J Clin Lab Investig. 1953;5:218–222.[PubMed][Google Scholar]
12. Gottschal J C, Kuenen J GMixotrophic growth of Thiobacillus A2 on acetate and thiosulfate as growth limiting substrates in the chemostat. Arch Microbiol. 1980;126:33–42.[PubMed][Google Scholar]
13. Grasshoff K, Erhardt M, Kremling K Methods of seawater analysis. Deerfield Beach, Fla: Basel Verlag Chemie; 1983. [PubMed][Google Scholar]
14. Griess-Romein van Eck . Physiological and chemical tests for drinking water, NEN 1056, IV-2. Rijswijk, The Netherlands: Nederlands Normalisatie-Instituut Rijswijk; 1966. [PubMed]
15. Hagen K D, Nelson D COrganic carbon utilization by obligately and facultatively autotrophic Beggiatoa strains in homogeneous and gradient cultures. Appl Environ Microbiol. 1996;62:947–953.[Google Scholar]
16. Hüttel M, Forster S, Klöser S, Fossing HVertical migration in the sediment-dwelling sulfur bacteria Thioploca spp. in overcoming diffusion limitation. Appl Environ Microbiol. 1996;62:1863–1872.[Google Scholar]
17. Jørgensen B B, Gallardo V A. Thioploca spp.: filamentous sulfur bacteria with nitrate vacuoles. FEMS Microbiol Ecol. 1999;28:301–313.[PubMed]
18. Kuenen J G, Veldkamp HEffects of organic compounds on growth of chemostat cultures of Thiomicrospira pelophila, Thiobacillus thioparus and Thiobacillus neapolitanus. Arch Microbiol. 1973;94:173–190.[PubMed][Google Scholar]
19. Maier S, Gallardo V ANutritional characteristics of two marine Thioplocas determined by autoradiography. Arch Microbiol. 1984;139:218–220.[PubMed][Google Scholar]
20. Maier S, Völker H, Beese M, Gallardo V AThe fine structure of Thioploca araucae and Thioploca chileae. Can J Microbiol. 1990;36:438–448.[PubMed][Google Scholar]
21. McHatton S C, Barry J P, Jannasch H W, Nelson D CHigh nitrate concentrations in vacuolate, autotrophic marine Beggiatoa spp. Appl Environ Microbiol. 1996;62:954–958.[Google Scholar]
22. Möller M M, Nielsen L P, Jørgensen B BOxygen responses and mat formation by Beggiatoa. Appl Environ Microbiol. 1985;50:373–382.[Google Scholar]
23. Nelson D C, Jørgensen B B, Revsbech N PGrowth pattern and yield of a chemoautotrophic Beggiatoa sp. in oxygen-sulfide microgradients. Appl Environ Microbiol. 1986;52:225–233.[Google Scholar]
24. Nelson D C, Revsbech N P, Jørgensen B BThe microoxic/anoxic niche of Beggiatoa spp.: a microelectrode survey of marine and freshwater strains. Appl Environ Microbiol. 1986;52:161–168.[Google Scholar]
25. Nelson D C, Wirsen C O, Jannasch H WCharacterization of large, autotrophic Beggiatoa spp. abundant at hydrothermal vents of Guaymas Basin. Appl Environ Microbiol. 1989;55:2909–2917.[Google Scholar]
26. Nielsen L PDenitrification in sediments determined from nitrogen isotope pairing. FEMS Microbiol Ecol. 1992;86:357–362.[PubMed][Google Scholar]
27. Paerl H WBacterial uptake of dissolved organic matter in relation to detrital aggregation in marine and freshwater systems. Limnol Oceanogr. 1974;19:966–972.[PubMed][Google Scholar]
28. Rethmaier J, Rabenstein A, Lang M, Fischer UDetection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high performance liquid chromatography methods. J Chromatogr A. 1997;760:295–302.[PubMed][Google Scholar]
29. Risgaard-Petersen N, Rysgaard S, Revsbech N PA sensitive assay for determination of ^N/^{N isotope distribution in NO}₃J Microbiol Methods. 1993;17:155–164.[PubMed][Google Scholar]
30. Risgaard-Petersen N, Rysgaard S, Revsbech N PCombined microdiffusion-hypobromite oxidation method for determining nitrogen-15 isotope in ammonium. Soil Sci Soc Am J. 1995;59:1077–1080.[PubMed][Google Scholar]
31. Schulz, HN. Unpublished results.
32. Schulz H N, Jørgensen B B, Fossing H A, Ramsing N BCommunity structure of filamentous, sheath-building sulfur bacteria, Thioploca spp., off the coast of Chile. Appl Environ Microbiol. 1996;62:1855–1862.[Google Scholar]
33. Stouthamer A H. Dissimilatory reduction of oxidized nitrogen compounds. In: Zehnder A J B, editor. Biology of anaerobic microorganisms. New York, N.Y: John Wiley & Sons, Inc.; 1988. pp. 245–303. [PubMed]
34. Strotmann, B. Unpublished results.
35. Sweerts J P R A, Beer D D, Nielsen L P, Verdouw H, Heuvel J C V, Cohen Y, Cappenberg T EDenitrification by sulfur oxidizing Beggiatoa spp. mats on freshwater sediments. Nature. 1990;334:762–763.[PubMed][Google Scholar]
36. Teske, A. Unpublished results.
37. Teske A, Ramsing N B, Küver J, Fossing H APhylogeny of Thioploca and related filamentous sulfide-oxidizing bacteria. Syst Appl Microbiol. 1995;18:517–526.[PubMed][Google Scholar]
38. Timmer-ten Hoor ACell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans. Antonie Leeuwenhoek. 1981;47:231–243.[PubMed][Google Scholar]
39. Vargas A, Strohl W RUtilization of nitrate by Beggiatoa alba. Arch Microbiol. 1985;142:279–284.[PubMed][Google Scholar]
40. Widdel, F. Personal communication.

Nitrogen, Carbon, and Sulfur Metabolism in Natural <em>Thioploca</em> Samples

MATERIALS AND METHODS

Collection.

Incubation.

Analytical procedures.

N experiments and mass spectrometry.

NaHCO3, [2-C]acetate, and [H]acetate incorporation experiments.

Microautoradiography.

Calculations. (i) N experiments: the ratio between added [N]nitrate and intravacuolar [N]nitrate.

(ii) Sulfide oxidation: the ratio between observed sulfide reoxidation rates and specific activity of Thioploca.

Collection.

Incubation.

Analytical procedures.

N experiments and mass spectrometry.

NaHCO3, [2-C]acetate, and [H]acetate incorporation experiments.

Microautoradiography.

Calculations. (i) N experiments: the ratio between added [N]nitrate and intravacuolar [N]nitrate.

(ii) Sulfide oxidation: the ratio between observed sulfide reoxidation rates and specific activity of Thioploca.

RESULTS

Cultivation and survival.

N metabolism.

N-labeling experiments.

Sulfur metabolism.

TABLE 1

Carbon metabolism.

TABLE 2

(i) NaHCO3.

(ii) [C]- and [H]acetate.

Cultivation and survival.

N metabolism.

N-labeling experiments.

Sulfur metabolism.

TABLE 1

Carbon metabolism.

TABLE 2

(i) NaHCO3.

(ii) [C]- and [H]acetate.

DISCUSSION

Nitrogen metabolism.

Sulfur metabolism.

Carbon metabolism.

Nitrogen metabolism.

Sulfur metabolism.

Carbon metabolism.

Abstract

ACKNOWLEDGMENTS

REFERENCES

References

^{N experiments and mass spectrometry.}

NaH^CO₃, [2-^{C]acetate, and [}^{H]acetate incorporation experiments.}

Calculations. (i) ^{N experiments: the ratio between added [}^{N]nitrate and intravacuolar [}^N]nitrate.

^{N experiments and mass spectrometry.}

NaH^CO₃, [2-^{C]acetate, and [}^{H]acetate incorporation experiments.}

Calculations. (i) ^{N experiments: the ratio between added [}^{N]nitrate and intravacuolar [}^N]nitrate.

^{N-labeling experiments.}

(i) NaH^CO₃.

(ii) [^{C]- and [}^H]acetate.

^{N-labeling experiments.}

(i) NaH^CO₃.

(ii) [^{C]- and [}^H]acetate.