Uptake Rates of Oxygen and Sulfide Measured with Individual Thiomargarita namibiensis Cells by Using Microelectrodes

Gradients of oxygen and sulfide measured towards individualcells of the large nitrate-storing sulfur bacterium Thiomargaritanamibiensis showed that in addition to nitrate oxygen is usedfor oxidation of sulfide. Stable gradients around the cellswere found only if acetate was added to the medium at low concentrations.

INTRODUCTION

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Introduction

The sulfur bacterium Thiomargarita namibiensis is a close relativeof the filamentous sulfur bacteria of the genera Beggiatoa andThioploca. It was only recently discovered off the Namibiancoast in fluid sediments rich in organic matter and sulfide(15). The large, spherical cells of Thiomargarita (diameter,100 to 300 µm) are held together in a chain by mucus thatsurrounds each cell (Fig. 1). Most of the cell volume is takenup by a central vacuole in which nitrate is stored at concentrationsof up to 800 mM. The ability to accumulate nitrate is also foundin larger, marine species of Beggiatoa (8) and Thioploca (3).The latter have been shown to use nitrate as an electron acceptorfor the oxidation of sulfide to elemental sulfur and then tosulfate while they reduce nitrate to ammonia (13). Both Beggiatoaand Thioploca spp. show a phobic reaction towards oxygen evenat low concentrations (5, 10). Higher oxygen concentrationsin the bottom water (5 to 35 µM), when they occur offthe Chilean coast during winter or El Niño events, dramaticallyreduce the Thioploca population (16). In contrast to this, Thiomargaritacells survive exposure to oxygen even at the concentrationsof air saturation (15), although the bottom water overlyingthe sediments off Walvis Bay is usually anoxic.

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FIG. 1. Experimental setup. A chain of T. namibiensis cells was placed in a chamber with 250 ml of artificial seawater. The oxygen concentration in the medium was controlled by bubbling the medium with either air or argon. Sulfide was added by prior bubbling through a wash bottle containing a concentrated solution of sulfide. To ensure three-dimensional diffusion towards the cells, the chain of cells was elevated above the bottom of the chamber by clamping it between two platinum wires. One microelectrode (either oxygen or sulfide) was used to measure gradients towards the cells, while the other recorded the concentration of oxygen or sulfide in the bulk medium. The inset shows an example of oxygen flux without sulfide in the medium.

Because Thiomargarita cells are not motile, the only way thatthe cells can come into contact with water containing nitrateis during intervals when the sediment gets resuspended in thewater column. This can happen, for example, due to large sedimentoutgassings of methane, which occur regularly in the area inhabitedby Thiomargarita (2, 17). During these events the highly fluidizedsulfidic mud containing Thiomargarita cells may get mixed withoxygenated water containing nitrate. The purpose of this studywas to investigate whether the cells merely survive such exposureto oxygen or whether Thiomargarita cells can use oxygen as anelectron acceptor in addition to nitrate for oxidation of sulfide.Like the nitrate-storing species of Beggiatoa and Thioploca,Thiomargarita spp. have not been isolated yet in pure culture.Nevertheless, cells may be kept alive and growing in their nativesediment for years. As Thiomargarita cells are not motile, itis not possible to draw conclusions about their physiology byobserving chemotactic behavior, as has been done successfullywith Beggiatoa and Thioploca filaments (5, 10). However, becauseof the large size of Thiomargarita cells, they develop, aroundindividual cells, measurable gradients of oxygen and sulfidethat can be used for calculating uptake rates of oxygen andsulfide. Thus, the physiological reactions of individual cellsto changes in oxygen and sulfide concentrations can be directlyobserved by observing changes in the uptake rates.

Experiments.

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Experiments.

The cells used for the experiments in this study were collectedoff Namibia during a cruise of the German RV Poseidon in May1999 and were kept in their natural sediment at 5°C formore than 1 year. At approximately 3-month intervals the overlyingwater was removed and the sediment was resuspended several timesin seawater enriched with nitrate (1 mM), which induced growthof Thiomargarita cells. During this treatment cells were exposedto oxygen. The experiments were conducted in a square polycarbonatechamber (7 by 7 by 7 cm) containing 250 ml of artificial seawater(36.4 g of NaCl per liter, 1 g of CaCl₂ per liter, 0.5 g ofK₂HPO₄ per liter, 0.1 g of NaH₂PO₄ per liter; pH 7.3). The chamberwas capped with a movable plate containing two holes for microelectrodeaccess (Fig. 1). Thiomargarita cells were washed in medium andtransferred to the chamber by sucking them up in the tip ofa Pasteur pipette. After the cells were placed in the experimentalchamber, acetate was added to a final concentration of 10 µM.The chamber was continuously flushed with either air or argonto control the oxygen concentration. Sulfide was added to themedium by passing the gas through a bottle containing 100 mlof carbonate buffer (pH 9.3) and sulfide at a high concentration(100-fold higher than the desired final concentration in theexperimental chamber). Thus, the gas was supplemented with H₂Sat the desired concentration before it was bubbled through theexperimental chamber. After a stable concentration of sulfidewas established, as measured with an H₂S microsensor, samples(1 ml) were taken, and the exact sulfide concentration was determinedspectrophotometrically (1). The bubbling was adjusted so thatthe liquid in the chamber was well mixed and only a thin diffusiveboundary layer (DBL) (approximately 140 µm) developedaround the cells. The DBL around the cells (diameter, 220 µm)was found by moving the microsensor in 10-µm intervalsaway from the cell until no change in concentration was found.To measure fluxes of oxygen and sulfide towards a single cell(Fig. 2), a chain of Thiomargarita cells was fixed between twoplatinum wires at a distance of ca. 1 cm from the bottom ofthe chamber (Fig. 1). By using a preparation needle the twoends of the chain were placed into the V-shaped ends of theplatinum wires. This design allowed three-dimensional diffusiontoward the cell investigated. Vertical profiles of oxygen orsulfide were measured through the DBL of the cells at 10-µmintervals by using a Clark-type oxygen microelectrode with aguard cathode (14) or a sulfide microelectrode (6). The gradientsthrough the DBL of the cells were found to be almost linear;therefore, the total flux towards the cell could be calculatedby multiplying the linear flux (J = D x dC/dr, where J is theflux, D is the diffusion coefficient, dC is the change in concentrationbetween two points, and dr is the distance between these twopoints) by the surface area of the cell (diameter, 220 µm).To calculate the surface area involved in uptake, either thesurface of a sphere (4 ${pi}$ r²) or the surface of the side of a cylinder(2 ${pi}$ rh, where r is the radius and h is the height) could be assumed.In our case the height of the cylinder was twice the radius(h = 2r), so the two assumptions gave the same value. Duringthe experiment whose results are shown in Fig. 3, the cellswere lying on the bottom of the experimental chamber. The oxygenelectrode was placed directly at the cell surface. Several timesduring the experiment, the electrode was moved 500 µmup and down to ensure that the bulk concentration of oxygenremained unchanged. In order to prove that oxygen and sulfidegradients resulted from physiological activity of the cells,selected active cells were exposed for 1 min to pure ethanoland returned to the chamber. These cells showed no gradientsof oxygen or sulfide towards the cell surface. During this shortexposure the cells were killed, but the internal sulfur inclusiondid not dissolve. Also, addition of methanol to a final concentrationof 1% in the medium led to immediate disappearance of sulfideand oxygen gradients around initially active cells. The abioticoxidation of sulfide with oxygen is a slow process that occurswith half times between 0.4 and 65 h (9). As we flushed themedium continuously with both gases, this process should nothave played a major role in these experiments.

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FIG. 2. Oxygen flux (A) and sulfide flux (B) towards an individual Thiomargarita cell with a diameter of 220 µm. The fluxes ( ${circ}$ ) were calculated from microprofiles measured through the 140-µm-thick DBL around the cell in the presence of a stable oxygen concentration of 150 µM (A) and a stable sulfide concentration of 320 µM (B). The changing bulk medium concentrations of sulfide (A) and oxygen (B) ( ${diamondsuit}$ ) were measured continuously with a separate electrode. The graph shows individual measurements. Therefore, the absolute numbers should be interpreted as examples of possible uptake rates found in Thiomargarita cells rather than as the typical rate for this genus. Numerous measurements showing the same type of response were obtained with cells lying on the bottom of the chamber, but quantification of fluxes from these profiles by using Fick's law may have led to overestimates and were therefore not included in the data set.

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FIG. 3. Response of an individual Thiomargarita cell to changes in sulfide concentration ( ${diamondsuit}$ ) under oxic conditions. The sulfide sensor was placed in the bulk medium, and the oxygen electrode was placed directly at the cell surface ( ${circ}$ ). Thus, enhanced oxygen uptake rates are reflected by a decrease in oxygen concentration at the cell surface. The dashed line indicates a change in the x-axis scale.

Addition of acetate.

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Addition of acetate.

In initial experiments freshly added cells showed pronouncedgradients of oxygen and sulfide that slowly disappeared within1 or 2 h. Only after sodium acetate was added to the mediumat a final concentration of 10 µM did the oxygen gradientsremain stable for at least 2 days, even when no sulfide waspresent in the medium. Further addition of 10 µM acetatedid not enhance the oxygen flux towards the cells (data notshown). If the 10 µM acetate were consumed by the celland used as an electron donor, this result would have correspondedto a maximum acetate flux of 6.5 pmol cm^-2 s^-1 and would haveincreased the oxygen flux by 13 pmol cm^-2 s^-1 (CH₃COOH + 2 O₂ $->$ 2 CO₂ + 2 H₂O). As the oxygen flux was stabilized at only 1.1pmol cm^-2 s^-1 and not increased even by the first addition ofacetate, it is likely that the Thiomargarita cells in this experimentdepended on acetate only as a carbon source and not as an electrondonor. Acetate has also been shown to stimulate the sulfideuptake of Thioploca spp. (13) and the thiosulfate uptake ofThiothrix spp. (12) and can be used as a supplemental or solecarbon source by lithotrophic marine Beggiatoa strains (4).Nevertheless, the possibility that T. namibiensis grows autotrophicallycannot be ruled out by the results of these experiments, becausethe special setup, which was designed to measure sulfide uptakein the presence of minimum oxygen concentrations and with astable pH, did not allow bicarbonate-carbonate to be presentin the medium. To maintain very low oxygen concentrations, itwas necessary to bubble the medium constantly with argon, whichwould have stripped CO₂ from the medium.

Use of oxygen.

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Use of oxygen.

The presence of sulfide in the medium clearly enhanced oxygenuptake (Fig. 2A), and likewise, sulfide uptake by the cellswas enhanced by oxygen (Fig. 2B). These results suggest thatThiomargarita cells not only are able to survive exposure tooxygen but also may use oxygen as an electron acceptor. Additionof nitrate to the medium had no effect on the oxygen uptake.As judged by the sulfide uptake rates, the cells remained physiologicallyactive even under oxygen concentrations close to saturation.This indicates that unlike Beggiatoa spp. (7, 10, 11), T. namibiensisis not obligately microaerophilic. The maximum sulfide fluxwas 7.5 pmol cm^-2 s^-1 and could not be increased by increasingthe oxygen concentration (Fig. 2B); likewise, the oxygen fluxdid not exceed 2.2 pmol cm^-2 s^-1 after further addition of sulfide(Fig. 2A). It seems that with the rigorous stirring resultingfrom bubbling with gas, the maximum uptake rates occurred atsulfide and oxygen concentrations that are low (100 µM)compared to the concentrations that T. namibiensis can tolerate(air saturation). The observed sulfide flux under anoxic conditions(approximately 5 pmol cm^-2 s^-1) (Fig. 2B) must have been supportedby internally stored nitrate. Addition of oxygen increased thesulfide flux by ca. 2.5 pmol cm^-2 s^-1. Thus, even though thesulfide flux could be significantly stimulated by oxygen, reductionof nitrate was still the more important process for sulfideuptake.

Cell response to sulfide under aerobic conditions.

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Cell response to sulfide...

It was possible to monitor the reaction of an individual cellto pulses of sulfide in the medium by placing the oxygen electrodedirectly at the cell surface. A decline in the oxygen concentrationrepresented higher uptake rates, and an increase indicated lowerrates of oxygen uptake (Fig. 3). The response of the cell couldbe observed with very high temporal time resolution, but onlyrelative changes in the uptake rate were visible. The fluxescould be obtained only from steady-state profiles. Two sequentialpulses of sulfide (60 and 120 µM) both resulted in a sharpdrop in the oxygen concentration, followed by an increase andthen a decrease in the oxygen tension at the cell surface (Fig.3). In both cases the minimum concentration at the cell surfacewas reached at sulfide concentrations of around 20 µM(24 and 18 µM, respectively). Together with the findingthat oxygen uptake rates did not increase with sulfide concentrationsabove 37 µM, this suggests that under our experimentalconditions T. namibiensis reached maximum sulfide fluxes ata sulfide concentration of around 20 µM even though ittolerates much higher concentrations. After both sulfide pulsesthe oxygen concentration at the cell begun to rise again afterthe sulfide concentration dropped below approximately 9 µM,indicating that this sulfide concentration was too low to supportmaximal oxygen uptake rates. The oxygen concentration returnedto the former value only 9 h after sulfide was completely removedfrom the medium (Fig. 3). Possibly, the accumulation of elementalsulfur under aerobic conditions with sulfide present led tothe long-term effect of the sulfide pulses on the uptake rateof oxygen. A similar experiment in which oxygen was added underanoxic, sulfidic conditions resulted in a single drop and risein the sulfide concentration at the cell surface (data not shown).

Implications.

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Implications.

The results of our experiments show that even though T. namibiensiscells store high concentrations of nitrate, they can take upoxygen in the presence or absence of nitrate. As the oxygenuptake is greatly stimulated by the presence of sulfide andvice versa, oxygen seems to be used as an electron acceptorfor the oxidation of sulfide, even though the sediments in whichT. namibiensis is found are permanently anoxic. These observationssuggest that even though most of the time T. namibiensis cellssurvive in sediments containing high sulfide concentrationswith internally stored nitrate as their sole electron acceptor,they may be physiologically most active during times when thesediment is suspended. Suspension of sulfidic mud in oxic seawatershould provide the cells with the opportunity to oxidize sulfidewith oxygen. Even though such events may be relatively infrequent,the rapid response to oxygen and the relatively high uptakerates suggest that these short periods might be the major timesof energy gain, whereas nitrate might be used primarily to survivethe time between sediment suspension events.

ACKNOWLEDGMENTS

This work was financed by the Max Planck Society.

We thank Anja Eggers, Gabi Eickert, and Ines Schröder forconstruction and help with the use of microelectrodes, HellePloug for help with calculations, and Doug Nelson for editingand for many helpful suggestions to improve the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Section of Microbiology, University of California, Davis, One Shields Avenue, Davis, CA 95616. Phone: (530) 752-0283. Fax: (530) 752-9014. E-mail: hschulz{at}ucdavis.edu.

REFERENCES

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