Coexistence of aerobic chemotrophic and anaerobic phototrophic sulfur bacteria under oxygen limitation

ELSEVIER

FEMS Microbiology Ecology I9 (1996) 14I- I5 1

Coexistence of aerobic chemotrophic and anaerobic phototrophic sulfur bacteria under oxygen limitation Frank P. van den Ende *, Anniet M. Laverman, Hans van Gemerden Department of Microbiolog!. Linkersi& of Groningen, Kerklaan 30. 9751 NN Hot-en. The Netherlands

Received 11August 1995: revised 2 November

1995; accepted 3 November

I995

Abstract The aerobic chemotrophic sulfur bacterium Thiobacillus fhioparus T5 and the anaerobic phototrophic sulfur bacterium Thiocupsa roseopersicina M 1 were co-cultured in continuously illuminated chemostats at a dilution rate of 0.05 h ‘. Sulfide was the only externally supplied electron donor, and oxygen and carbon dioxide served as electron acceptor and carbon source, respectively. Steady states were obtained with oxygen supplies ranging from non-limiting amounts (1.6 mol O2 per mol sulfide, resulting in sulfide limitation) to severe limitation (0.65 mol O2 per mol sulfide). Under sulfide limitation Thiocapsa was competitively excluded by Thiobacillus and washed out. Oxygen/sulfide ratios between 0.65 and 1.6 resulted in stable coexistence. It could be deduced that virtually all sulfide was oxidized by Thiohacillus. The present experiments showed that Thiocupsa is able to grow phototrophically on the partially oxidized products of Thiobacilltts. In pure Thiohucillus cultures in steady state extracellular zerovalent sulfur accumulated, in contrast to mixed cultures. This suggests that a soluble form of sulfur at the oxidation state of elemental sulfur is formed by Thiobacillus as intermediate. As a result, under oxygen limitation colorless sulfur bacteria and purple sulfur bacteria do not competitively exclude each other but can coexist. It was shown that its ability to use partially oxidized sulfur compounds, formed under oxygen limiting conditions by Thiohucillus, helps explain the bloom formation of Thiocupsu in marine microbial mats. Kemmds:

Thiobacillus;

Thiocapsa:

Oxygen

limitation:

Sulfide oxidation;

1. Introduction In microbial sulfidic layers,

Simultaneously, dantly present reduced sulfur petition would low substrate Jergensen and

mats, purple

provided light sulfur bacteria

penetrates to often bloom.

colorless sulfur bacteria are abunin these systems. Both groups exploit compounds and, consequently, combe expected to occur, particularly at concentrations. Jorgensen [I], and Des Marais [2] evaluated the competi-

* Corresponding author 016%6496/96/$15.00 0 1996 Federation SDf 0168-6496(95)00082-S

of European

Microbiological

Marine microbial

mat

tion between colorless sulfur bacteria and purple sulfur bacteria in marine microbial mats (Kal@ Vig, Denmark and Guerrero Negro, Mexico) in relation to environmental parameters. The mats which were studied, situated in areas without tidal movements, were permanently overlain by a thin layer of water and dominated by the purple sulfur bacterium C/zromatium and the colorless sulfur bacterium Beggiatna. These organisms exhibited diurnal migration patterns, and positioned themselves in narrow bands where the prevailing conditions were’ optimal for their development. As a result of their different nutritional preferences, particularly with-’ respect to Societies.

AH rights reserved

142

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Microbiology

oxygen, Chromatium and Beggiatoa were often spatially separated, and direct competition for reduced sulfur compounds thus appears to be of less paramount importance. In microbial mats developing on sandy intertidal sediments in temperate zones the dominant purple sulfur bacterium often is Thiocapsa roseopersicina. This has been reported for the North Sea barrier islands of Mellum [3], Schiermonnikoog [4], and Texel [5], as well as for salt marshes in Cape Cod, Massachusetts [6], and sandy beaches on the Orkney Islands [7]. The type of aerobic sulfide-oxidizing bacteria which dominate these systems is less well documented. Data collected on North Sea barrier islands showed small motile thiobacilli to develop profusely, whereas only a few Beggiatoa filaments were found ([5], unpublished observations). Contrary to Chromatium. Thiocapsa is an immobile organism. Cells are known to produce a slime layer [8] and have been reported to cement sand grains together thus forming small clumps [9,10]. In mats developing on the barrier island of Schiermonnikoog aggregates up to 0.2 mm were observed (unpublished). In intertidal environments. aggregate formation is an advantage, since it effectively prevents the organism from being washed away with the tidal currents [9,1 I]. A disadvantage, however, is that immobile organisms cannot actively position themselves towards layers with optimal conditions. Microelectrode measurements have shown that strongly fluctuating conditions occur at the depth horizons where the organisms were found [4,12]. As a consequence. in these ecosystems the organisms were not concentrated in a narrow band, but rather were distributed over a layer of several millimeters thickness [4]. In these microbial mats purple sulfur bacteria and colorless sulfur bacteria thrive in the same depth layers and thus will compete for mutual substrates under a wide range of conditions. However, data collected on the depth distributions of these organisms unambiguously show that their coexistence is a common phenomenon. Previously, experiments were carried out with pure cultures of the obligate aerobic chemotrophic sulfide oxidizer Thiobacillus thioparus TS and the facultatively anaerobic phototrophic sulfide oxidizer Thiocapsa roseopersicina Ml. These studies have shown that Thiobacillus has a higher affinity for

Ecalog~

19 llYY61

i-f-15/

thiosulfate [5], and the same is true for sulfide ([ 131, M.T.J. van der Meer. unpublished observations). Therefore it was expected that in the presence of oxygen Thiocapsa would be outcompeted by Thiobacillus. However, at oxygen-sulfide interfaces the activity of aerobic sulfide-oxidizing bacteria is restricted by the limited availability of oxygen. Sulfide oxidation particularly takes place at the oxygen sulfide interface [12,14-161. For that reason it was anticipated that oxygen limitation could play an important role in the interactions between chemotrophic and phototrophic sulfide-oxidizing bacteria. Oxygen limitation experiments carried out with pure cultures of Thiobacillus thioparus T5 have shown that this organism is able to lower the concentration of sulfide to undetectable levels, even when oxygen is severely limiting [16]. However, under the latter conditions sulfide is not fully oxidized to sulfate, thus yielding reduced sulfur compounds other than sulfide, which are potential electron donors for purple sulfur bacteria [16]. In this study, experiments were performed in continuous culture with mixed populations of the chemotrophic sulfur bacterium T. thioparus T5 and the phototrophic sulfur bacterium T. roseopersicina MI. In these experiments the rates of supply of oxygen and sulfide were varied in order to obtain different oxygen/sulfide ratios. The experiments were carried out to find a clue to the better understanding of the coexistence of colorless sulfur bacteria and purple sulfur bacteria in microbial mats.

2. Materials

and methods

2.1. Bacterial strains Experiments were performed with the colorless sulfur bacterium Thiobacillus thioparus strain T5, isolated from a marine microbial mat on the island of Texel, The Netherlands [5], and the purple sulfur bacterium Thiocapsa roseopersicina strain Ml, isolated from a marine microbial mat on the island of Mellum, Germany [ 171. 2.2. Culture conditions The organisms were grown in continuous culture at a constant dilution rate of 0.05 h-’ with sulfide as

F.P. fan den Ende et al. / FEMS Microbiology

the only externally supplied electron donor, and oxygen and carbon dioxide as electron acceptor and carbon source, respectively. The culture was continuously illuminated at an intensity of 40 PE m-’ s-‘. The composition of the mineral medium was as described before [16], except for the sulfide concentration (see Table 2). The liquid culture volume was 1000 ml, with a headspace of 500 ml. The headspace was flushed with an adjustable water-saturated air/nitrogen gas mixture at a rate of 40 1 hh’. The medium was pumped from two reservoir bottles at equal rates. One bottle contained a double-strength solution of sulfide and carbonate at pH 11, the other contained a double-strength solution of the remaining constituents at pH 4.5. Temperature of the culture was kept constant at 25 f O.l”C, and the pH was maintained at 8 f 0.04. Stirring speed and gas flow rate were kept constant. Thiobacillus was maintained in continuous culture, whereas Thiocapsa was routinely grown in batch culture. To obtain a mixed culture, half the volume of the Thiobacillus culture was removed and replaced by the same volume of a growing batch culture of Thiocupsa. 2.3. Oxygen /sulfide

ratios

Mixed cultures of Thiobacillus and Thiocapsa were subjected to different oxygen to sulfide supply ratios, and were sampled after a steady-state situation had established. By applying a fixed dilution rate and a constant concentration of sulfide in the reservoir solution (SR_\u,fide), a constant sulfide supply rate was maintained. The oxygen supply rate was regulated by the composition of the air/nitrogen gas mixture in the headspace. Oxygen consumption was calculated according to (G,o,s,rxru,, - G,.cu,,u,,) X K,a X H, in which O,.,,, M,XTt,REis the percentage of air in the gas-mixture. 02_cuLTuRE is the oxygen concentration in the culture fluid as percentage of air saturation, K,a is the gas-transfer coefficient (in min- ’ ), and H is the conversion factor from air saturation (%) to oxygen concentration (in PM). The oxygen concentration in the culture was determined with an oxygen electrode (Ingold, Switzerland). Since the presence of Thiocapsa appeared to influence the gas-transfer coefficient, K,a was determined sepa-

Ecology

19 (1996) 141-151

143

rately after each steady state, according to Pirt [ 181. H was determined using the Winkler titration [19]. 2.4. Analytical

procedures

Sulfide was measured calorimetrically using the methylene-blue method of Pachmayr [20]. Sulfate was measured calorimetrically as chloranilate according to Bertolacini and Barney [21]. Zerovalent sulfur and bacteriochlorophyll a (BChla) were determined spectrophotometrically in methanol extracts [22]. Thiosulfate, tetrathionate and polysulfides were measured calorimetrically after cyanolysis of 0.2 pm filtered samples [23,24]. Incubations were as described previously [16]. The same method was used to check for the presence of non-pelletable zerovalent sulfur, using 60 min incubation at 90°C at pH 8.7. Protein was assayed with the Folin phenol reagent [25] on cell pellets solubilized in 1 M NaOH, after removal of zerovalent sulfur by methanol extraction. Glycogen was measured as total hexose with the anthrone reagent [26]. Corrections for nonglycogen sugars derived from structural cell material were made based on protein measurements. Conversion factors applied were 0.176 mg sugar mg- ’ protein for Thiocupsa cells [17], and 0.045 mg sugar cells. Cells were mgg ’ protein for Thiobacillus counted using an electronic particle counter (Coulter Counter, model ZM, equipped with a Chanalyzer 256). Control counts were done with a phase-contrast microscope in a Biirker-Turk counting chamber for Thiocapsa, and by epifluorescence microscopy after staining with DAPI 1271 for Thiobacillus. 2.5. Redox conversion factors The following conversion factors construct the redox balances: 8 mol sulfide, 6 mol e- per mol zerovalent mol e- per g cell-protein [28], 0.136 glycogen [29].

were used to e- per mol sulfur, 0.456 mol e- per g

3. Results Attempts were made to co-culture Thiobacillus thioparus T5 and Thiocapsa roseopersicina Ml at four different oxygen/sulfide ratios: three of them resulted in stable coexistence of the two organisms.

I44

F.P. LYW den Ende et al./ FEMS Microbiology

Ecology 19 f 1996) 14/L151

Table 1 Rates of oxygen and sulfide supply applied to mixed cultures of Thiobncillus and Thiocnpsa grown at a dilution rate of 0.05 l-0.053 and the resulting steady state concentrations of BChln, protein. glycogen. and the number of cells Ratio 02/S’_

Supply rates (FMmin-‘)

tmmol/mmolf

02

I .56 1.33 0.91 0.65

13.0 10.6 7.0 4.9

BChlcr (/Lgl_‘)

Protein (mg I-‘)

Glycogen (mg I-‘)

0 166 625 1066

25.5 35.4 440 51.8

0 12.5 28.9 37.9

Supplying the culture with 100% air in the headspace, which allowed a maximum attainable oxygen supply rate of 16.6 Frnol min- ’ , in combination with a concentration of sulfide in the reservoir solution of 9.7 mmol l-‘, which gave a sulfide supply rate of 8.3 pmol min-‘, resulted in complete wash out of the phototroph. Under these conditions. the limiting factor for T~ziobacillus was sulfide (steady-state concentration < 1.5 PM), rather than oxygen (steady-state concentration 47 PM). The observed rate of oxygen consumption (13.0 pmol min-‘) was lower than the maximum supply rate, whereas the sulfide uptake rate equalled the supply rate, resulting in an oxygen/sulfide ratio of 1.56 mm01 mmol- ’ (Table 1). Most of the sulfide supplied was oxidized to sulfate, a low concentration of zerovalent sulfur was also encountered, whereas the concentrations of polysulfide (SE-), thiosulfate (S,OiP) and tetrathionate (S,Oi-) were even lower (Table 2). At oxygen/sulfide ratios of 1.33 and lower, resulting in oxygen limitation rather than in sulfide limitation, stable coexistence was observed. With decreasing oxygen availability. population densities

Table 2 Steady state concentrations

of sulfur compounds s’-

( PM)

( FM)

St(firno

1.56 1.33 0.91 0.65

9720 9060 8528 8453

< < < <

6 4 2 2

The concentration

1.5 1.5 1.5 1.5

of sulfide in the in-flowing

S I-‘)

medium (S,)

Tiziobaciif~ts

Thioccqxw

12 10 3.8 3.1

0

I .5 2.1 3.3

of Thiobacillus decreased concomitant with an increasing cell number of Thiocupsa and increasing concentrations of BChla (Table 1). Under these conditions sulfur speciation changed from almost complete oxidation to sulfate to increasing concentrations of zerovalent sulfur, reaching a maximum concentration of 1.9 mmol So 1-l at O,/H,S of 0.65 mmol mm01 - ’ (Table 21, but sulfate remained the main product of sulfide oxidation. Concentrations of sulfide in the cultures were below the detection limit at all conditions. The recovery data show that no other sulfur species were present (Table 2). 3. I. Su&r

balances

In order to facilitate a proper comparison between data collected with slightly different sulfide concentration in the in-flowing medium, sulfur species were calculated as a percentage of SR_\u,fide (Fig. 1, top panel). Under sulfide limitation, resulting in the competitive exclusion of Thiocupsa, 96% of the in-flowing sulfide was oxidized to sulfate, and 4% to zerovalent sulfur. With decreasing oxygen supply rates product formation shifted to sulfur, which max-

in mixed cultures of Thiobacillus

Ratio o?/sz~mmol/mmol)

sR

Cell number (NX 10” 1-1)

S’8.3 8.0 1.5 1.5

h-' ,

and Thiocapsa at different oxygen/sulfide

supply ratios

s,of( /X)

s,o;( /.LM)

S” (/*mol S I-‘)

so;( /AM)

S-recovery (%ofSa)

15 6 9 9

9 5 10 10

348 500 2240 1930

9300 8700 6200 6500

100 102 100 101

and sulfur recovery

balance are also shown.

F.P. can den Ende et al./ FEM.7 Microbiology Ecology 19 (1996) 141-151

145

imally was 26% of SR_ru,fide,whereas sulfur present in thiosulfate, polysulfide, and tetrathionate together not exceeded 0.8% of SR__ifide (Fig. 1, top panel). As shown in Table 1, decreased oxygen availability resulted in increased Thiocapsa cell numbers. Microscopical observation of these co-cultures revealed that virtually all zerovalent sulfur was present as intracellular sulfur in Thiocapsa (Fig. 2B). Extracellular sulfur globules were not observed, nor did sulfur precipitate on the wall of the culture vessel, a well-known nuisance in pure cultures of Thiobacillus. No visible extracellular sulfur was pelleted after centrifugation of samples, and the negative results of cyanolysis after 0.2 pm-membrane filtration showed that very small granules of sulfur were absent as well. It thus appears that in co-cultures subjected to

1.5

1.5

1.4

1.4

1.3

1.3

1.2

1.1

1

0.9

0.8

1.2 1.1 1 0.9 0.8 /Sulfide (mmol / mmol)

0.7

0.7

RatioOxygen

Fig. 1. Effect of increasing oxygen limitation on speciation of sulfur compounds formed during sulfide oxidation in mixed cultures of Thiobacillus and Thiocapsa (top panel). and pure cultures of Thiobacillus (bottom panel). Values are calculated from steady-state concentrations (Table 2 and [16]) and expressed as a percentage of the sulfide concentration in the in-flowing medium to allow direct comparison. The degree of oxygen limitation is quantified as the amount of oxygen supplied per sulfide supplied.

Fig. 2. Light micrographs of sulfide oxidizing cultures grown under oxygen limitation. (A). Pure culture of Thiobacillus. Amorphous clumps of extracellular zerovalent sulfur can be seen between cells. (B). Mixed culture of Thiobacillus and Thiocapsa. Intracellular zerovalent sulfur can be seen as black globules inside Thiocapsa cells, whereas extracellular zerovalent sulfur is absent (bar = 10 Km).

oxygen limitation, Thiocupsa was growing, at least in part, on sulfur species that resulted in intracellular sulfur formation. The lower panel of Fig. 1 shows data collected in pure cultures of Thiobacillus subjected to similar conditions. These data show that with decreasing oxygen availability, increasing amounts of sulfide were not oxidized beyond the level of zerovalent sulfur, which in this case was exclusively present as extracellular sulfur (Fig. 2A). Maximally 15-20% of SR_su,fidewas oxidized to thiosulfate. As was the case in the co-cultures, sulfide was below the detection limit at all times. It remains to be elucidated which sulfur species resulted in the

growth of and glycogen formation when co-cultured with Thiobacillus.

by

Thiocapsa

3.2. Redox balances When the oxygen supply rate was reduced to values below 1.6 mol oxygen per mol sulfide, the oxygen concentration in the culture was below the detection limit. Thus, all oxygen supplied was completely consumed. Under these conditions a stable coexistence of Thiobacillus and Thiocapsa was observed. Like Thiobacillus, Thiocapsa can use oxygen for sulfide oxidation [ 17,301. However, when the specific content of BChla is sufficiently high. Thiocapsa will grow fully phototrophically even in the presence of oxygen [28]. At light saturation the threshold value lies between 1.9 and 4 pug BChla mg-’ protein [31,32]. Even if all protein in the oxygen-limited steady states were accounted to Thiocapsa, the BChl u/protein ratio would never be lower than 4.7 ,ug mg-’ (Table l), thus indicating that BChla concentrations are high enough for full phototrophic growth of Thiocupsa. It thus could be deduced that all oxygen was consumed by Thiobacillus. In order to establish the fate of reducing equivalents in co-cultures of Thiobacillus and Thiocapsa, redox balances were calculated (Fig. 3). The protein assay does not discriminate between Thiocapsa and Thiobacillus material. Cell numbers (Table 1) cannot easily be transferred to biomass due to differences in cell size (Fig. 4). Direct conversion of biovolume to biomass is hampered by inaccuracy of volume determination of the small Thiobacillus cells. However, the contribution of Thiobacillus to total protein can be calculated from the measured oxygen consumption rates (Table 1). In the sulfide-limited steady states, Thiocapsa was washed out completely, and the protein formed (25.5 mg 1-l > thus originated exclusively from Thiobacillus. The oxygen consumption rate was 13.0 pmol mini ’ (780 pmol hh ’ ). Dividing this figure by the dilution rate, the oxygen consumption per liter culture medium (15.3 mmol O2 1-l > was obtained. Since four reducing equivalents are needed to reduce molecular oxygen, the yield of Thiobacillus was calculated to be 0.42 mg protein per mol reducing equivalents transferred to oxygen (25.5/(4 x 15.3)). P revious pure culture experiments

2 90 .k

60

1

”-

1.5

1.4

1.3

1.2

1.1

Ratio Oxygen /Sulfide

1 0.9 0.8 (mmol I mmol)

0.7

Fig. 3. Fate of reducing equivalents available from sulfide oxidation with increasing oxygen limitation in mixed cultures of Tbiobucillus and Thiocapsa. 100% (solid circles) represents full oxidation of all sulfide supplied to sulfate. Upper panel: reducing equivalents associated with Thiobacillus. Values are presented cumulative for biomass and respiration. Solid squares represent reducing equivalents used for respiration and biomass in pure cultures of Thiobacillus [16]. Lower panel: reducing equivalents associated with Thiocapsa. Values are presented cumulative for biomass, glycogen and intracellular zerovalent sulfur. Solid squares represent reducing equivalents in extracellular zerovalent sulfur and thiosulfate in pure cultures of Thiobacillus [ 161.

with T. thioparus T5 under oxygen limitation have shown that this yield is independent of the degree of oxygen limitation [16]. In combination with the measured oxygen consumption rates, this yield allowed for the calculation of the Thiobacillus protein in the oxygen-limited mixed cultures. The remainder of the protein was attributed to Thiocapsa. Thiocapsa protein figures obtained in this way agreed well with those based on the concentration of BChl a (Table I > using the average BChla content in light-saturated anoxic sulfide-limited cultures of T. roseopersicina Ml (21.4 pg BChla mgg’ protein) ([13], supplemented with unpublished BChla data). A conversion

F.P. mn den Ende et al./FEMS

Microbiology

factor of 0.456 mmol e- mgg ’ protein was applied to calculate the amount of reducing power used for biomass formation in Thiocupsa [28]. The results (Fig. 3) show that all reducing equivalents that could not be used by Thiobacillus due to electron acceptor (oxygen) limitation are found associated with Thiocapsa. As shown in the top panel of Fig. 3, most of the reducing equivalents that allow for the synthesis of biopolymers of Thiocapsa are the result of reduced respiration rates of Thiobacillus and to a far lesser extent the result of a lower biomass of the colorless sulfur bacterium. The in-

CELL NUMBER

Ecology 19 (1996) 141-151

147

creased availability of reducing equivalents for Thiocapsa not only results in increased concentrations of structural cell material, but as well in increased storage of elemental sulfur and glycogen, as is observed in other anoxygenic phototrophs [33]. Thiobacillus and Thiocapsa are sufficiently different in size to allow for electronic sizing. As shown in the left panels of Fig. 4, a decrease in the cell number of Thiobacillus with decreasing O,/H,S ratios was accompanied by an increase in Thiocapsa cell numbers. The average cell size of Thiobacillus reduced with increasing oxygen limitation, whereas

:: ;:..., T

(N*lO%

:o;:;*s;i11;6

:::. ..,, :::: ::::

:: :

i

j

jj;;

:

:

: : : :

: : :

: :::: ; jjjj jiiii

: :

:.:::: :j::::

;

jjijii

: j

:::: :::: ::::

:::: :::: : ::: ::::

j

j

;jj;

: :

: :

: : : : ::::

:::

: :::::::

“’ ::: :::

: ::

:::: ::::

\

::::

,I

; 02 HzS=0.65 ::: ::.: : ::;j: :: ::::: :::: :::: j ; ::j:;; jjjj :::: .:: ::::: ::.: :: : : : : : : i : ::::: i:ji ; : :::, j:ji :::: : j ii;; :::: __,, jj::

_jibTr :

::: “’

::: ,,. ..: ::: ::: ::: ::: ::.

1

CELL SIZE (urn?

10

k

0.1

CELL SIZE f.um9)

Fig. 4. Cell number and biovolume of Thiobacillus and Thiocapsa in mixed cultures at different ratios of oxygen supplied per sulfide. Left panels: distribution of cell numbers (N lo9 I- ‘) over 138 size classes. Data presented in left panels were used to calculate biovolume ( ~1 I- ’ ) shown in right panels.

148

F.P. ran den Ende et al./FEMS

Microbiology

Thiocupsa cell size increased. Taking the cellular volume into account, the total biovolume of each population in the co-cultures can be calculated (Fig. 4, right panels). Changes in the total Thiocupsa biovolume agreed with the calculated biomass changes (Fig. 3). Although the biovolume determination of Thiobacillus cells is somewhat less accurate due to the small individual cell size of these chemotrophs, the data clearly show that the increase in Thiocupsu biomass is much larger than the decrease in Thiobucillus biomass, again reflecting the large proportion of reducing equivalents used by Thiobucillus for respiration.

4. Discussion 4.1. Competition for sulfide Thiobucillus and Thiocupsu

in

co-cultures

qf

Both Thiobucillus and Thiocapsa can oxidize sulfide. During steady-state growth in continuous culture the outcome of competition for the limiting substrate can be explained by the affinity of the competing organisms to this substrate. Substrate affinity during balanced growth is determined by the initial slope of the Monod-equation /.L,,,/( K, - s), with s approaching zero. Thiobucillus has a maximum specific growth rate ( /_L,,,) on sulfide of 0.58 hh’, and a saturation constant (K,) of 2.35 PM (M.T.J. van der Meer, unpublished), resulting in an affinity to sulfide of 0.247 hh’ pmol-’ 1. Thiocupsu has a pmax on sulfide of 0.09 hP ‘, and a K, of 8.0 PM [ 131, resulting in an affinity to sulfide of 0.011 hh ’ pmoll’ 1. Thiobucillus thus has a 20-fold higher affinity for sulfide than Thiocupsn. It is therefore not surprising that Thiobucillus completely outcompeted Thiocupsa under sulfide limiting conditions (O,/H,S = 1.56, Fig. 4). Under oxygen limiting conditions a stable coexistence of Thiobacillus and Thiocupsa developed. Sulfide was consumed completely in these mixed cultures (Table 2). Since the affinity for sulfide of Thiobucillus under oxygen limitation is not exactly known, a direct comparison of affinities as in the case of sulfide limitation is difficult. However, sufficient data are available from pure culture studies to allow an interpretation of the fate of sulfide in the

Ecology 19 (1996) 141-151

mixed cultures. In sulfide grown pure cultures of Thiobacillus subjected to oxygen limitation virtually all sulfide was consumed; however, part of the sulfide was oxidized to sulfur species less oxidized than sulfate [ 161. Steady-state sulfide concentrations were below 1 PM at oxygen/sulfide ratios exceeding 0.6 mmol mmol- ’ [ 161. In sulfide-limited. phototrophitally growing cultures of Thiocupsa the steady-state sulfide concentration at a dilution rate of 0.05 h-’ was 10 PM [ 131. The concentration of sulfide was below the detection limit (1.5 PM) in all steady-states of the mixed cultures (Table 2). Substituting pmax and K, in the Monod-equation with the values for Thiocupsu mentioned earlier. it can be calculated that with a sulfide concentration of 1.5 PM Thiocupsa maximally can attain a growth rate of 0.0014 hh’, which is insufficient to maintain itself in the cultures at the dilution rate of 0.05 hh ’ used here. For kinetic reasons it is therefore expected that in mixed cultures Thiobucillus was not hindered in the use of sulfide as a substrate by the presence of Thiocupsu. Apparently, the observed population of Thiocupsu is to be explained by the utilization of other sources of reducing equivalents than sulfide. However, given its biomass, Thiocupsu will use some sulfide, especially at strong oxygen limitation. 4.2. Substrates Thiobucillus

used by Thiocupsu in co-cultures

with

Considering the conclusion that Thiobucillus used most of the sulfide supplied, it appears that the Thiocupsu biomass is the result of the consumption of partial oxidation products formed by Thiobacillus. If Thiobucillus would form the same products in mixed cultures as in pure cultures a small population of Thiocupsu could be expected to develop as the result of thiosulfate oxidation, and possibly by oxidizing part of the zerovalent sulfur formed. Instead, a substantial population of Thiocupsu was found, and most of the sulfide supplied was found to be completely oxidized to sulfate (Fig. 1). As in pure cultures of Thiobucillus, zerovalent sulfur was present in the mixed culture, however in this case exclusively as intracellular reserve in Thiocupsu. Judged from the absence of extracellular sulfur and thiosulfate in the mixed culture (Fig. 1, top panel), Thiocups~7 was apparently able to use all of the remaining

F.P. can den Ende et al./ FEMS Microbiology Ecology 19 (1996) 141-151

reducing equivalents that could not be used by Thiobacillus due to shortage of oxygen (Fig. 3). The utilization of these product(s) by Thiocapsa predominantly resulted in the formation of biomass and glycogen, but intracellular sulfur was present as well (Fig. 1, top panel). In pure cultures of Thiobacillus extracellular zerovalent sulfur was the main product of sulfide oxidation under oxygen limitation. This sulfur was present as globules associated with individual Thiobacillus cells, after some time resulting in large detached aggregates (Fig. 2A). In mixed cultures of Thiobacillus and Thiocapsa extracellular zerovalent sulfur globules only were observed transiently after reduction of the rate of supply of oxygen and not during steady states (Fig. 2B). T. roseopersicina is known to be able to grow on particulate S” [8], however, growth on this substrate is extremely slow and, more importantly, does not result in storage of intracellular sulfur. It thus can be ruled out that Thiocapsa was using particulate S” as electron donor. In addition, if formed in mixed cultures, at least some sulfur particles should have been visible, or, when too small to observe microscopically, a measurable amount should have been found in the culture fluid. Since this was not the case it can be concluded that Thiobacillus in oxygen-limited steady states of mixed cultures formed a soluble reduced sulfur compound, rather than particulate S”. The nature of this soluble product, formed by Thiobacillus and consecutively used by Thiocapsa, remains unknown. In view of their role as precursor in the formation of elemental sulfur, polysulfides (H-S,-H) and thionates (H-S,SO,) are possible candidates (R. Steudel, personal communication). A schematic representation of the interactions is given in Fig. 5. 4.3. Why is Thiocapsa successful bial mats?

in intertidal micro-

Thiocapsa is an immobile organism. Its depth distribution in a marine microbial mat was studied in detail by De Wit et al. [4] where it was reported to occur from 1.5 to 5 mm depth, with 85% of the population concentrated between 2 and 3 mm depth. At night oxygen penetration in the sediment was less than 1 mm. However, when surface irradiance exceeded 200 PE mm2 s- ’ (one hour after sunrise to

149

Fig. 5. Schematic representation of the interactions between Thiobacillus and Thiocapsa under oxygen limitation. See text for details.

one hour before sunset), oxygen was detected down to 3-3.5 mm depth. Thus, the majority of the population was facing the presence of oxygen during most of day, whereas anoxic conditions were mostly prevailing at night. Regarding a 24-h period, Thiocapsa is hindered in one way or another for most of the time. During the night the uptake of substrates is not feasible. In the presence of oxygen (daytime) dissimilatory sulfate reduction continues, although at lower rates than under fully anoxic conditions [35,36]. However, little of the sulfide produced can be expected to reach the anoxygenic phototrophs because of their low affinity compared to that of colorless sulfur bacteria [5,37,38]. This competitive disadvantage is not eliminated before anoxia coincides with the availability of light. In the intertidal microbial mats studied on the North Sea barrier islands, such favorable conditions occur only for short periods just after sunrise and just before sunset [4,12]. During these periods Thiocapsa takes full advantage of its hoarding capacity, i.e. sulfide is oxidized no further than sulfur, which is stored inside the cells, and CO, fixation results in the intracellular deposition of glycogen rather than in the formation of structural biomass. With respect to the utilization of small organic compounds (i.e. acetate) it is anticipated that anoxygenic phototrophs are unable to compete successfully with aerobic heterotrophs.

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Although during the oxic light period growth of T&~upsa on external substrates is restricted it has the capacity to grow phototrophically in the presence of oxygen using intracellularly stored zerovalent sulfur as electron donor [5]. During the anoxic dark conditions it has the ability to synthesize BChlcr and to grow at the expense of stored glycogen [34]. Thus for most of the day growth of Thiocapsa depends on intracellular storage compounds rather than external supply of reducing power. In order to be able to use these capacities it is fully dependable on the short favorable periods at sunrise and sunset to replenish the stock of zerovalent sulfur and glycogen. However, the present experiments show that not only anoxic light periods but also the periods of oxygen limitation can be used to this end. Thus, the period that can be used to load the cells with reserve materials is extended. These observations help to understand the blooming of Thiocapsa in marine microbial mats.

Acknowledgements The authors would like to thank Bart E.M. Schaub for cell volume measurements. This study was carried out as part of the EC MAST II project “Oxicanoxic interfaces as productive sites” (contract no. MAST2-CT-93-0058).

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