H Vangemerden

The sulfide affinity of phototrophic bacteria in relation to the location of elemental sulfur

Seventeen strains of phototrophic bacteria (4 strains of Chromatium spp., 2 strains of Thiocapsa sp., 4 strains of Ectothiorhodospira spp., 2 strains of Rhodopseudomonas sp., and 5 strains of Chlorobium spp.) have been grown in sulfide-limited continuous cultures to assess the affinity for sulfide. It was found that the affinity (calculated as the initial slope of the specific growth rate versus the concentration of sulfide) is higher in those phototrophic bacteria that deposit elemental sulfur outside the cells, than in those bacteria that store the sulfur inside the cells. A hypothesis is presented to explain this correlation.

Impact of light/dark regimen on growth rate, biomass formation and bacteriochlorophyll synthesis in Erythromicrobium hydrolyticum

The impact of illumination on specific growth rate, biomass formation, and synthesis of photopigment was studied in Erythromicrobium hydrolyticum, an obligately aerobic heterotrophic bacterium having the ability to synthesize bacteriochlorophyll a. In dark-grown continuous cultures the concentration of protein increased with increasing dilution rate, the concentration of bacteriochlorophyll a showed the opposite effect. At a dilution rate of 0.08 h-1 (68% of μmax in the dark) and SR-acetate of 11.8 mM, the concentration of BChla of illuminated cultures in steady-state was 11–22 nM, compared to 230–241 nM in cultures incubated in darkness. No significant differences were observed in the concentration of protein. A shift from darkness to light conditions resulted in increased specific growth rates resulting in increased biomass formation, thus showing that light enhances growth by serving as an additional energy source. This phenomenon, however, was temporary because bacteriochlorophyll synthesis is inhibited by light. In contrast to incubation in continuous light or dark, incubation under light/dark regimen resulted in permanently enhanced biomass formation. In the dark periods, bacteriochlorophyll was synthesized at elevated rates (compared to constant darkness), thus compensating the inhibitory effect of light in the preceding period. It thus appears that the organism is well-adpated to life in environments with alternating light/dark conditions. The ecological relevance of the observations is discussed.

SEARCH FOR POLYTHIONATES IN CULTURES OF CHROMATIUM-VINOSUM AFTER SULFIDE INCUBATION

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S6, S7 S8 rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (Sx2−), polythionates (SnO62−, n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.

GROWTH AND METABOLISM OF THE PURPLE SULFUR BACTERIUM THIOCAPSA-ROSEOPERSICINA UNDER COMBINED LIGHT DARK AND OXIC ANOXIC REGIMENS

The dominant purple sulfur bacterium of laminated sediment ecosystems in temperate environments, Thiocapsa roseopersicina, was cultivated in sulfide-limited continuous cultures (D=0.03 h-1) subjected to various combined diel regimen of aeration and illumination in order to simulate environmental conditions in microbial mats. For comparison, cultures were grown under similar illumination regimens but continuously anoxic conditions.Bacteriochlorophyll a (BChla) and carotenoid synthesis was restricted to anoxic-dark periods and did not occur during oxic-light periods. An increase in the length of the oxic-light periods resulted in decreased pigment contents. However, phototrophic growth remained possible even at 20 h oxic-light/4 h anoxic-dark regimens. When anoxic conditions were maintained throughtout, BChla synthesis occurred both during light and dark periods.Glycogen was synthesized in the light and degraded in the dark. Calculations showed that degradation of 1/4–1/5 of the glycogen is sufficient to account for the BChla and carotenoid synthesis in the dark.The data showed that T. roseopersicina is very well adapted to cope with the combined oxygen and light regimes as they occur in microbial mats, which may explain the dominance of this bacterium in the purple layer of these sediment ecosystems.

SPECIFIC RATES OF SUBSTRATE OXIDATION AND PRODUCT FORMATION IN AUTOTROPHICALLY GROWING CHROMATIUM-VINOSUM CULTURES

Kinetics ofChromatium metabolism under autotrophic conditions were studied in batch culture. Description of various over-all metabolic rates in equal dimensions enabled direct comparison of such different processes as electron donor oxidation, storage-polymer synthesis, and growth. In the presence of sulfide, the specific rate of sulfur oxidation is partly depressed. Upon sulfide depletion, the latter rate showed an instantaneous increase of 22%. Simultaneously, glycogen was degraded. These two modifications fully compensate for the absence of sulfide, thus enabling growth to continue withμmax (0.12 h-1). These findings indicate that the potential rate of supply of electrons exceeds the biosynthetic demand for reducing power. The synthesis of glycogen in the presence of sulfide may be regarded overflow metabolism, assuming theμmax is intrinsically limited at 0.12 h-1. An alternative hypothesis is based on the assumption of an ineffective glycogen-synthesis regulation. Conceivably, growth and glycogen synthesis are competing for biosynthetic intermediates, the supply of which is limited by the maximum specific rate of e.g. the Calvin cycle. If so, both growth and glycogen synthesis are performed at submaximal rates. The rate of glycogen synthesis is greatly enhanced by the addition of growth inhibitors, and an increasedμmax value is found in the presence of acetate. These two findings together are consistent with the second hypothesis.

ACTUAL AND POTENTIAL RATES OF SUBSTRATE OXIDATION AND PRODUCT FORMATION IN CONTINUOUS CULTURES OF CHROMATIUM-VINOSUM

Kinetics of electron-donor oxidation, storage-polymer formation and growth were studied in continuous cultures ofChromatium under conditions of balanced growth as well as during transient states.Under steady-state conditions, glycogen was accumulated at all dilution rates. This observation is consistent with previously postulated ideas about an ineffective glycogen-synthesis regulation.Upon perturbing the steady states, brought about by injection of extra sulfide into steady-state cultures, the following phenomena were observed immediately, irrespective of the dilution rate: the specific rate of sulfide oxidation increased to the value found in batch cultures, the sulfur-oxidation rate was decreased, the specific glycogen-synthesis rate increased, the increment being higher the lower the dilution rate, but an increase in the specific growth rate, if any, was below the limit of detection. The inverse relationship between the specific rates of glycogen synthesis and growth after removing the substrate limitation is to be explained by a shortage of intermediates, rather than by a growth-rate dependent intrinsic glycogen-synthesis limitation, because upon complete inhibition of growth a further increase in the rate of glycogen synthesis was observed. Essayed in this way, identical glycogen-synthesis rates were found at all dilution rates.Competitive advantages of such an apparently not adapted metabolism in environments with diurnal fluctuations in substrate concentrations are discussed.

PRODUCTION OF ELEMENTAL SULFUR BY GREEN AND PURPLE SULFUR BACTERIA

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (SR).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherSR values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.

COEXISTENCE OF CHLOROBIUM AND CHROMATIUM IN A SULFIDE-LIMITED CONTINOUS CULTURE

Competition experiments between Chromatium vinosum and Chlorobium limicola in sulfide-limited continuous culture under photolithoautotrophic conditions resulted in the coexistence of both organisms. The ratio between the two bacteria was dilution-rate as well as pH dependent. The observed coexistence can be explained as a hitherto not reported form of dual substrate limitation. The two substrates involved are the electron donors sulfide (growth-limiting substrate in the reservoir vessel) and extracellular elemental sulfur (formed by Chlorobium as a result of sulfide oxidation). It is argued that, although Chlorobium may have the better affinity for both substrates involved, Chromatium can compete successfully on the basis of its intracellular storage of sulfur. Ecological implication of the observed coexistence with respect to natural blooms are discussed.

PHOSPHATE-LIMITED GROWTH OF CHROMATIUM-VINOSUM IN CONTINUOUS CULTURE

Chromatium vinosum DSM 185 was grown in continuous culture at a constant dilution rate of 0.071 h-1 with sulfide as the only electron donor. The organism was subjected to conditions ranging from phosphate limitation (SR-phosphate=2.7 μM and SR-sulfide=1.8 mM) to sulfide limitation (SR-phosphate=86 μM and SR-sulfide=1.8 mM). At values of SR-phosphate below 7.5 μM the culture was washed out, whereas SR-phosphate above this value resulted in steady states. The saturation constant (Kμ) for growth on phosphate was estimated to be between 2.6 and 4.1 μM. The specific phosphorus content of the cells increased from 0.30 to 0.85 μmol P mg-1 protein with increasing SR-phosphate. The specific rate of phosphate uptake increased with increasing SR-phosphate, and displayed a non-hyperbolic saturation relationship with respect to the concentration of phosphate in the inflowing medium. Approximation of a hyperbolic saturation function yielded a maximum uptake rate (Vmax) of 85 nmol P mg-1 protein h-1, and a saturation constant for uptake (Kt) of 0.7 μM. When phosphate was supplied in excess 8.5% of the phosphate taken up by the cells was excreted as organic phosphorus at a specific rate of 8 nmol P mg-1 protein h-1.

THE INFLUENCE OF ACETATE ON THE OXIDATION OF SULFIDE BY RHODOPSEUDOMONAS-CAPSULATA

The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO2) grown Rhodopseudomonas capsulata was investigated.The capacity to oxidize sulfide and the influence of the simultaneous presence of acetate in heterotrophically (acetate) and autotrophically (sulfide/CO2) grown Rhodopseudomonas capsulata was investigated.Sulfide oxidation of acetate-limited cultures was found inversely related to the specific growth rate. Upon acetate deprevation (metering pump stopped) increased rates of sulfide oxidation were observed. This points to the existence of a constitutive acceptor for the electrons from sulfide. It is suggested that a carrier functional in the light-induced cyclic electron flow operates as such. The rate of sulfide oxidation, however, is low when compared to autotrophically-grown cells. This is probably due to the low levels of Calvin cycle enzymes present in the acetate-grown cells.In cells growing on sulfide/CO2, the addition of acetate resulted in less sulfide being oxidized. Upon depletion of the acetate, the rate of sulfide oxidation again increased, however, insufficiently to maintain the accelerated growth rate. This indicates that under mixotrophic conditions the enzymes of the Calvin cycle are being synthesized to a far lesser extent.