Motility of Marichromatium gracile in Response to Light, Oxygen, and Sulfide

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an ~500-μm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at ~10 μM O₂. Flux calculations yielded a molar conversion ratio S_tot/O₂ of 2.03:1 (S_tot = [H₂S] + [HS⁻] + [S²⁻]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 μM O₂. The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 μM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Microoxic-Anoxic Niche of Beggiatoa spp.: Microelectrode Survey of Marine and Freshwater Strains

Beggiatoa spp. grow optimally in media containing opposed gradients of oxygen and soluble sulfide, although some strains also require an organic substrate. By using microelectrodes, we characterized oxygen and sulfide gradients during their initial development in uninoculated media and in cultures of marine and freshwater strains. In gradient media, Beggiatoa strains always grew some distance below the air/agar interface as a dense “plate” of constantly gliding filaments with sharply demarcated upper and lower boundaries. Within established plates, the maximum oxygen partial pressure was 0.6 to 6.0% of air saturation and not significantly lower if filaments were fixing nitrogen. Oxygen penetrated only 100 to 300 μm into the plate, and the anoxic fraction increased from less than 10% to approximately 90% during later stages of growth. For lithoautotrophically grown marine strains, the linearity of the oxygen profile above the plate plus its drop to zero therein indicated that oxygen uptake for the entire tube occurred only within the Beggiatoa plate. Consequently, oxygen consumption could be predicted solely from the distance between the air/agar interface and the top of a plate, given the diffusion coefficient for oxygen. By contrast, for freshwater strains grown heterotrophically (with sulfide also in the medium), oxygen profiles were frequently nonlinear because of nonbiological reaction with sulfide which had diffused past the aggregated filaments. For all strains tested, microoxic aggregation also occurred in the absence of sulfide, apparently reflecting a step-up phobic response to oxygen.     

CO2 Uptake and Fixation by Endosymbiotic Chemoautotrophs from the Bivalve Solemya velum

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with ¹⁴C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO₂ and not with HCO₃⁻ concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO₂ was 28 ± 3 μM, and the average maximal velocity was 50.8 ± 7.1 μmol min⁻¹ g of protein⁻¹. The reliance of S. velum symbionts on extracellular CO₂ is consistent with their intracellular lifestyle, since HCO₃⁻ utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO₂ may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Complexities of complex II: Sulfide metabolism in vivo

High levels of H₂S produced by gut microbiota can block oxygen utilization by inhibiting mitochondrial complex IV. Kumar et al. have shown how cells respond to this inhibition by using the mitochondrial sulfide oxidation pathway and reverse electron transport. The reverse activity of mitochondrial complex II (succinate-quinone oxidoreductase, i.e., fumarate reduction) generates oxidized coenzyme Q, which is then reduced by the mitochondrial sulfide quinone oxidoreductase to oxidize H₂S. This newly identified redox circuitry points to the importance of complex II reversal in mitochondria during periods of hypoxia and cellular stress.     

Experimental manipulation of sediment organic content and water column aeration reduces Zostera marina (eelgrass) growth and survival

The effect of synergy between sediment organic enrichment and lack of night oxygen renewal in the water column on growth and survival of Zostera marina, and how it is reflected in the sulfur parameters in the plants (δ³⁴S, TS and S⁰) was studied experimentally. An experiment consisting of Z. marina mesocosms with different levels of organic enrichment and water column aeration was established, and the effects on sediment conditions, sulfide invasion and growth and survival of Z. marina were examined over a 4 week period. Shoots growing in Ambient Organic matter-sediments showed signs of sulfide invasion, as TS increased in all plant compartments and δ³⁴S of the plant tissues decreased during the experiment, but neither growth rate nor survival were significantly affected. The lack of night oxygen renewal had no evident effects in non-enriched sediments as porewater sulfide concentrations, AVS- and CRS-pools were not different from the corresponding 24 h aeration treatment. Plant growth rate and survival were neither different from the corresponding 24 h aeration treatment. On the contrary, shoots growing on High Organic matter-sediments suffered a massive sulfide invasion and it was directly correlated to the observed decrease in growth rates. Even though the lack of night oxygen renewal had no evident effects on sediment variables there were, however, strong indications that the different aeration levels affected plant performance, suggesting a lower sulfide oxidation capacity and confirming that low water column oxygen concentrations reduces the defense capacity of the shoots against sulfide invasion.Although δ³⁴S, TS and S⁰ concentrations together provided a powerful set of indicators to detect the invasion of sulfide in Z. marina shoots, this study enlightens the need for a deeper investigation of sulfide intrusion in seagrasses and the relation between plant sulfur parameters and sediment conditions.     

NO- and H2S brain systems of the Japanese shore crab Hemigrapsus sanguineus under conditions of anoxia

The topography and dynamics of the activity of the enzymes of the synthesis of nitric oxide (NO) and hydrogen sulfide (H₂S) in the brain of the shore crab Hemigrapsus sanguineus after 1, 6, and 12 h of anoxia was studied histochemically and immunocytochemically. Changes in the activity and number of NO- and CBS-immune-positive cells that take place due to anoxia and the intensity of which depends on the duration of the influence were revealed. The fact that the balance between the nitric oxide and hydrogen sulfide systems in the brain of the crabs H. sanguineus is preserved indicates the joint participation of those systems in the central regulation of adaptive mechanisms under the influence of anoxia and, apparently, plays an important role in the adaptation of these hydrobionts to oxygen deficit.     

Respiration Strategies Utilized by the Gill Endosymbiont from the Host Lucinid Codakia orbicularis (Bivalvia: Lucinidae)

The large tropical lucinid clam Codakia orbicularis has a symbiotic relationship with intracellular, sulfide-oxidizing chemoautotrophic bacteria. The respiration strategies utilized by the symbiont were explored using integrative techniques on mechanically purified symbionts and intact clam-symbiont associations along with habitat analysis. Previous work on a related symbiont species found in the host lucinid Lucinoma aequizonata showed that the symbionts obligately used nitrate as an electron acceptor, even under oxygenated conditions. In contrast, the symbionts of C. orbicularis use oxygen as the primary electron acceptor while evidence for nitrate respiration was lacking. Direct measurements obtained by using microelectrodes in purified symbiont suspensions showed that the symbionts consumed oxygen; this intracellular respiration was confirmed by using the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). In the few intact chemosymbioses tested in previous studies, hydrogen sulfide production was shown to occur when the animal-symbiont association was exposed to anoxia and elemental sulfur stored in the thioautotrophic symbionts was proposed to serve as an electron sink in the absence of oxygen and nitrate. However, this is the first study to show by direct measurements using sulfide microelectrodes in enriched symbiont suspensions that the symbionts are the actual source of sulfide under anoxic conditions.     

Antioxidant enzyme activity in Acidithiobacillus ferrooxidans LR maintained in contact with chalcopyrite

The total protein content and activity of the enzymes glutathione reductase (GR), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were evaluated in Acidithiobacillus ferrooxidans LR cells maintained in contact with the metal sulfide chalcopyrite for 1 and 10 days. A significant decrease in total protein content was observed in cells maintained for 10 days in the presence of chalcopyrite, suggesting proteolytic breakdown due to exposure to the metal sulfide. Following 10 days of contact with chalcopyrite, increases in GR, SOD and TrxR activities were detected, suggesting the formation of reactive oxygen species. After ten days, there was a fivefold increase in GR activity, of which, isoenzyme IV represented approximately 82% of the total. An increase in Fe-SOD activity following ten days exposure to chalcopyrite was also determined, as measured on non-denaturing polyacrylamide gels. Also, after 10 days, an approximately 31-fold increase was observed for TrxR activity. The presence of oxidative stress when A. ferrooxidans is in the presence of chalcopyrite could have a negative impact on bioleaching.     

Uptake rates of oxygen and sulfide measured with individual Thiomargarita namibiensis cells by using microelectrodes

Gradients of oxygen and sulfide measured towards individual cells of the large nitrate-storing sulfur bacterium Thiomargarita namibiensis showed that in addition to nitrate oxygen is used for oxidation of sulfide. Stable gradients around the cells were found only if acetate was added to the medium at low concentrations.     

Motility of Marichromatium gracile in response to light, oxygen, and sulfide.

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an approximately 500-microm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at approximately 10 microM O(2). Flux calculations yielded a molar conversion ratio S(tot)/O(2) of 2.03:1 (S(tot) = [H(2)S] + [HS(-)] + [S(2-)]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 microM O(2). The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 microM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Taxonomic distribution,structure/function relationship and metabolic context of the two families of sulfide dehydrogenases: SQR and FCSD

Hydrogen sulfide (H₂S) is a versatile molecule with different functions in living organisms: it can work as a metabolite of sulfur and energetic metabolism or as a signaling molecule in higher Eukaryotes. H₂S is also highly toxic since it is able to inhibit heme cooper oxygen reductases, preventing oxidative phosphorylation. Due to the fact that it can both inhibit and feed the respiratory chain, the immediate role of H₂S on energy metabolism crucially relies on its bioavailability, meaning that studying the central players involved in the H₂S homeostasis is key for understanding sulfide metabolism.Two different enzymes with sulfide oxidation activity (sulfide dehydrogenases) are known: flavocytochrome c sulfide dehydrogenase (FCSD), a sulfide:cytochrome c oxidoreductase; and sulfide:quinone oxidoreductase (SQR).In this work we performed a thorough bioinformatic study of SQRs and FCSDs and integrated all published data. We systematized several properties of these proteins: (i) nature of flavin binding, (ii) capping loops and (iii) presence of key amino acid residues. We also propose an update to the SQR classification system and discuss the role of these proteins in sulfur metabolism.     

Kinetics of net photosynthetic oxygen production of a microalgae suspension at small doses of sulfide

A new empirical model for the net oxygen production rate of an alkaliphilic microalgae consortium (AMC) with prominent members of Picochlorum and Pseudoanabaena was developed as a function of sulfide at concentrations up to 1.50 mM. The kinetic model consists of a non-continuous function with two domains for sulfide concentration, which describes the enhancement and the inhibition of net photosynthetic oxygen production. Small doses of sulfide can foster the photosynthetic activity evaluated by a Gaussian type of kinetic model; while, at a total sulfide concentration higher than 1.00 mM, the photosynthetic activity was inhibited following a linear inverse response. This study shows that small sulfide concentrations around 0.60 mM improved the photosynthetic activity by up to 90% compared to assays without sulfide. Moreover, the sulfide influence on the oxygenic photosynthetic activity of the AMC was confirmed after one year, suggesting that the kinetic model could be helpful for the design and operation of photobioreactors to improve the performance of microalgae cells exposed to hydrogen sulfide.

     

Sulfide and cyanide induced mortality and anaerobic metabolism in the arcid blood clam Scapharca inaequiv alvis

1. The survival and metabolic adjustments of the blood clam S. inaequivalvis have been determined at environmental anoxia and tissue anoxia induced by sulfide and cyanide.2. Times to 50% mortality were established in clams placed in oxygenated seawater with and without dissolved sulfide or free cyanide or deoxygenated seawater with and without dissolved sulfide.3. Anaerobic metabolism was studied in live animals and in red blood cells incubated in vitro. Tissue anoxia due to sulfide and cyanide caused greater changes in the levels of aspartate and the pyruvate derivatives, compared to environmental anoxia.     

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO₂ fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO₂ fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.     

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.     

Enhancing denitrifying sulfide removal with functional strains under micro-aerobic condition

The biological denitrifying sulfide reaction (DSR) frequently proceeds in anaerobic environments since excess oxygen inhibits the activity of the denitrifiers. This study isolated a consortium H7, comprising two strains, Penibacillus sp. and Aneurinibacillus aneurinilyticus. It indicated that the DSR performance with the H7 in a mixotrophic medium is significantly enhanced at high sulfide concentrations. The H7 was inhibited by adding >200 mg l⁻¹ of S²⁻ under anaerobic conditions. However, when 280 mg ⁻¹ of S²⁻ was added, H7 could still degrade some of the sulfide, nitrate and acetate under micro-aerobic conditions. Micro-aerobic conditions stimulated the activity of sulfide oxidase and increased the removal rate of highly concentrated sulfide, reducing the inhibition of sulfide on denitrifiers and improving DSR performance.     

Anaerobic versus Aerobic Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments

Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry · h⁻¹), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N₂ or H₂ atmosphere (0.37 and 0.32 nmol per ml of sediment slurry · h⁻¹, respectively). Incubations under experimental conditions which mimic the in situ conditions (i.e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H₂ atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparent K_m values (6 to 8 μM) for aerobic dimethyl sulfide degradation which are comparable to those reported for Thiobacillus spp., Hyphomicrobium spp., and other methylotrophs. Apparent K_m values determined for anaerobic degradation of dimethyl sulfide (3 to 8 μM) were of the same order of magnitude. The low apparent K_m values obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.     

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.     

Electric coupling between distant nitrate reduction and sulfide oxidation in marine sediment

Filamentous bacteria of the Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4–6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.