Coupling between anammox and autotrophic denitrification for simultaneous removal of ammonium and sulfide by enriched marine sediments

In the present study, the capacity of enrichments derived from marine sediments collected from different sites of the Mexican littoral to perform anaerobic ammonium oxidation (anammox) coupled to sulfide-dependent denitrification for simultaneous removal of ammonium and sulfide linked to nitrite reduction was evaluated. Sulfide-dependent denitrification out-competed anammox during the simultaneous oxidation of sulfide and ammonium. Significant accumulation of elemental sulfur (ca. 14–30 % of added sulfide) occurred during the coupling between the two respiratory processes, while ammonium was partly oxidized (31–47 %) due to nitrite limitation imposed in sediment incubations. Nevertheless, mass balances revealed up to 38 % more oxidation of the electron donors available (ammonium and sulfide) than that expected from stoichiometry. Recycling of nitrite, from nitrate produced through anammox, is proposed to contribute to extra oxidation of sulfide, while additional ammonium oxidation is suggested by sulfate-reducing anammox (SR-anammox). The complex interaction between nitrogenous and sulfurous compounds occurring through the concomitant presence of autotrophic denitrification, conventional anammox and SR-anammox may significantly drive the nitrogen and sulfur fluxes in marine environments.     

Novel groups of Gammaproteobacteria catalyse sulfur oxidation and carbon fixation in a coastal, intertidal sediment

The oxidation of hydrogen sulfide is essential to sulfur cycling in marine habitats. However, the role of microbial sulfur oxidation in marine sediments and the microorganisms involved are largely unknown, except for the filamentous, mat‐forming bacteria. In this study we explored the diversity, abundance and activity of sulfur‐oxidizing prokaryotes (SOP) in sulfidic intertidal sediments using 16S rRNA and functional gene sequence analyses, fluorescence in situ hybridization (FISH) and microautoradiography. The 16S rRNA gene analysis revealed that distinct clades of uncultured Gammaproteobacteria are important SOP in the tidal sediments. This was supported by the dominance of gammaproteobacterial sequences in clone libraries of genes encoding the reverse dissimilatory sulfite reductase (rDSR) and the adenosine phosphosulfate reductase (APR). Numerous sequences of all three genes grouped with uncultured autotrophic SOP. Accordingly, Gammaproteobacteria accounted for 40–70% of all ¹⁴CO₂‐incorporating cells in surface sediments as shown by microautoradiography. Furthermore, phylogenetic analysis of all three genes consistently suggested a discrete population of SOP that was most closely related to the sulfur‐oxidizing endosymbionts of the tubeworm Oligobrachia spp. FISH showed that members of this population (WS‐Gam209 group) were abundant, reaching up to 1.3 × 10⁸ cells ml^?1 (4.6% of all cells). Approximately 25% of this population incorporated CO₂, consistent with a chemolithoautotrophic metabolism most likely based on sulfur oxidation. Thus, we hypothesize that novel, gammaproteobacterial SOP attached to sediment particles may play a more important role for sulfide removal and primary production in marine sediments than previously assumed.     

Sulfide oxidation under chemolithoautotrophic denitrifying conditions

Chemolithoautotrophic denitrifying microorganisms oxidize reduced inorganic sulfur compounds coupled to the reduction of nitrate as an electron acceptor. These denitrifiers can be applied to the removal of nitrogen and/or sulfur contamination from wastewater, groundwater, and gaseous streams. This study investigated the physiology and kinetics of chemolithotrophic denitrification by an enrichment culture utilizing hydrogen sulfide, elemental sulfur, or thiosulfate as electron donor. Complete oxidation of sulfide to sulfate was observed when nitrate was supplemented at concentrations equal or exceeding the stoichiometric requirement. In contrast, sulfide was only partially oxidized to elemental sulfur when nitrate concentrations were limiting. Sulfide was found to inhibit chemolithotrophic sulfoxidation, decreasing rates by approximately 21-fold when the sulfide concentration increased from 2.5 to 10.0 mM, respectively. Addition of low levels of acetate (0.5 mM) enhanced denitrification and sulfate formation, suggesting that acetate was utilized as a carbon source by chemolithotrophic denitrifiers. The results of this study indicate the potential of chemolithotrophic denitrification for the removal of hydrogen sulfide. The sulfide/nitrate ratio can be used to control the fate of sulfide oxidation to either elemental sulfur or sulfate.     

Significance of archaeal nitrification in hypoxic waters of the Baltic Sea

Ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread, and their abundance in many terrestrial and aquatic ecosystems suggests a prominent role in nitrification. AOA also occur in high numbers in oxygen-deficient marine environments, such as the pelagic redox gradients of the central Baltic Sea; however, data on archaeal nitrification rates are scarce and little is known about the factors, for example sulfide, that regulate nitrification in this system. In the present work, we assessed the contribution of AOA to ammonia oxidation rates in Baltic deep basins and elucidated the impact of sulfide on this process. Rate measurements with ¹⁵N-labeled ammonium, CO₂ dark fixation measurements and quantification of AOA by catalyzed reporter deposition–fluorescence in situ hybridization revealed that among the three investigated sites the highest potential nitrification rates (122–884 nmol l⁻¹per day) were measured within gradients of decreasing oxygen, where thaumarchaeotal abundance was maximal (2.5–6.9 × 10⁵ cells per ml) and CO₂ fixation elevated. In the presence of the archaeal-specific inhibitor GC₇, nitrification was reduced by 86–100%, confirming the assumed dominance of AOA in this process. In samples spiked with sulfide at concentrations similar to those of in situ conditions, nitrification activity was inhibited but persisted at reduced rates. This result together with the substantial nitrification potential detected in sulfidic waters suggests the tolerance of AOA to periodic mixing of anoxic and sulfidic waters. It begs the question of whether the globally distributed Thaumarchaeota respond similarly in other stratified water columns or whether the observed robustness against sulfide is a specific feature of the thaumarchaeotal subcluster present in the Baltic Deeps.     

Effect of sulfide concentration on autotrophic denitrification from nitrate and nitrite in vertical fixed-bed reactors

Autotrophic denitrification coupled with sulfide oxidation represents an interesting alternative for the simultaneous removal of nitrate/nitrite and sulfide from wastewaters. The applicability of such bioprocess is especially advantageous for the post treatment of effluents from anaerobic reactors, since they usually produce sulfides, which can be used as endogenous electron donor for autotrophic denitrification. This study evaluated the effect of sulfide concentration on this bioprocess using nitrate and nitrite as electron acceptors in vertical fixed-bed reactors. The results showed that intermediary sulfur compounds were mainly produced when excess of electron donor was applied, which was more evident when nitrate was used. Visual evidences suggested that elemental sulfur was the intermediary compound produced. There was also evidence that the elemental sulfur previously formed was being used when sulfide was applied in stoichiometric concentration relative to nitrate/nitrite. Nitrite was more readily consumed than nitrate. For both electron acceptors and sulfide concentrations tested, autotrophic denitrification was not affected by residual heterotrophic denitrification via endogenic activity, occurring as a minor additional nitrogen removal process.     

Community Shift from Phototrophic to Chemotrophic Sulfide Oxidation following Anoxic Holomixis in a Stratified Seawater Lake

Most stratified sulfidic holomictic lakes become oxygenated after annual turnover. In contrast, Lake Rogoznica, on the eastern Adriatic coast, has been observed to undergo a period of water column anoxia after water layer mixing and establishment of holomictic conditions. Although Lake Rogoznica''s chemistry and hydrography have been studied extensively, it is unclear how the microbial communities typically inhabiting the oxic epilimnion and a sulfidic hypolimnion respond to such a drastic shift in redox conditions. We investigated the impact of anoxic holomixis on microbial diversity and microbially mediated sulfur cycling in Lake Rogoznica with an array of culture-independent microbiological methods. Our data suggest a tight coupling between the lake''s chemistry and occurring microorganisms. During stratification, anoxygenic phototrophic sulfur bacteria were dominant at the chemocline and in the hypolimnion. After an anoxic mixing event, the anoxygenic phototrophic sulfur bacteria entirely disappeared, and the homogeneous, anoxic water column was dominated by a bloom of gammaproteobacterial sulfur oxidizers related to the GSO/SUP05 clade. This study is the first report of a community shift from phototrophic to chemotrophic sulfide oxidizers as a response to anoxic holomictic conditions in a seasonally stratified seawater lake.     

A new method of autotrophic denitrification with spent sulfidic caustic as substrate and alkalinity source

A new method based on sulfide utilizing autotrophic denitrification was adopted to remove nitrate from wastewater and to reuse spent sulfidic caustic containing high sulfide and alkalinity levels. The experiments were performed using a bench-scale upflow anoxic hybrid growth reactor (UAHGR) and an upflow anoxic suspended growth reactor (UASGR) to characterize the stoichiometric relationship between sulfur and nitrate in the process as well as the performance of the reactors. The level of nitrate removal from the UAHGR and UASGR were maintained at over 90% at a nitrate loading rate ranging from 0.15∼0.40 kgNO₃ ⁻/m³·d and no significant nitrite accumulation was observed in either reactor. Although the influent pH values were higher than the optimum range of autotrophic denitrification at 8.7∼10.1, the effluent pH was stable at 7.2∼7.9 due to the production of hydrogen ions during operation. The stoichiometric ratio of sulfate production to nitrate removal was 1.5∼2.1 mgSO₄ ²⁻/mgNO₃ ⁻ in both reactors. A comparison of the reactor performance revealed that the chemical parameters of the UAHGR operation corresponded to a plug flow like type reactor while the chemical parameters of the UASGR operation corresponded to a completely stirred tank reactor like type reactor. UAHGR did not require sludge recycling due to the packed media while UASGR required 300∼700% sludge recycling. Therefore, spent sulfidic caustic could be used in the sulfur utilizing autotrophic denitrification processes as substrate and alkalinity sources.     

Physical, biogeochemical and isotopic processes related to heterogeneity of a shallow crystalline rock aquifer

This study deals with the chemical characterization of the biogeochemical processes occurring in a shallow aquifer in crystalline rocks. The influence of rock heterogeneity and the related physical processes on the aquifer biogeochemistry have been investigated. A hydrochemical survey (major anion and cation analysis) shows that rock heterogeneity leads to a stronger spatial than temporal variability. Some rapidly recharged and low- mineralized waters are present at the soil/rock interface. However the pumped well intersects a preferential flow path and pumps nitrate-rich water. Sulfur and oxygen isotope data from sulfates in the pumped water clearly show sulfide oxidation with only 20–30% of the oxygen atoms in sulfates formed by sulfide oxidation coming from atmospheric oxygen. This low contribution of molecular oxygen in sulfide oxidation, associated with the drastic decrease in nitrate concentration, involves a marked relationship between the nitrogen and sulfur cycles through denitrification, coupled with sulfide oxidation. Conversely, for rapidly recharged waters, the rock physical heterogeneity allows sulfide oxidation by molecular oxygen indicated by a contribution of atmospheric oxygen of nearly 70% in the newly formed sulfate. As the aquifer biogeochemistry is controlled by the physical characteristics of the rocks, pumping may overcome the natural flux pattern described previously. This anthropogenic disturbance leads to a modification of water pathways (spatial mixing or relative contribution of the fracture/matrix waters to the global fluxes) and, consequently, to a modification of the physical and biogeochemical processes occurring in the aquifer.     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

Not so old Archaea – the antiquity of biogeochemical processes in the archaeal domain of life

Since the archaeal domain of life was first recognized, it has often been assumed that Archaea are ancient, and harbor primitive traits. In fact, the names of the major archaeal lineages reflect our assumptions regarding the antiquity of their traits. Ancestral state reconstruction and relaxed molecular clock analyses using newly articulated oxygen age constraints show that although the archaeal domain itself is old, tracing back to the Archean eon, many clades and traits within the domain are not ancient or primitive. Indeed many clades and traits, particularly in the Euryarchaeota, were inferred to be Neoproterozoic or Phanerozoic in age. Both Eury- and Crenarchaeota show increasing metabolic and physiological diversity through time. Early archaeal microbial communities were likely limited to sulfur reduction and hydrogenotrophic methanogenesis, and were confined to high-temperature geothermal environments. However, after the appearance of atmospheric oxygen, nodes containing a wide variety of traits (sulfate and thiosulfate reduction, sulfur oxidation, sulfide oxidation, aerobic respiration, nitrate reduction, mesophilic methanogenesis in sedimentary environments) appear, first in environments containing terrestrial Crenarchaeota in the Meso/Neoproterozoic followed by environments containing marine Euryarchaeota in the Neoproterozoic and Phanerozoic. This provides phylogenetic evidence for increasing complexity in the biogeochemical cycling of C, N, and S through geologic time, likely as a consequence of microbial evolution and the gradual oxygenation of various compartments within the biosphere. This work has implications not only for the large-scale evolution of microbial communities and biogeochemical processes, but also for the interpretation of microbial biosignatures in the ancient rock record.     

Denitrification in a nitrogen-limited stream ecosystem

Denitrification was measured in hyporheic, parafluvial, and bank sediments of Sycamore Creek, Arizona, a nitrogen-limited Sonoran Desert stream. We used three variations of the acetylene block technique to estimate denitrification rates, and compared these estimates to rates of nitrate production through nitrification. Subsurface sediments of Sycamore Creek are typically well-oxygenated, relatively low in nitrate, and low in organic carbon, and therefore are seemingly unlikely sites of denitrification. However, we found that denitrification potential (C & N amended, anaerobic incubations) was substantial, and even by our conservative estimates (unamended, oxic incubations and field chamber nitrous oxide accumulation), denitrification consumed 5–40% of nitrate produced by nitrification. We expected that denitrification would increase along hyporheic and parafluvial flowpaths as dissolved oxygen declined and nitrate increased. To the contrary, we found that denitrification was generally highest at the upstream ends of subsurface flowpaths where surface water had just entered the subsurface zone. This suggests that denitrifiers may be dependent on the import of surface-derived organic matter, resulting in highest denitrification rate at locations of surface-subsurface hydrologic exchange. Laboratory experiments showed that denitrification in Sycamore Creek sediments was primarily nitrogen limited and secondarily carbon limited, and was temperature dependent. Overall, the quantity of nitrate removed from the Sycamore Creek ecosystem via denitrification is significant given the nitrogen-limited status of this stream.     

湖泊氮素氧化及脱氮过程研究进展

自然界中氮的生物地球化学循环主要由微生物驱动,由固氮作用、硝化作用、反硝化作用和氨化作用来完成。过去数十年间,随着异养硝化、厌氧氨氧化和古菌氨氧化作用的发现,人们对环境中氮素循环认识逐步深入,提出了多种脱氮途径新假说。对湖泊生态系统中氮素的输入、输出及其在水体、沉积物和水土界面的迁移转化过程进行了概括,对湖泊生态系统中反硝化和厌氧氨氧化脱氮机理及脱氮效率的最新研究进展进行了探讨,并对以后的氮素循环研究进行了展望。     

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.     

Using modern low-oxygen marine ecosystems to understand the nitrogen cycle of the Paleo- and Mesoproterozoic oceans

During the productive Paleoproterozoic (2.4–1.8 Ga) and less productive Mesoproterozoic (1.8–1.0 Ga), the ocean was suboxic to anoxic and multicellular organisms had not yet evolved. Here, we link geologic information about the Proterozoic ocean to microbial processes in modern low-oxygen systems. High iron concentrations and rates of Fe cycling in the Proterozoic are the largest differences from modern oxygen-deficient zones. In anoxic waters, which composed most of the Paleoproterozoic and ~40% of the Mesoproterozoic ocean, nitrogen cycling dominated. Rates of N₂ production by denitrification and anammox were likely linked to sinking organic matter fluxes and in situ primary productivity under anoxic conditions. Additionally autotrophic denitrifiers could have used reduced iron or methane. 50% of the Mesoproterozoic ocean may have been suboxic, promoting nitrification and metal oxidation in the suboxic water and N₂O and N₂ production by partial and complete denitrification in anoxic zones in organic aggregates. Sulfidic conditions may have composed ~10% of the Mesoproterozoic ocean focused along continental margins. Due to low nitrate concentrations in offshore regions, anammox bacteria likely dominated N₂ production immediately above sulfidic zones, but in coastal regions, higher nitrate concentrations probably promoted complete S-oxidizing autotrophic denitrification at the sulfide interface.     

Motility patterns of filamentous sulfur bacteria, Beggiatoa spp

The large sulfur bacteria, Beggiatoa spp., live on the oxidation of sulfide with oxygen or nitrate, but avoid high concentrations of both sulfide and oxygen. As gliding filaments, they rely on reversals in the gliding direction to find their preferred environment, the oxygen-sulfide interface. We observed the chemotactic patterns of single filaments in a transparent agar medium and scored their reversals and the glided distances between reversals. Filaments within the preferred microenvironment glided distances shorter than their own length between reversals that anchored them in their position as a microbial mat. Filaments in the oxic region above the mat or in the sulfidic, anoxic region below the mat glided distances longer than the filament length between reversals. This reversal behavior resulted in a diffusion-like spreading of the filaments. A numerical model of such gliding filaments was constructed based on our observations. The model was applied to virtual filaments in the oxygen- and sulfide-free zone of the sediment, which is a main habitat of Beggiatoa in the natural environment. The model predicts a long residence time of the virtual filament in the suboxic zone and explains why Beggiatoa accumulate high nitrate concentrations in internal vacuoles as an alternative electron acceptor to oxygen.     

An investigation of a process for partial nitrification and autotrophic denitrification combined desulfurization in a single biofilm reactor

In this study, a vertical submerged biofilm reactor was applied to investigate autotrophic partial nitrification/denitrification and simultaneous sulfide removal by using synthetic wastewater. The appropriate influent ratios of ammonia and sulfide needed to achieve partial autotrophic nitrification and denitrification were evaluated with influent ammonium nitrogen ranging from 54.6 to 129.8 mg L^?1 and sulfide concentrations ranging from 52.7 to 412.4 mg S L^?1. The results demonstrated that the working parameter was more stable when the sulfur/nitrogen ratio was set at 3:2, which yielded the maximum sulfur conversion. Batch experiments with different phosphate concentrations proved that a suitable phosphate buffer solution to control pH values could improve synchronous desulfurization denitrification process performance.