Niels Peter Revsbech

Microelectrodes: Their Use in Microbial Ecology

Among the fundamental goals of microbial ecology is the development of methods that will enable the identification and counting of the important microorganisms in nature, the determination of their physical and chemical microenvironment, and the analysis of their metabolic processes and interactions. Due to the small size of the organisms, much effort has been devoted to the development of high-resolution techniques for the observation and understanding of the world of bacteria on a microscale. Scanning and transmission electron microscopy and fluorescent staining, immunofluorescence and other techniques for light microscopy have been the most successful in terms of reaching a high spatial resolution. With respect to our understanding of the microbial microenvironments and of the nature of the microorganisms that carry out the measured metabolic activities, there is still a long way to go. Most chemical and radiotracer techniques in use today operate on a centimeter or at best on a millimeter scale and in most cases their results cannot be directly related to the relevant microorganisms. One notable exception to this is the combined use of autoradiography and fluorescence microscopy on microbial communities.

A Cryptic Sulfur Cycle in Oxygen-Minimum–Zone Waters off the Chilean Coast

Cryptic Sulfur Cycling Aerobic bacteria and ocean circulation patterns control the formation and distribution of oxygen-minimum zones at moderate depth in the oceans. These habitats host microorganisms that thrive on other metabolic substrates in the absence of oxygen—most commonly, metabolizing thermodynamically favorable nitrogen compounds like nitrate. Off the coast of Chile, however, Canfield et al. (p. 1375, published online 11 November; see the Perspective by Teske) suggest that bacteria may often reduce sulfate as well. Metagenomic sequencing revealed the presence of both sulfate-reducing and sulfide-oxidizing bacteria. With the coincidence of sulfate and nitrate reduction, the sulfur and nitrogen cycles may be intimately linked; for example, sulfate reduction could provide nitrogen-rich ammonium for bacteria that ultimately transform it into nitrogen gas. Bacterial sulfur reduction and oxidation accompanies nitrogen cycling where oxygen levels at depth are low. Nitrogen cycling is normally thought to dominate the biogeochemistry and microbial ecology of oxygen-minimum zones in marine environments. Through a combination of molecular techniques and process rate measurements, we showed that both sulfate reduction and sulfide oxidation contribute to energy flux and elemental cycling in oxygen-free waters off the coast of northern Chile. These processes may have been overlooked because in nature, the sulfide produced by sulfate reduction immediately oxidizes back to sulfate. This cryptic sulfur cycle is linked to anammox and other nitrogen cycling processes, suggesting that it may influence biogeochemical cycling in the global ocean.

Biomarkers for In Situ Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria

The existence of anaerobic ammonium oxidation (anammox) was hypothesized based on nutrient profiles and thermodynamic calculations (5, 31, 44). It was first discovered about 1 decade ago (25) in a pilot plant treating wastewater from a yeast-producing company in Delft, The Netherlands. The anammox reaction is the oxidation of ammonium under anoxic conditions with nitrite as the electron acceptor and dinitrogen gas as the product. Hydroxylamine and hydrazine were identified as important intermediates (51). Due to their very low growth rates (doubling time in enrichments is at best 11 days) the cultivation of the anammox bacteria proved to be tedious and required very efficient biomass retention (41, 43). A physical purification of anammox organisms from enrichment cultures was achieved with percoll density centrifugation (42). The purified cells performed the anammox reaction after activation by hydrazine. Based on phylogenetic analysis, the discovered anammox organism branched deep in the Planctomycetes phylum (Fig. 1A and B, [42]) and was named “Candidatus Brocadia anammoxidans” (19). FIG. 1. (A) 16S rRNA gene-based phylogenetic tree reflecting the relationship of “Ca. Scalindua,” “Ca. Brocadia,” and “Ca. Kuenenia” to other Planctomycetes and other reference organisms. Tree reconstruction was ... After the first discovery, nitrogen losses, which could only be explained by the anammox reaction, were reported in other wastewater treatment facilities including landfill leachate treatment plants in Germany, Switzerland, and England (11, 14, 15, 36), as well as in semitechnical wastewater treatment plants in Germany (34), Belgium (30), Japan (12), Australia (48), and the United States (10, 45). Molecular techniques showed the presence of organisms affiliated with the anammox branch within the Planctomycetes in all these wastewater treatment plants. Nutrient profiles and 15N tracer studies in suboxic marine and estuarine environments indicated that anammox is also a key player in the marine nitrogen cycle (8, 46, 49). In addition, 16S rRNA gene analysis, fluorescence in situ hybridization (FISH), the distribution of specific anammox membrane lipids, nutrient profiles, and tracer experiments with [15N]ammonia showed the link between the anammox reaction and the occurrence of the anammox bacterium “Candidatus Scalindua sorokinii” in the suboxic zone of the Black Sea (20). The anammox reaction has also been tested for implementation for full-scale removal of ammonia in wastewater treatment (13, 52, 53). The detection and identification of active anammox organisms in environmental samples combined with information on environmental conditions can facilitate the search for possible biomass sources to be used as an inoculum for laboratory, semitechnical, or full-scale anammox reactors. Additionally, such information could provide insights into the niche differentiation of anammox organisms. This review summarizes the recent advances made in the 16S rRNA gene-based techniques for the detection of anammox bacteria. A convenient PCR detection method for anammox organisms is presented in which anammox-specific FISH probes were used as primers. Furthermore, methods which link activity and the detection of anammox bacteria, such as the combination of FISH and microautoradiography (FISH-MAR) (22) as well as FISH targeting the intergenic spacer region (ISR) between the 16S and 23S rRNA are discussed and compared to conventional methods to detect anammox activity. Each of these approaches by itself only addresses limited aspects, such as abundance, activity, or physiology. Thus, a combination of rRNA-based and non-rRNA-based methods is necessary to allow a comprehensive study of anammox bacteria in their ecosystems.

Evidence for complete denitrification in a benthic foraminifer

Benthic foraminifera are unicellular eukaryotes found abundantly in many types of marine sediments. Many species survive and possibly reproduce in anoxic habitats, but sustainable anaerobic metabolism has not been previously described. Here we demonstrate that the foraminifer Globobulimina pseudospinescens accumulates intracellular nitrate stores and that these can be respired to dinitrogen gas. The amounts of nitrate detected are estimated to be sufficient to support respiration for over a month. In a Swedish fjord sediment where G. pseudospinescens is the dominant foraminifer, the intracellular nitrate pool in this species accounted for 20% of the large, cell-bound, nitrate pool present in an oxygen-free zone. Similarly high nitrate concentrations were also detected in foraminifera Nonionella cf. stella and a Stainforthia species, the two dominant benthic taxa occurring within the oxygen minimum zone of the continental shelf off Chile. Given the high abundance of foraminifera in anoxic marine environments, these new findings suggest that foraminifera may play an important role in global nitrogen cycling and indicate that our understanding of the complexity of the marine nitrogen cycle is far from complete.

Diffusive and total oxygen uptake of deep-sea sediments in the eastern South Atlantic Ocean:in situ and laboratory measurements

Abstract Total O 2 uptake rates were measured by the benthic flux chamber lander ELINOR, and O 2 microprofiles were measured by the profiling lander PROFILUR in the eastern South Atlantic. Diffusive O 2 fluxes through the diffusive boundary layer and the depth distribution of O 2 consumption rates within the sediment were calculated from the obtained microprofiles. The depth integrated O 2 consumption rate agreed closely with the diffusive O 2 uptake at all stations. Total O 2 uptake was 1.2–4.2 times the diffusive O 2 uptake, and the difference correlated with the abundance of macrofauna in the sediment. Diffusive O 2 uptake and O 2 -penetration depths correlated with the organic content of the sediments and exhibited an inverse correlation with water depth. Total and diffusive rates of in situ O 2 uptake were higher than previously published data for shelf and abyssal sediments in the Atlantic, but were comparable to rates from upwelling areas in the eastern Pacific. Laboratory measurements on recovered sediment cores showed lower O 2 penetration depths and higher diffusive uptake rates than in situ measurements. The differences increased with increasing water depth. We primarily ascribe this compression of O 2 profiles to a transiently increased temperature during recovery and enhanced microbial activity in decompressed sediment cores. Total O 2 uptake rates measured in the laboratory on macrofauna-rich stations were, in contrast, lower than those measured in situ because of underrepresentation and disturbance of the macrofauna.

Oxygen in the Sea Bottom Measured with a Microelectrode

The depth ofpenetration ofoxygen into coastal marine sediments (water depth, 4 to 44 meters; 60 to 10°C) variedfrom I to 5.5 millimeters, as measured with membrane-covered oxygen microelectrodes. Below these upperfew millimeters, oxygen was present only in the immediate vicinity ofanimal burrows. The depth ofoxygen penetration is related to the rate at which oxygen is consumed in the sediment.

A comparison of oxygen, nitrate, and sulfate respiration in coastal marine sediments

Aerobic respiration with oxygen and anaerobic respiration with nitrate (denitrification) and sulfate (sulfate reduction) were measured during winter and summer in two coastal marine sediments (Denmark). Both aerobic respiration and denitrification took place in the oxidized surface layer, whereas sulfate reduction was most significant in the deeper, reduced sediment. The low availability of nitrate apparently limited the activity of denitrification during summer to less than 0.2 mmoles NO3− m−2 day−1, whereas activities of 1.0–3.0 mmoles NO3− m−2 day−1 were measured during winter. Sulfate reduction, on the contrary, increased from 2.6–7.6 mmoles SO42− m−2 day−1 during winter to 9.8–15.1 mmoles SO42− m−2 day−1 during summer. The aerobic respiration was high during summer, 135–140 mmoles O2 m−2 day−1, as compared to estimated winter activities of about 30 mmoles O2 m−2 day−1. The little importance of denitrification relative to aerobic respiration and sulfate reduction is discussed in relation to the availability and distribution of oxygen, nitrate, and sulfate in the sediments and to the detritus mineralization.

Denitrification in soil and sediment

I. Biochemistry and Genetics of Denitrification.- 1. Bio-inorganic Aspects of Denitrification: Structures and Reactions of NxOy Compounds and their Interaction with Iron and Copper Proteins.- 2. Distribution and Diversity of Dissimilatory NO2- Reductases in Denitrifying Bacteria.- 3. Metabolism of Nitrous Oxide.- 4. Physiology, Biochemistry and Genetics of Nitrate Dissimilation to Ammonia.- II. Ecophysiology of Denitrification.- 5. Role of Environmental Factors in Regulating Nitrate Respiration in Intertidal Sediments.- 6. Physiological and Ecological Aspects of Aerobic Denitrification, a Link with Heterotrophic Nitrification?.- III. Emission of NxOy Compounds.- 7. Flux of NOx between Soil and Atmosphere: Importance and Soil Microbial Metabolism.- 8. Emissions of N2O from Various Environments - the Use of Stable Isotope Composition of N2O as a Tracer for the Studies of N2O Biogeochemical Cycling.- IV. Denitrification in Soil.- 9. Acetylene Inhibition Technique: Development, Advantages and Potential Problems.- 10. The Use of Acetylene for the Quantification of N2 and N2O Production from Biological Processes in Soil.- 11. Measuring Denitrification in Soils Using 15N Techniques.- 12. Rhizosphere Denitrification a Minor Process but Indicator of Decomposition Activity.- 13. Characterizing the Variability of Soil Denitrification.- 14. Anaerobic Zones and Denitrification in Soil: Modeling and Measurement.- 15. Diffusion-Reaction Models of Denitrification in Soil Microsites.- V. Denitrification in Biofilm and Sediment.- 16. Combined Use of the Acetylene Inhibition Technique and Microsensors for Quantification of Denitrification in Sediments and Biofilms.- 17. Denitrification in Stream Biofilm and Sediment: In Situ Variation and Control Factors.- 18. Measurement of Sediment Denitrification Using 15N Tracer Method.- 19. Denitrification in Aquatic Sediments.- 20. Denitrification Model for Marine Sediment.- Contributors.

Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida

Benthic foraminifers inhabit a wide range of aquatic environments including open marine, brackish, and freshwater environments. Here we show that several different and diverse foraminiferal groups (miliolids, rotaliids, textulariids) and Gromia, another taxon also belonging to Rhizaria, accumulate and respire nitrates through denitrification. The widespread occurrence among distantly related organisms suggests an ancient origin of the trait. The diverse metabolic capacity of these organisms, which enables them to respire with oxygen and nitrate and to sustain respiratory activity even when electron acceptors are absent from the environment, may be one of the reasons for their successful colonization of diverse marine sediment environments. The contribution of eukaryotes to the removal of fixed nitrogen by respiration may equal the importance of bacterial denitrification in ocean sediments.

Denitrification and oxygen respiration in biofilms studied with a microsensor for nitrous oxide and oxygen

Depth distributions of O2 respiration and denitrification activity were studied in 1- to 2-mm thick biofilms from nutrient-rich Danish streams. Acetylene was added to block the reduction of N2O, and micro-profiles of O2 and N2O in the biofilm were measured simultaneously with a polarographic microsensor. The specific activities of the two respiratory processes were calculated from the microprofiles using a one-dimensional diffusion-reaction model. Denitrification only occurred in layers where O2 was absent or present at low concentrations (of a fewμM). Introduction of O2 into deeper layers inhibited denitrification, but the process started immediately after anoxic conditions were reestablished. Denitrification activity was present at greater depth in the biofilm when the NO3− concentration in the overlying water was elevated, and the deepest occurrence of denitrification was apparently determined by the depth penetration of NO3−. The denitrification rate within each specific layer was not affected by an increase in NO3− concentration, and the half-saturation concentration (Km) for NO3− therefore considered to be low (<25μM). Addition of 0.2% yeast extract stimulated denitrification only in the uppermost 0.2 mm of the denitrification zone indicating a very efficient utilization of the dissolved organic matter within the upper layers of the biofilm.