Habitat diversity and type govern potential nitrogen loss by denitrification in coastal sediments and differences in ecosystem-level diversities of disparate N2O reducing communities.

In coastal sediments, excess nitrogen is removed primarily by denitrification. However, losses in habitat diversity may reduce the functional diversity of microbial communities that drive this important filter function. We examined how habitat type and habitat diversity affects denitrification and the abundance and diversity of denitrifying and N2O reducing communities in illuminated shallow-water sediments. In a mesocosm experiment, cores from four habitats were incubated in different combinations, representing ecosystems with different habitat diversities. We hypothesized that habitat diversity promotes the diversity of N2O reducing communities and genetic potential for denitrification, thereby influencing denitrification rates. We also hypothesized that this will depend on the identity of the habitats. Habitat diversity positively affected ecosystem-level diversity of clade II N2O reducing communities, however neither clade I nosZ communities nor denitrification activity were affected. The composition of N2O reducing communities was determined by habitat type, and functional gene abundances indicated that silty mud and sandy sediments had higher genetic potentials for denitrification and N2O reduction than cyanobacterial mat and Ruppia maritima meadow sediments. These results indicate that loss of habitat diversity and specific habitats could have negative impacts on denitrification and N2O reduction, which underpin the capacity for nitrogen removal in coastal ecosystems.

Habitat diversity and ecosystem multifunctionality-The importance of direct and indirect effects.

Ecosystems worldwide are facing habitat homogenization due to human activities. Although it is commonly proposed that such habitat homogenization can have negative repercussions for ecosystem functioning, this question has yet to receive explicit scientific attention. We expand on the framework for evaluating the functional consequences of biodiversity loss by scaling up from the level of species to the level of the entire habitats. Just as species diversity generally fosters ecosystem functioning through positive interspecies interactions, we hypothesize that different habitats within ecosystems can facilitate each other through structural complementarity and through exchange of material and energy across habitats. We show that experimental ecosystems comprised of a diversity of habitats show higher levels of multiple ecosystem functions than ecosystems with low habitat diversity. Our results demonstrate that the effect of habitat diversity on multifunctionality varies with season; it has direct effects on ecosystem functioning in summer and indirect effects, via changes in species diversity, in autumn, but no effect in spring. We propose that joint consideration of habitat diversity and species diversity will prove valuable for both environmental management and basic research.

Habitat partitioning of marine benthic denitrifier communities in response to oxygen availability.

Denitrification is of global significance for the marine nitrogen budget and the main process for nitrogen loss in coastal sediments. This facultative anaerobic respiratory pathway is modular in nature and the final step, the reduction of nitrous oxide (N2 O), is performed by microorganisms with a complete denitrification pathway as well as those only capable of N2 O reduction. Fluctuating oxygen availability is a significant driver of denitrification in sediments, but the effects on the overall N2 O-reducing community that ultimately controls the emission of N2 O from marine sediments is not well known. To investigate the effects of different oxygen regimes on N2 O reducing communities, coastal marine surface sediment was incubated in microcosms under oxic, anoxic or oscillating oxygen conditions in the overlying water for 137 days. Quantification of the genetic potential for denitrification, anammox and respiratory ammonification indicated that denitrification supported nitrogen removal in these sediments. Furthermore, denitrifiers with a complete pathway were identified as the dominant community involved in N2 O reduction, rather than organisms that are only N2 O reducers. Specific lineages within each group were associated with different oxygen regimes suggesting that oxygen availability in the overlying water is associated with habitat partitioning of N2 O reducers in coastal marine surface sediments.

Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus.

Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels.

Subcellular localization of an ATPase in anammox bacteria using proteomics and immunogold electron microscopy.

Anaerobic ammonium oxidation (anammox) has received significant attention during optimization of waste-water treatment and constitutes an important pathway for the removal of bioavailable nitrogen from natural environments. Studies of key catabolic enzymes indicate that the anammox reaction takes place inside the anammoxosome, an organelle-like membranous compartment of anammox bacteria. The anammoxosome has also been suggested as a site for ATP synthesis. A lipid-based protein immobilization technique, previously used to identify proteins essential for the anammox reaction, was in this study used to select linear epitopes for antibodies specifically targeted against an identified ATPase. The approach of using proteomics and bioinformatics as tools for selecting antibody targets for immunolocalization provides an important alternative to traditional methods for selection of specific antibodies. Immunogold electron microscopy and statistical evaluations indicated that the antibodies against the ATPase were exclusively found associated with the anammoxosome membrane. This provides strong evidence for ATP synthesis by an intracellular proton motive force in anammox bacteria. Within prokaryotes, an ATP synthase associated with an intracellular compartment is a feature unique for anammox bacteria.

A potential tool for high-resolution monitoring of ocean acidification.

Current anthropogenic carbon dioxide emissions generate besides global warming unprecedented acidification rates of the oceans. Recent evidence indicates the possibility that ocean acidification and low oceanic pH may be a major reason for several mass extinctions in the past. However, a major bottleneck for research on ocean acidification is long-term monitoring and the collection of consistent high-resolution pH measurements. This study presents a low-power (<1 W) small sample volume (25 µL) semiconductor based fluorescence method for real-time ship-board pH measurements at high temporal and spatial resolution (approximately 15 s and 100 m between samples). A 405 nm light emitting diode and the blue and green channels from a digital camera was used for swift detection of fluorescence from the pH sensitive dye 6,8-Dihydroxypyrene-1,3-disulfonic acid in real-time. Main principles were demonstrated by automated continuous measurements of pH in the surface water across the Baltic Sea and the Kattegat region with a large range in salinity (~3-30) and temperature (~0-25°C). Ship-board precision of salinity and temperature adjusted pH measurements were estimated as low as 0.0001 pH units.

Functioning of a shallow-water sediment system during experimental warming and nutrient enrichment.

Effects of warming and nutrient enrichment on intact unvegetated shallow-water sediment were investigated for 5 weeks in the autumn under simulated natural field conditions, with a main focus on trophic state and benthic nitrogen cycling. In a flow-through system, sediment was exposed to either seawater at ambient temperature or seawater heated 4°C above ambient, with either natural or nutrient enriched water. Sediment-water fluxes of oxygen and inorganic nutrients, nitrogen mineralization, and denitrification were measured. Warming resulted in an earlier shift to net heterotrophy due to increased community respiration; primary production was not affected by temperature but (slightly) by nutrient enrichment. The heterotrophic state was, however, not further strengthened by warming, but was rather weakened, probably because increased mineralization induced a shortage of labile organic matter. Climate-related warming of seawater during autumn could therefore, in contrast to previous predictions, induce shorter but more intensive heterotrophic periods in shallow-water sediments, followed by longer autotrophic periods. Increased nitrogen mineralization and subsequent effluxes of ammonium during warming suggested a preferential response of organisms driving nitrogen mineralization when compared to sinks of ammonium such as nitrification and algal assimilation. Warming and nutrient enrichment resulted in non-additive effects on nitrogen mineralization and denitrification (synergism), as well as on benthic fluxes of phosphate (antagonism). The mode of interaction appears to be related to the trophic level of the organisms that are the main drivers of the affected processes. Despite the weak response of benthic microalgae to both warming and nutrient enrichment, the assimilation of nitrogen by microalgae was similar in magnitude to rates of nitrogen mineralization. This implies a sustained filter function and retention capacity of nutrients by the sediment.

A multi-proxy study of anaerobic ammonium oxidation in marine sediments of the Gullmar Fjord, Sweden.

Anaerobic ammonium oxidation (anammox) is an important process for nitrogen removal in marine pelagic and benthic environments and represents a major sink in the global nitrogen cycle. We applied a suite of complementary methods for the detection and enumeration of anammox activity and anammox bacteria in marine sediments of the Gullmar Fjord, and compared the results obtained with each technique. (15) N labelling experiments showed that nitrogen removal through N2 production was essentially limited to the upper 2 cm of the sediment, where anammox contributed 23-47% of the total production. The presence of marine anammox bacteria belonging to the genus 'Candidatus Scalindua' was shown by 16S rRNA gene sequence comparison. FISH counts of anammox bacteria correlated well with anammox activity, while quantitative PCR may have underestimated the number of anammox bacterial 16S rRNA gene copies at this site. Potential nitrogen conversion by anammox ranged from 0.6 to 4.8 fmol N cell(-1) day(-1) , in agreement with previous measurements in the marine environment and in bioreactors. Finally, intact ladderane glycerophospholipid concentrations better reflected anammox activity and abundance than ladderane core lipid concentrations, most likely because the core lipid fraction contained a substantial fossil component, especially deeper in the sediment.

Analytical performance during ratiometric long-term imaging of pH in bioturbated sediments.

In this study, the long-term analytical performance of a high-resolution ratiometric imaging sensor for pH was quantitatively determined. The sensor was applied in an experimental microcosm to illustrate biogeochemical consequences from mining activities by the chemosymbiotic bivalve Thyasira sarsi. Utilizing time-correlated pixel-by-pixel calibration protocols during imaging, close to 90% of the pixels were associated with a precision (S.D.) of <0.05 pH units at the end of an experimental period of 17 days. For comparison, a precision of <0.05 pH units was achieved for less than 50% of the pixels throughout experiments using conventional pre-sample calibration procedures. The average standard deviation of pixels was 0.01 pH units. Image analysis of single pixel derivatives and pH measurements over time suggested that T. sarsi affect pH distributions and general sediment geochemistry more than would be expected based on the small size of the bivalves. A significant decrease of pH in the overlying water suggested a considerable release of reduced compounds from the exhalent stream of the thyasirids. Strong pH gradients were demonstrated not only across the sediment-water interface but, also associated with bioturbation activities immediately adjacent to T. sarsi burrowing tracts, inhalant tubes and pedal tracts in the sediment matrix. Gradients of up to 1.16 pH units per mm were observed.

A high-performance fluorosensor for pH measurements between 6 and 9.

This study presents a high-performance ratiometric pH optode based on the fluorophore 6,8-dihydroxypyrene-1,3-disulfonic acid (DHPDS). The two pH-sensitive terminal hydroxy groups of DHPDS facilitated dual excitation/dual emission (F(1): lambda(1,ex)=420 nm, lambda(1,em)=462 nm; F(2): lambda(2,ex)=470 nm, lambda(2,em)=498 nm) properties for ratiometric (R(F1,F2)=F(1)/F(2)) normalization of sensor signal. The sensor demonstrated an exponentially decreasing ratiometric response with increasing pH, with a linear correlation (R(2)=0.9936) between (10)log(R(F1,F2)) and pH within the pH interval 6-9. Precision determined as the IUPAC pooled standard deviation for the pH values 6.00, 7.01 and 9.01, was 0.0057 pH units for the fluorosensor and 0.0054 for a commercially available pH electrode used for comparison. Between the end-points of calibration at pH 7.01, the precision of the sensor was 0.0037 pH units. Effects from changes in ionic strength (I(tot), 10-700 mM) were more pronounced for the electrode, with a linear (R(2)=0.9976) increase in response (deltaE/deltapH) with increasing I(tot). The DHPDS-based fluorosensor, however, retained sensitivity (delta(10)log(R(F1,F2))/deltapH=0.8024+/-0.0145), though with an overall increase in ratiometric signal with increasing I(tot). The preserved sensitivity despite changes in ionic strength was possibly a consequence from the dual photo-acidic properties of DHPDS. Analytical characteristics of immobilized DHPDS therefore not only facilitated high-performance measurements over a wide pH range, but also opened for straightforward simultaneous measurements of pH and ionic strength.

Identification of key proteins involved in the anammox reaction.

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N(2). Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N(2) production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N(2) and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.

Biogeochemistry in highly reduced mussel farm sediments during macrofaunal recolonization by Amphiura filiformis and Nephtys sp.

Mussel farming is considered a viable means for reducing coastal eutrophication. This study assessed the importance of bioturbation by recolonizing fauna for benthic solute fluxes and porewater distributions in manipulated mussel farm sediments. Three consecutive time-series flux incubations were performed during an experimental period of three weeks in sieved farm sediment treated with the brittle star Amphiura filiformis and the polychaete Nephtys sp. The functional behavior of Nephtys sp. and interactions between Nephtys sp. and the spontaneously colonizing spionid Malacoceros fuliginosus determined the biogeochemical response in the Nephtys sp. treatment. For example, the oxic zone was restricted and benthic nitrate and silicate fluxes were reduced compared to the brittle star treatment. A. filiformis seemed to enhance the bioadvective solute transport, although an increased supply of oxygen was due to the highly reducing conditions of the sediment mainly seen as secondary effects related to porewater distributions and benthic nutrient fluxes.

A high-precision ratiometric fluorosensor for pH: implementing time-dependent non-linear calibration protocols for drift compensation.

We present a versatile time-dependent non-linear calibration protocol for optical sensors, implemented on the pH sensitive ratiometric fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) immobilized in ethyl-cellulose. The calibration protocol individually compensated for the progressive drift of calibration parameters, whereby sensor precision and accuracy, as well as applicable lifetime were improved. A severely reduced photoacidity was observed for the immobilized fluorophore, for which excited state dynamics was characterized and benefited from during measurements. Due to the significantly reduced photoacidity of HPTS immobilized in the ethyl-cellulose sensing membrane, a dual excitation/dual emission (F(1), ex/em: 405/440 nm and F(2), ex/em: 465/510 nm) ratiometric (R(F(1), F(2) =F(1)/F(2)) sensing scheme could be used to amplify sensor response. The signal to noise (S/N) ratio was enhanced by approximately 400% utilizing the dual excitation/dual emission ratiometric sensing scheme, rather than the more commonly used protocol of dual excitation/single emission for HPTS fluorescence. Apparent pK(a) of the fluorophore ranged from 6.74 to 8.50, mainly determined by the immobilization procedure. The repeatability (IUPAC, pooled standard deviation) over three pH values (6.986, 7.702 and 7.828) was 0.0044 pH units for the optical sensor, compared to 0.0046 for the electrode used for standardization. Sensor analytical characteristics were thereby in principle limited by the performance of the standardization procedure.

Anaerobic ammonium-oxidizing bacteria in marine environments: widespread occurrence but low diversity.

Laboratory and field studies have indicated that anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. In this study 11 additional anoxic marine sediment and water column samples were studied to substantiate this claim. In a combined approach using the molecular methods, polymerase chain reaction (PCR), qualitative and quantitative fluorescence in situ hybridization (FISH), as well as (15)N stable isotope activity measurements, it was shown that anammox bacteria were present and active in all samples investigated. The anammox activity measured in the sediment samples ranged from 0.08 fmol cell(-1) day(-1) N(2) in the Golfo Dulce (Pacific Ocean, Costa Rica) sediment to 0.98 fmol cell(-1) day(-1) N(2) in the Gullmarsfjorden (North Sea, Sweden) sediment. The percentage of anammox cell of the total population (stained with DAPI) as assessed by quantitative FISH was highest in the Barents Sea (9% +/- 4%) and in most of the samples well over 2%. Fluorescence in situ hybridization and phylogenetic analysis of the PCR products derived from the marine samples indicated the exclusive presence of members of the Candidatus'Scalindua' genus. This study showed the ubiquitous presence of anammox bacteria in anoxic marine ecosystems, supporting previous observations on the importance of anammox for N cycling in marine environments.