Copper Catalysis-Based Amperometric Microsensors for Carbon Dioxide Monitoring.

A fast response microsensor that can detect the distribution of CO2 at the microscale level is essential for the observation of biophysiological activity, carbon flux, and carbon burial. Inspired by the previous success of Cu catalysis, we attempted to use this metal Cu material to develop an amperometric microsensor that can meet the requirements. Specifically, the ambient gases diffuse through a silicone membrane into a trap casing filled with an acidic CrCl2 solution, where the otherwise interfering O2 interferent is removed by a redox with Cr2+. The gases then diffuse through a second silicone membrane into an electrolyte, where CO2 is selectively reduced to methanol (CH3OH) at a Cu cathode through a carbon monoxide (CO) pathway. Due to the use of Cu catalysis at the WE tip, CO2 can be reduced at a less negative polarization (-470 mV) instead of the previously reported -1200 mV, thus avoiding hydrogen-evolution interference due to water from the byproduct or from water diffusion through the silicone membrane. This moderate polarization results in a stable baseline, making the microsensor suitable for long-term monitoring. Interferences from other gases, such as N2O, which may be of much concern in environmental monitoring, can be ignored. Applications and limitations are also discussed with a view to further improvement in the future.

Microsensor for total dissolved sulfide (TDS).

Total Dissolved Sulfide (TDS) concentrations can either be derived from simultaneous measurement of pH and one of the sulfide species or determined indirectly in samples following an acidification step. Here we report a microsensor that allows for direct measurement of TDS in aquatic media without the need for pH monitoring. An acidic chamber placed in front of a commercial, amperometric H2S microsensor allows for the in-situ conversion of dissolved ionic sulfide species to H2S, which in turn is oxidized at the transducer anode. A typical sensor had a tip opening of 30 µm, a response time of <50 s and linear range between 0.5 and 650 µM. The sensor performance can be largely tuned by altering the geometry of the chamber. Sensors of different sensitivity (0.04-2.93 pA/µM) showed no noticeable change in zero current and sensitivity during continuous polarization over 7 weeks. The sensor was successfully applied to resolve microscale TDS gradients in freshwater and marine sediments. Other avenues of application include the online monitoring of industrial and urban sewers.

Total Dissolved Inorganic Carbon Sensor Based on Amperometric CO2 Microsensor and Local Acidification.

We present a dipping probe total dissolved inorganic carbon (DIC) microsensor based on a localized acidic microenvironment in front of an amperometric CO2 microsensor. The acidic milieu facilitates conversion of bicarbonate and carbonate to CO2, which in turn is reduced at a silver cathode. Interfering oxygen is removed by an acidic CrCl2 oxygen trap. Theoretical simulations of microsensor functioning were performed to find a suitable compromise between response time and near-complete conversion of bicarbonate to CO2. The sensor exhibited a linear response over a wide range of 0-8 mM DIC, with a calculated LOD of 5 µM and a 90% response time of 150 s. The sensor was successfully tested in measuring DIC in bottled mineral water and seawater. This DIC microsensor holds the potential to become an important tool in environmental sensing and beyond for measurements of DIC at high spatial and temporal resolution.

Microaerobic Lifestyle at Nanomolar O2 Concentrations Mediated by Low-Affinity Terminal Oxidases in Abundant Soil Bacteria.

High-affinity terminal oxidases (TOs) are believed to permit microbial respiration at low oxygen (O2) levels. Genes encoding such oxidases are widespread, and their existence in microbial genomes is taken as an indicator for microaerobic respiration. We combined respiratory kinetics determined via highly sensitive optical trace O2 sensors, genomics, and transcriptomics to test the hypothesis that high-affinity TOs are a prerequisite to respire micro- and nanooxic concentrations of O2 in environmentally relevant model soil organisms: acidobacteria. Members of the Acidobacteria harbor branched respiratory chains terminating in low-affinity (caa3-type cytochrome c oxidases) as well as high-affinity (cbb3-type cytochrome c oxidases and/or bd-type quinol oxidases) TOs, potentially enabling them to cope with varying O2 concentrations. The measured apparent Km (Km(app)) values for O2 of selected strains ranged from 37 to 288 nmol O2 liter-1, comparable to values previously assigned to low-affinity TOs. Surprisingly, we could not detect the expression of the conventional high-affinity TO (cbb3 type) at micro- and nanomolar O2 concentrations but detected the expression of low-affinity TOs. To the best of our knowledge, this is the first observation of microaerobic respiration imparted by low-affinity TOs at O2 concentrations as low as 1 nM. This challenges the standing hypothesis that a microaerobic lifestyle is exclusively imparted by the presence of high-affinity TOs. As low-affinity TOs are more efficient at generating ATP than high-affinity TOs, their utilization could provide a great benefit, even at low-nanomolar O2 levels. Our findings highlight energy conservation strategies that could promote the success of Acidobacteria in soil but might also be important for as-yet-unrevealed microorganisms. IMPORTANCE Low-oxygen habitats are widely distributed on Earth, ranging from the human intestine to soils. Microorganisms are assumed to have the capacity to respire low O2 concentrations via high-affinity terminal oxidases. By utilizing strains of a ubiquitous and abundant group of soil bacteria, the Acidobacteria, and combining respiration kinetics, genomics, and transcriptomics, we provide evidence that these microorganisms use the energetically more efficient low-affinity terminal oxidases to respire low-nanomolar O2 concentrations. This questions the standing hypothesis that the ability to respire traces of O2 stems solely from the activity of high-affinity terminal oxidases. We propose that this energetically efficient strategy extends into other, so-far-unrevealed microbial clades. Our findings also demonstrate that physiological predictions regarding the utilization of different O2 concentrations based solely on the presence or absence of terminal oxidases in bacterial genomes can be misleading.

Urea Biosensor Based on a CO2 Microsensor.

Urea sensors based on electrodes in direct contact with the medium have limited long-term stability when exposed to complex media. Here, we present a urea biosensor based on urease immobilized in an alginate polymer, buffered at pH 6, and placed in front of a newly developed fast and sensitive CO2 microsensor, where the electrodes are shielded by a gas-permeable membrane. The CO2 produced by the urease in the presence of urea diffuses into the microsensor and is reduced at a Ag cathode. Oxygen interference is prevented by a Cr2+ trap. The 95% response time to changes in urea concentration was 120 s with a linear calibration curve in the range 0-1000 µM and a detection limit of 1 µM. The Ni2+ cofactor to improve sensor performance was continuously supplied from a reservoir behind the sensor tip. The stability of the urea sensor was optimized by the addition of bovine serum albumin as a stabilizer to the urease/alginate mixture that was cross-linked with glutaraldehyde and Ca2+ ions. This immobilization strategy resulted in about 70% of the initial urea sensor sensitivity after two weeks of continuous operation. The sensor was successfully tested in blood serum.

Ion Selective Amperometric Biosensors for Environmental Analysis of Nitrate, Nitrite and Sulfate.

Inorganic ions that can be redox-transformed by living cells can be sensed by biosensors, where the redox transformation gives rise to a current in a measuring circuit. Such biosensors may be based on enzymes, or they may be based on application of whole cells. In this review focus will be on biosensors for the environmentally important ions NO3-, NO2-, and SO42-, and for comparison alternative sensor-based detection will also be mentioned. The developed biosensors are generally characterized by a high degree of specificity, but unfortunately also by relatively short lifetimes. There are several investigations where biosensor measurement of NO3- and NO2- have given new insight into the functioning of nitrogen transformations in man-made and natural environments such as sediments and biofilms, but the biosensors have not become routine tools. Future modifications resulting in better long-term stability may enable such general use.

Fast Responding Amperometric CO2 Microsensor with Ionic Liquid-Aprotic Solvent Electrolytes.

Knowledge about the microscale distribution of CO2 is essential in many environmental and technical settings, and electrochemical CO2 sensing may be optimized to yield such information. The performance of a Clark-type CO2 sensor was greatly improved by adding 20% dimethylformamide (DMF) to the ionic liquid 1-ethyl-3-methylimidazolium dicyanamide (EMIM-DCA) previously used as an electrolyte. The addition of DMF resulted in a much faster response to increasing (95% response of about 100 s) or decreasing CO2 concentration, a negligible interference from low concentrations of N2O, and a signal temperature dependence similar to that of O2 microsensors. The use of 80% EMIM-DCA/20% DMF as an electrolyte leads to CO2 reduction at -0.72 V (vs standard hydrogen electrode), reducing the overpotential by 0.2 V as compared to the use of 100% EMIM-DCA. The CO2 microsensor has a calculated limit of detection of 0.5 Pa CO2, and sensors optimized for high sensitivity exhibited a linear response within the range of 0-4.6 kPa (0-1.7 mM) CO2. A set of four sensors exhibited no noticeable change of zero current and CO2 sensitivity during 4 months of continuous polarization.

Microsensors in plant biology: in vivo visualization of inorganic analytes with high spatial and/or temporal resolution.

This Expert View provides an update on the recent development of new microsensors, and briefly summarizes some novel applications of existing microsensors, in plant biology research. Two major topics are covered: (i) sensors for gaseous analytes (O2, CO2, and H2S); and (ii) those for measuring concentrations and fluxes of ions (macro- and micronutrients and environmental pollutants such as heavy metals). We show that application of such microsensors may significantly advance understanding of mechanisms of plant-environmental interaction and regulation of plant developmental and adaptive responses under adverse environmental conditions via non-destructive visualization of key analytes with high spatial and/or temporal resolution. Examples included cover a broad range of environmental situations including hypoxia, salinity, and heavy metal toxicity. We highlight the power of combining microsensor technology with other advanced biophysical (patch-clamp, voltage-clamp, and single-cell pressure probe), imaging (MRI and fluorescent dyes), and genetic techniques and approaches. We conclude that future progress in the field may be achieved by applying existing microsensors for important signalling molecules such as NO and H2O2, by improving selectivity of existing microsensors for some key analytes (e.g. Na, Mg, and Zn), and by developing new microsensors for P.

Amperometric sensor for nanomolar nitrous oxide analysis.

Nitrous oxide is an important greenhouse gas and there is a need for sensitive techniques to study its distribution in the environment at concentrations near equilibrium with the atmosphere (9.6 nM in water at 20 °C). Here we present an electrochemical sensor that can quantify N2O in the nanomolar range. The sensor principle relies on a front guard cathode placed in front of the measuring cathode. This cathode is used to periodically block the flux of N2O towards the measuring cathode, thereby creating an amplitude in the signal. This signal amplitude is unaffected by drift in the baseline current and can be read at very high resolution, resulting in a sensitivity of 2 nM N2O for newly constructed sensors. Interference from oxygen is prevented by placing the front guard cathode in oxygen-consuming electrolyte. The sensor was field tested by measuring an N2O profile to a depth of 120 m in the oxygen minimum zone of the Eastern Tropical North Pacific Ocean (ETNP) off the coast of Mexico.

Strong leaf surface basification and CO2 limitation of seagrass induced by epiphytic biofilm microenvironments.

Coastal eutrophication is a growing problem worldwide, leading to increased epiphyte overgrowth of seagrass leaves. Yet little is known about how epiphytes affect key biogeochemical conditions and processes in the seagrass phyllosphere. We used electrochemical microsensors to measure microgradients of O2 , pH, and CO2 at the bare and epiphyte-covered leaf surface of seagrass (Zostera marina L.) to determine effects of epiphytes on the leaf chemical microenvironment. Epiphytes result in extreme daily fluctuations in pH, O2 , and inorganic carbon concentrations at the seagrass leaf surface severely hampering the plant's performance. In light, leaf epiphyte biofilms and their diffusive boundary layer lead to strong basification, markedly reducing the CO2 and HCO3- availability at the leaf surface, leading to reduced photosynthetic efficiency as a result of carbon limitation and enhanced photorespiration. With epiphytes, leaf surface pH increased to >10, thereby exceeding final pH levels (~9.62) and CO2 compensation points for active photosynthesis. In darkness, epiphyte biofilms resulted in increased CO2 and hypoxia at the leaf surface. Epiphytes can lead to severe carbon limitation in seagrasses owing to strong phyllosphere basification leading to CO2 depletion and costly, yet limiting, HCO3- utilization, increasing the risk of plant starvation.

Root O2 consumption, CO2 production and tissue concentration profiles in chickpea, as influenced by environmental hypoxia.

Roots in flooded soils experience hypoxia, with the least O2 in the vascular cylinder. Gradients in CO2 across roots had not previously been measured. The respiratory quotient (RQ; CO2 produced : O2 consumed) is expected to increase as O2 availability declines. A new CO2 microsensor and an O2 microsensor were used to measure profiles across roots of chickpea seedlings in aerated or hypoxic conditions. Simultaneous, nondestructive flux measurements of O2 consumption, CO2 production, and thus RQ, were taken for roots with declining O2 . Radial profiling revealed severe hypoxia and c. 0.8 kPa CO2 within the root vascular cylinder. The distance penetrated by O2 into the roots was shorter at lower O2 . The gradient in CO2 was in the opposite direction to that of O2 , across the roots and diffusive boundary layer. RQ increased as external O2 was lowered. For chickpea roots in solution at air equilibrium, O2 was very low and CO2 was elevated within the vascular cylinder; the extent of the severely hypoxic core increased as external O2 was reduced. The increased RQ in roots in response to declining external O2 highlighted the shift from respiration to ethanolic fermentation as the severely hypoxic/anoxic core became a progressively greater proportion of the root tissues.

Biogas upgrading with hydrogenotrophic methanogenic biofilms.

Hydrogen produced from periodic excess of electrical energy may be added to biogas reactors where it is converted to CH4 that can be utilized in the existing energy grid. The major challenge with this technology is gas-to-liquid mass transfer limitation. The microbial conversions in reactors designed for hydrogenotrophic methanogenesis were studied with microsensors for H2, pH, and CO2. The H2 consumption potential was dependent on the CO2 concentration, but could partially recover after CO2 depletion. Reactors with 3-dimensional biofilm carrier material and a large gas headspace allowed for a methanogenic biofilm in direct contact with the gas phase. A high density of Methanoculleus sp. in the biofilm mediated a high rate of CH4 production, and it was calculated that a reactor filled with 75% carrier material could mediate a biogas upgrading from 50 to 95% CH4 within 24 h when an equivalent amount of H2 was added.

Anammox and partial nitritation in the mainstream of a wastewater treatment plant in a temperate region (Denmark).

The Marselisborg WWTP (Aarhus, Denmark) fed the mainstream nitrification/denitrification tanks with excess sludge from a sidestream DEMON tank for more than three years to investigate if anammox can supplement conventional nitrification/denitrification in a mainstream of a temperate region. To evaluate this long-term attempt, anammox and also denitrification rates were measured in activated sludge from the main- and sidestream at 10, 20 and 30 °C using 15N-labelling (stable isotope) experiments. The results show that anammox contributes by approximately 1% of the total nitrogen removal in the mainstream tanks and that anammox conversion rates there are approximately 800-900 times lower than in the DEMON. A distinct temperature dependence of both anammox and denitrification rates was also confirmed, however, results from different temperatures did not significantly alter relative shares, e.g. anammox rates in activated sludge from the nitrification/denitrification tanks are also negligible at 30 °C. This indicates that the anammox bacteria abundance in the nitrification/denitrification tanks is too low to play an important role and that an adaptation to lower temperatures had not occurred. Additional in situ measurements in the nitrification/denitrification tanks further revealed that full nitrification dominates over partial nitritation. Dominant nitritation-anammox is therefore excluded per se and also nitrite shunt activities are not particularly supported.

Metabolic preference of nitrate over oxygen as an electron acceptor in foraminifera from the Peruvian oxygen minimum zone.

Benthic foraminifera populate a diverse range of marine habitats. Their ability to use alternative electron acceptors-nitrate (NO3-) or oxygen (O2)-makes them important mediators of benthic nitrogen cycling. Nevertheless, the metabolic scaling of the two alternative respiration pathways and the environmental determinants of foraminiferal denitrification rates are yet unknown. We measured denitrification and O2 respiration rates for 10 benthic foraminifer species sampled in the Peruvian oxygen minimum zone (OMZ). Denitrification and O2 respiration rates significantly scale sublinearly with the cell volume. The scaling is lower for O2 respiration than for denitrification, indicating that NO3- metabolism during denitrification is more efficient than O2 metabolism during aerobic respiration in foraminifera from the Peruvian OMZ. The negative correlation of the O2 respiration rate with the surface/volume ratio is steeper than for the denitrification rate. This is likely explained by the presence of an intracellular NO3- storage in denitrifying foraminifera. Furthermore, we observe an increasing mean cell volume of the Peruvian foraminifera, under higher NO3- availability. This suggests that the cell size of denitrifying foraminifera is not limited by O2 but rather by NO3- availability. Based on our findings, we develop a mathematical formulation of foraminiferal cell volume as a predictor of respiration and denitrification rates, which can further constrain foraminiferal biogeochemical cycling in biogeochemical models. Our findings show that NO3- is the preferred electron acceptor in foraminifera from the OMZ, where the foraminiferal contribution to denitrification is governed by the ratio between NO3- and O2.

Gene expression of terminal oxidases in two marine bacterial strains exposed to nanomolar oxygen concentrations.

The final step of aerobic respiration is carried out by a terminal oxidase transporting electrons to oxygen (O2). Prokaryotes harbor diverse terminal oxidases that differ in phylogenetic origin, structure, biochemical function, and affinity for O2. Here we report on the expression of high-affinity (cytochrome cbb3 oxidase), low-affinity (cytochrome aa3 oxidase), and putative low-affinity (cyanide-insensitive oxidase (CIO)) terminal oxidases in the marine bacteria Idiomarina loihiensis L2-TR and Marinobacter daepoensis SW-156 upon transition to very low O2 concentrations (<200 nM), measured by RT-qPCR. In both strains, high-affinity cytochrome cbb3 oxidase showed the highest expression levels and was significantly up-regulated upon transition to low O2 concentrations. Low-affinity cytochrome aa3 oxidase showed very low transcription levels throughout the incubation. Surprisingly, however, it was also up-regulated upon transition to low O2 concentrations. In contrast, putative low-affinity CIO had much lower expression levels and markedly different regulation patterns between the two strains. These results demonstrate that exposure to low O2 concentrations regulates the gene expression of different types of terminal oxidases, but also that the type and magnitude of transcriptional response is species-dependent. Therefore, in situ transcriptome data cannot, without detailed knowledge of the transcriptional regulation of the species involved, be translated into relative respiratory activity.

Cryptic oxygen cycling in anoxic marine zones.

Oxygen availability drives changes in microbial diversity and biogeochemical cycling between the aerobic surface layer and the anaerobic core in nitrite-rich anoxic marine zones (AMZs), which constitute huge oxygen-depleted regions in the tropical oceans. The current paradigm is that primary production and nitrification within the oxic surface layer fuel anaerobic processes in the anoxic core of AMZs, where 30-50% of global marine nitrogen loss takes place. Here we demonstrate that oxygenic photosynthesis in the secondary chlorophyll maximum (SCM) releases significant amounts of O2 to the otherwise anoxic environment. The SCM, commonly found within AMZs, was dominated by the picocyanobacteria Prochlorococcus spp. Free O2 levels in this layer were, however, undetectable by conventional techniques, reflecting a tight coupling between O2 production and consumption by aerobic processes under apparent anoxic conditions. Transcriptomic analysis of the microbial community in the seemingly anoxic SCM revealed the enhanced expression of genes for aerobic processes, such as nitrite oxidation. The rates of gross O2 production and carbon fixation in the SCM were found to be similar to those reported for nitrite oxidation, as well as for anaerobic dissimilatory nitrate reduction and sulfate reduction, suggesting a significant effect of local oxygenic photosynthesis on Pacific AMZ biogeochemical cycling.

Ammonium and nitrite oxidation at nanomolar oxygen concentrations in oxygen minimum zone waters.

A major percentage of fixed nitrogen (N) loss in the oceans occurs within nitrite-rich oxygen minimum zones (OMZs) via denitrification and anammox. It remains unclear to what extent ammonium and nitrite oxidation co-occur, either supplying or competing for substrates involved in nitrogen loss in the OMZ core. Assessment of the oxygen (O2) sensitivity of these processes down to the O2 concentrations present in the OMZ core (<10 nmol⋅L(-1)) is therefore essential for understanding and modeling nitrogen loss in OMZs. We determined rates of ammonium and nitrite oxidation in the seasonal OMZ off Concepcion, Chile at manipulated O2 levels between 5 nmol⋅L(-1) and 20 µmol⋅L(-1) Rates of both processes were detectable in the low nanomolar range (5-33 nmol⋅L(-1) O2), but demonstrated a strong dependence on O2 concentrations with apparent half-saturation constants (Kms) of 333 ± 130 nmol⋅L(-1) O2 for ammonium oxidation and 778 ± 168 nmol⋅L(-1) O2 for nitrite oxidation assuming one-component Michaelis-Menten kinetics. Nitrite oxidation rates, however, were better described with a two-component Michaelis-Menten model, indicating a high-affinity component with a Km of just a few nanomolar. As the communities of ammonium and nitrite oxidizers were similar to other OMZs, these kinetics should apply across OMZ systems. The high O2 affinities imply that ammonium and nitrite oxidation can occur within the OMZ core whenever O2 is supplied, for example, by episodic intrusions. These processes therefore compete with anammox and denitrification for ammonium and nitrite, thereby exerting an important control over nitrogen loss.

Effects of Bacterial Community Members on the Proteome of the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain Is79.

UNLABELLED: Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi, in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi, several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism. IMPORTANCE: Aerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbe-microbe interactions, in particular between ammonia oxidizers and their neighboring microbial community.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle.

UNLABELLED: Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30 µmol H2 liter(-1) at about 1-mm depth below the mat surface, slowly decreasing to about 11 µmol H2 liter(-1) just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 µmol H2 liter(-1), was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE: Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H2, it is now possible to study the microdistribution of H2 in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H2 profiles within the mat compared to previous measurements of H2 emission gives much more detailed information about the sources and sinks of H2 in such communities, and it was demonstrated that the high rates of H2 formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H2 is formed as a by-product.

Respiratory Kinetics of Marine Bacteria Exposed to Decreasing Oxygen Concentrations.

During aerobic respiration, microorganisms consume oxygen (O2) through the use of different types of terminal oxidases which have a wide range of affinities for O2. The Km values for O2 of these enzymes have been determined to be in the range of 3 to 200 nmol liter(-1). In this study, we examined the time course of development of aerobic respiratory kinetics of four marine bacterial species (Dinoroseobacter shibae, Roseobacter denitrificans, Idiomarina loihiensis, and Marinobacter daepoensis) during exposure to decreasing O2 concentrations. The genomes of all four species have genes for both high-affinity and low-affinity terminal oxidases. The respiration rate of the bacteria was measured by the use of extremely sensitive optical trace O2 sensors (range, 1 to 1,000 nmol liter(-1)). Three of the four isolates exhibited apparent Km values of 30 to 60 nmol liter(-1) when exposed to submicromolar O2 concentrations, but a decrease to values below 10 nmol liter(-1) was observed when the respiration rate per cell was lowered and the cell size was decreased due to starvation. The fourth isolate did not reach a low respiration rate per cell during starvation and exhibited apparent Km values of about 20 nmol liter(-1) throughout the experiment. The results clearly demonstrate not only that enzyme kinetics may limit O2 uptake but also that even individual cells may be diffusion limited and that this diffusion limitation is the most pronounced at high respiration rates. A decrease in cell size by starvation, due to limiting organic carbon, and thereby more efficient diffusion uptake may also contribute to lower apparent Km values.