Emilio Garcia-Robledo

NC10 bacteria in marine oxygen minimum zones

Bacteria of the NC10 phylum link anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophy pathway. A niche for NC10 in the pelagic ocean has not been confirmed. We show that NC10 bacteria are present and transcriptionally active in oceanic oxygen minimum zones (OMZs) off northern Mexico and Costa Rica. NC10 16S rRNA genes were detected at all sites, peaking in abundance in the anoxic zone with elevated nitrite and methane concentrations. Phylogenetic analysis of particulate methane monooxygenase genes further confirmed the presence of NC10. rRNA and mRNA transcripts assignable to NC10 peaked within the OMZ and included genes of the putative nitrite-dependent intra-aerobic pathway, with high representation of transcripts containing the unique motif structure of the nitric oxide (NO) reductase of NC10 bacteria, hypothesized to participate in O2-producing NO dismutation. These findings confirm pelagic OMZs as a niche for NC10, suggesting a role for this group in OMZ nitrogen, methane and oxygen cycling.

LUMOS - A Sensitive and Reliable Optode System for Measuring Dissolved Oxygen in the Nanomolar Range

Most commercially available optical oxygen sensors target the measuring range of 300 to 2 μmol L-1. However these are not suitable for investigating the nanomolar range which is relevant for many important environmental situations. We therefore developed a miniaturized phase fluorimeter based measurement system called the LUMOS (Luminescence Measuring Oxygen Sensor). It consists of a readout device and specialized “sensing chemistry” that relies on commercially available components. The sensor material is based on palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin embedded in a Hyflon AD 60 polymer matrix and has a KSV of 6.25 x 10-3 ppmv-1. The applicable measurement range is from 1000 nM down to a detection limit of 0.5 nM. A second sensor material based on the platinum(II) analogue of the porphyrin is spectrally compatible with the readout device and has a measurement range of 20 μM down to 10 nM. The LUMOS device is a dedicated system optimized for a high signal to noise ratio, but in principle any phase flourimeter can be adapted to act as a readout device for the highly sensitive and robust sensing chemistry. Vise versa, the LUMOS fluorimeter can be used for read out of less sensitive optical oxygen sensors based on the same or similar indicator dyes, for example for monitoring oxygen at physiological conditions. The presented sensor system exhibits lower noise, higher resolution and higher sensitivity than the electrochemical STOX sensor previously used to measure nanomolar oxygen concentrations. Oxygen contamination in common sample containers has been investigated and microbial or enzymatic oxygen consumption at nanomolar concentrations is presented.

Cryptic oxygen cycling in anoxic marine zones

Significance Anoxic marine zones (AMZs) create expansive habitats for microbes whose anaerobic metabolisms help drive global nutrient cycles, for example, by removing nitrogen from the oceans by producing N2 gas. AMZ cycles may also be shaped by oxygen intrusion from outside the AMZ, creating opportunities for aerobic microbial metabolisms. Here we show that aerobic processes in AMZs are linked to oxygen production within the anoxic zone. Oxygen is produced during daytime in a layer of photosynthetic cyanobacteria near the top of the AMZ and then rapidly consumed by aerobic processes without accumulating. Oxygen turnover and carbon fixation rates are comparable to those of microbial N2 production, suggesting an important role for internal oxygen cycling in AMZ transformations of matter and energy. 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.

Effects of green macroalgal blooms on the meiofauna community structure in the Bay of Cádiz.

The effect of macroalgal blooms on the abundance and community structure of intertidal sediment meiofauna was studied using an in situ enclosure experiments (Bay of Cádiz, Spain). Meiofaunal abundance (3500-41,000 ind 10 cm⁻²) was three to sevenfold higher in the presence of macroalgae. Nematoda were the dominant taxon both in Control (52-82%) and Macroalgae plots (92-96%), followed by Harpacticoida Copepoda and Ostracoda. Non-metric Multi-Dimensional Scaling (MDS) analysis clearly separated the meiofaunal community from Control and Macroalgae plots. Organic matter, organic carbon, total nitrogen, chlorophyll a and freeze-lysable inorganic nutrients were higher in Macroalgae plots, and were highly correlated with the horizontal MDS axis separating Control and Macroalgae meiofaunal communities. Meiofaunal abundance and taxonomic composition in the Bay of Cádiz seem to be bottom-up controlled either through a grazer system based on microphytobenthos in bare sediments or through a decomposer system in macroalgae affected sediments.

A New Highly Sensitive Method to Assess Respiration Rates and Kinetics of Natural Planktonic Communities by Use of the Switchable Trace Oxygen Sensor and Reduced Oxygen Concentrations

Oxygen respiration rates in pelagic environments are often difficult to quantify as the resolutions of our methods for O2 concentration determination are marginal for observing significant decreases during bottle incubations of less than 24 hours. Here we present the assessment of a new highly sensitive method, that combine Switchable Trace Oxygen (STOX) sensors and all-glass bottle incubations, where the O2 concentration was artificially lowered. The detection limit of respiration rate by this method is inversely proportional to the O2 concentration, down to <2 nmol L−1 h−1 for water with an initial O2 concentration of 500 nmol L−1. The method was tested in Danish coastal waters and in oceanic hypoxic waters. It proved to give precise measurements also with low oxygen consumption rates (∼7 nmol L−1 h−1), and to significantly decrease the time required for incubations (≤14 hours) compared to traditional methods. This method provides continuous real time measurements, allowing for a number of diverse possibilities, such as modeling the rate of oxygen decrease to obtain kinetic parameters. Our data revealed apparent half-saturation concentrations (Km values) one order of magnitude lower than previously reported for marine bacteria, varying between 66 and 234 nmol L−1 O2. Km values vary between different microbial planktonic communities, but our data show that it is possible to measure reliable respiration rates at concentrations ∼0.5–1 µmol L−1 O2 that are comparable to the ones measured at full air saturation.

Dynamics of Inorganic Nutrients in Intertidal Sediments: Porewater, Exchangeable, and Intracellular Pools.

The study of inorganic nutrients dynamics in shallow sediments usually focuses on two main pools: porewater (PW) nutrients and exchangeable (EX) ammonium and phosphate. Recently, it has been found that microphytobenthos (MPB) and other microorganisms can accumulate large amounts of nutrients intracellularly (IC), highlighting the biogeochemical importance of this nutrient pool. Storing nutrients could support the growth of autotrophs when nutrients are not available, and could also provide alternative electron acceptors for dissimilatory processes such as nitrate reduction. Here, we studied the magnitude and relative importance of these three nutrient pools (PW, IC, and EX) and their relation to chlorophylls (used as a proxy for MPB abundance) and organic matter (OM) contents in an intertidal mudflat of Cadiz Bay (Spain). MPB was localized in the first 4 mm of the sediment and showed a clear seasonal pattern; highest chlorophylls content was found during autumn and lowest during spring-summer. The temporal and spatial distribution of nutrients pools and MPB were largely correlated. Ammonium was higher in the IC and EX fractions, representing on average 59 and 37% of the total ammonium pool, respectively. Similarly, phosphate in the IC and EX fractions accounted on average for 40 and 31% of the total phosphate pool, respectively. Nitrate in the PW was low, suggesting low nitrification activity and rapid consumption. Nitrate accumulated in the IC pool during periods of moderate MPB abundance, being up to 66% of the total nitrate pool, whereas it decreased when chlorophyll concentration peaked likely due to a high nitrogen demand. EX-Nitrate accounted for the largest fraction of total sediment nitrate, 66% on average. The distribution of EX-Nitrate was significantly correlated with chlorophyll and OM, which probably indicates a relation of this pool to an increased availability of sites for ionic adsorption. This EX-Nitrate pool could represent an alternative nitrate source with significant concentrations available to the microbial community, deeper in the sediment below the oxic layer.

Micro-scale H2-CO2 Dynamics in a Hydrogenotrophic Methanogenic Membrane Reactor.

Biogas production is a key factor in a sustainable energy supply. It is possible to get biogas with very high methane content if the biogas reactors are supplied with exogenous hydrogen, and one of the technologies for supplying hydrogen is through gas permeable membranes. In this study the activity and stratification of hydrogen consumption above such a membrane was investigated by use of microsensors for hydrogen and pH. A hydrogenotrophic methanogenic community that was able to consume the hydrogen flux within 0.5 mm of the membrane with specific rates of up to 30 m3 H2 m-3 day-1 developed within 3 days in fresh manure and was already established at time zero when analyzing slurry from a biogas plant. The hydrogen consumption was dependent on a simultaneous carbon dioxide supply and was inhibited when carbon dioxide depletion elevated the pH to 9.2. The activity was only partially restored when the carbon dioxide supply was resumed. Bioreactors supplied with hydrogen gas should thus be carefully monitored and either have the hydrogen supply disrupted or be supplemented with carbon dioxide when the pH rises to values about 9.

Photosynthetic activity and community shifts of microphytobenthos covered by green macroalgae

Macroalgae blooms, a frequent consequence of eutrophication in coastal areas, affect the photosynthetic activity of sediments dominated by microphytobenthos (MPB). Light spectra, steady-state (after 1 h) microprofiles of O2 , gross photosynthesis (Pg ), community respiration in light (RL ) and net community photosynthesis (Pn ) were measured in diatom- and cyanobacteria-dominated communities below increasing layers of Ulva. Photosynthetic photon flux (PPF) decreased exponentially with increasing layers of algae and the light spectrum was increasingly enriched in the green and deprived in blue and red regions. Sediment Pg , Pn and RL decreased as the number of Ulva layers increased; however, 1.6 times higher macroalgal density was necessary to fully inhibit cyanobacteria Pg compared with diatoms, indicating that cyanobacteria were better adapted to this light environment. Long-term (3 weeks) incubations of diatom-dominated sediments below increasing layers of Ulva resulted in a shift in the taxonomic composition of the MPB towards cyanobacteria. Hence, changes in the light climate below macroalgal accumulations can negatively affect the photosynthetic activity of sediments. However, spectral niche differentiation of MPB taxonomic groups and concurrent changes in the MPB community may provide sediments with increased resilience to the detrimental effects of eutrophication.

Respiratory Kinetics of Marine Bacteria Exposed to Decreasing Oxygen Concentrations

ABSTRACT 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.

Different Types of Diatom-Derived Extracellular Polymeric Substances Drive Changes in Heterotrophic Bacterial Communities from Intertidal Sediments

Intertidal areas support extensive diatom-rich biofilms. Such microphytobenthic (MPB) diatoms exude large quantities of extracellular polymeric substances (EPS) comprising polysaccharides, glycoproteins and other biopolymers, which represent a substantial carbon pool. However, degradation rates of different EPS components, and how they shape heterotrophic communities in sediments, are not well understood. An aerobic mudflat-sediment slurry experiment was performed in the dark with two different EPS carbon sources from a diatom-dominated biofilm: colloidal EPS (cEPS) and the more complex hot-bicarbonate-extracted EPS. Degradation rate constants determined over 9 days for three sediment fractions [dissolved organic carbon (DOC), total carbohydrates (TCHO), and (cEPS)] were generally higher in the colloidal-EPS slurries (0.105–0.123 d−1) compared with the hot-bicarbonate-extracted-EPS slurries (0.060–0.096 d−1). Addition of hot-bicarbonate-EPS resulted in large increases in dissolved nitrogen and phosphorous by the end of the experiment, indicating that the more complex EPS is an important source of regenerated inorganic nutrients. Microbial biomass increased ~4–6-fold over 9 days, and pyrosequencing of bacterial 16S rRNA genes revealed that the addition of both types of EPS greatly altered the bacterial community composition (from 0 to 9 days) compared to a control with no added EPS. Bacteroidetes (especially Tenacibaculum) and Verrucomicrobia increased significantly in relative abundance in both the hot-bicarbonate-EPS and colloidal-EPS treatments. These differential effects of EPS fractions on carbon-loss rates, nutrient regeneration and microbial community assembly improve our understanding of coastal-sediment carbon cycling and demonstrate the importance of diverse microbiota in processing this abundant pool of organic carbon.