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Background and Aims: Leaf tissue CO2 partial pressure (pCO2) shows contrasting dynamics over a diurnal cycle in C3 and Crassulacean Acid Metabolism (CAM) plants. However, simultaneous and continuous monitoring of pCO2 and pO2 in C3 and CAM plants under the same conditions was lacking. Our aim was to use a new CO2 microsensor and an existing O2 microsensor for non-destructive measurements of leaf pCO2 and pO2 dynamics to compare a C3 and a CAM plant in an aquatic environment. Methods: A new amperometric CO2 microsensor and an O2 microsensor elucidated with high temporal resolution the dynamics in leaf pCO2 and pO2 during light-dark cycles for C3Lobelia dortmanna and CAM Littorella uniflora aquatic plants. Underwater photosynthesis, dark respiration, tissue malate concentrations and sediment CO2 and O2 were also measured. Key Results: During the dark period, for the C3 plant, pCO2 increased to approx. 3.5 kPa, whereas for the CAM plant CO2 was mostly below 0.05 kPa owing to CO2 sequestration into malate. Upon darkness, the CAM plant had an initial peak in pCO2 (approx. 0.16 kPa) which then declined to a quasi-steady state for several hours and then pCO2 increased towards the end of the dark period. The C3 plant became severely hypoxic late in the dark period, whereas the CAM plant with greater cuticle permeability did not. Upon illumination, leaf pCO2 declined and pO2 increased, although aspects of these dynamics also differed between the two plants. Conclusions: The continuous measurements of pCO2 and pO2 highlighted the contrasting tissue gas compositions in submerged C3 and CAM plants. The CAM leaf pCO2 dynamics indicate an initial lag in CO2 sequestration to malate, which after several hours of malate synthesis then slows. Like the use of O2 microsensors to resolve questions related to plant aeration, deployment of the new CO2 microsensor will benefit plant ecophysiology research.

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Hydrogen may accumulate to micromolar concentrations in cyanobacterial mat communities from various environments, but the governing factors for this accumulation are poorly described. We used newly developed sensors allowing for simultaneous measurement of H2S and H2 or O2 and H2 within the same point to elucidate the interactions between oxygen, sulfate reducing bacteria, and H2 producing microbes. After onset of darkness and subsequent change from oxic to anoxic conditions within the uppermost ∼1 mm of the mat, H2 accumulated to concentrations of up to 40 µmol L-1 in the formerly oxic layer, but with high variability among sites and sampling dates. The immediate onset of H2 production after darkening points to fermentation as the main H2 producing process in this mat. The measured profiles indicate that a gradual disappearance of the H2 peak was mainly due to the activity of sulfate reducing bacteria that invaded the formerly oxic surface layer from below, or persisted in an inactive state in the oxic mat during illumination. The absence of significant H2 consumption in the formerly oxic mat during the first ∼30 min after onset of anoxic conditions indicated absence of active sulfate reducers in this layer during the oxic period. Addition of the methanogenesis inhibitor BES led to increase in H2, indicating that methanogens contributed to the consumption of H2. Both H2 formation and consumption seemed unaffected by the presence/absence of H2S.

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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 m(3) 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.

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We used a novel amperometric microsensor for measuring hydrogen gas production and consumption at high spatio-temporal resolution in cyanobacterial biofilms and mats dominated by non-heterocystous filamentous cyanobacteria (Microcoleus chtonoplastes and Oscillatoria sp.). The new microsensor is based on the use of an organic electrolyte and a stable internal reference system and can be equipped with a chemical sulfide trap in the measuring tip; it exhibits very stable and sulfide-insensitive measuring signals and a high sensitivity (1.5-5 pA per µmol L(-1) H2). Hydrogen gas measurements were done in combination with microsensor measurements of scalar irradiance, O2, pH, and H2S and showed a pronounced H2 accumulation (of up to 8-10% H2 saturation) within the upper mm of cyanobacterial mats after onset of darkness and O2 depletion. The peak concentration of H2 increased with the irradiance level prior to darkening. After an initial build-up over the first 1-2 h in darkness, H2 was depleted over several hours due to efflux to the overlaying water, and due to biogeochemical processes in the uppermost oxic layers and the anoxic layers of the mats. Depletion could be prevented by addition of molybdate pointing to sulfate reduction as a major sink for H2. Immediately after onset of illumination, a short burst of presumably photo-produced H2 due to direct biophotolysis was observed in the illuminated but anoxic mat layers. As soon as O2 from photosynthesis started to accumulate, the H2 was consumed rapidly and production ceased. Our data give detailed insights into the microscale distribution and dynamics of H2 in cyanobacterial biofilms and mats, and further support that cyanobacterial H2 production can play a significant role in fueling anaerobic processes like e.g., sulfate reduction or anoxygenic photosynthesis in microbial mats.

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The importance of extracellular DNA (eDNA) in biofilm formation has become increasingly clear from research on clinically relevant bacteria. This study aimed to determine whether the quantity of eDNA produced can be linked to the ability to form biofilm. We systematically quantified eDNA over time during planktonic growth and biofilm formation in Reinheimera sp. F8 and three other environmental isolates belonging to the genera Pseudomonas, Microbacterium and Serratia. eDNA in biofilms was visualised by fluorescence microscopy and quantified by PicoGreen(®) labelling without further sample preparation, whereas eDNA in planktonic cultures was precipitated before labelling and quantification. The effect of eDNA removal was investigated by DNase treatment. eDNA appeared in the early exponential growth phase of planktonic batch cultures and the concentration peaked in the stationary phase. The concentration in biofilms differed substantially between strains and over time during biofilm development. eDNA was important for the initial attachment in all strains, and DNase treatment reduced biofilm formation in three of four strains. The extent to which eDNA accumulated in planktonic cultures or biofilms did not reflect its significance to biofilm formation, and even very low concentrations of eDNA affected biofilm formation strongly. The significance of eDNA for biofilm formation in nature may thus be more widespread than previously anticipated.

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Organic crusts on liquid manure storage tanks harbor ammonia- and nitrite-resistant methane oxidizers and may significantly reduce methane emissions. Methane oxidation potential (0.6 mol CH(4) m(-2) day(-1)) peaked during fall and winter, after 4 months of crust development. Consequences for methane mitigation potential of crusts are discussed.

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Floating, organic crusts on liquid manure, stored as a result of animal production, reduce emission of ammonia (NH3) and other volatile compounds during storage. The occurrence of NO2- and NO3- in the crusts indicate the presence of actively metabolizing NH3-oxidizing bacteria (AOB) which may be partly responsible for this mitigation effect. Six manure tanks with organic covers (straw and natural) were surveyed to investigate the prevalence and potential activity ofAOB and its dependence on the O2 availability in the crust matrix as studied by electrochemical profiling. Oxygen penetration varied from <1 mm in young, poorly developed natural crusts and old straw crusts, to several centimeters in the old natural crusts. The AOB were ubiquitously present in all crusts investigated, but nitrifying activity could only be detected in old natural crusts and young straw crust with high O2 availability. In old natural crusts, total potential NH3 oxidation rates were similar to reported fluxes of NH3 from slurry without surface crust. These results indicate that old, natural surface crusts may develop into a porous matrix with high O2 availability that harbors an active population of aerobic microorganisms, including AOB. The microbial activity may thus contribute to a considerable reduction of ammonia emissions from slurry tanks with well-developed crusts.

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Liquid manure (slurry) storages are sources of gases such as ammonia (NH(3)) and methane (CH(4)). Danish slurry storages are required to be covered to reduce NH(3) emissions and often a floating crust of straw is applied. This study investigated whether physical properties of the crust or crust microbiology had an effect on the emission of the potent greenhouse gases CH(4) and nitrous oxide (N(2)O) when crust moisture was manipulated ("dry", "moderate", and "wet"). The dry crust had the deepest oxygen penetration (45 mm as compared to 20 mm in the wet treatment) as measured with microsensors, the highest amounts of nitrogen oxides (NO(2)(-) and NO(3)(-)) (up to 36 mumol g(-1) wet weight) and the highest emissions of N(2)O and CH(4). Fluorescent in situ hybridization and gene-specific polymerase chain reaction (PCR) were used to detect occurrence of bacterial groups. Ammonia-oxidizing bacteria (AOB) were abundant in all three crust types, whereas nitrite-oxidizing bacteria (NOB) were undetectable and methane-oxidizing bacteria (MOB) were only sparsely present in the wet treatment. A change to anoxia did not affect the CH(4) emission indicating the virtual absence of aerobic methane oxidation in the investigated 2-mo old crusts. However, an increase in N(2)O emission was observed in all crusted treatments exposed to anoxia, and this was probably a result of denitrification based on NO(x)(-) that had accumulated in the crust during oxic conditions. To reduce overall greenhouse gas emissions, floating crust should be managed to optimize conditions for methanotrophs.

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