Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria.

Recently, two fresh water species, " Candidatus Brocadia anammoxidans" and " Candidatus Kuenenia stuttgartiensis", and one marine species, " Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. " Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle--the anammoxosome--in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

On-line determination of nitrite in wastewater treatment by use of a biosensor.

A newly developed biosensor for nitrite having a 90% response time of about 1 min was used to monitor nitrite concentration in activated sludge exposed to oxic/anoxic cycles. The NO2- biosensor contains bacteria that reduce NO2-, but not NO3-, to N2O that is subsequently monitored by a built-in electrochemical sensor. Nitrite plus nitrate (NOx-) was simultaneously monitored by a NOx- biosensor. The maximum operational lifetime of the NO2- biosensor was 6 weeks, but much longer lifetimes can be expected as malfunctioning by the 3 sensors used for longer periods was due to either mechanical damage or ineffective internal sterilization during the construction. Insufficiently sterilized sensors became sensitive also to NO3- after some time due to development of NO3(-)-reducing bacterial populations within the sensor. The fraction of NO2- as compared to NO3- in the activated sludge was very dependent on prehistory, actual loading, and aeration. During balanced operation with NH4+ being exhausted during the later parts of the aerobic cycle, NO2- increased in concentration up to about 50 microM during the early part of the aeration cycle until NH4+ became limiting. At that time the NO2- concentration decreased to low levels. Under some operating conditions a peak of NO2- also appeared in the beginning of the anoxic period. NO2- and NO3- were depleted simultaneously during the anoxic period.

Methane microprofiles in a sewage biofilm determined with a microscale biosensor.

Microprofiles of the methane concentration in a 3.5-mm-thick sewage outlet biofilm were measured at high spatial and temporal resolution using a microscale biosensor for methane. In the freshly collected biofilm, methane was building up to a concentration of 175 mumol l-1 at 3 mm depth with a total methanogenesis of 0.14 mumol m-2 s-1, as compared to an aerobic respiration (including methane oxidation) of 0.80 mumol m-2 s-1. A model biofilm was established by homogenisation of an in situ biofilm and 12 days of incubation with surplus sodium acetate. The homogenised biofilm was able to maintain 50% of the methanogenic activity in the absence of external electron donor. Oxygen had only a minor effect on the methane production, but aerobic respiration consumed a substantial part of the produced methane and was thus an important control on methane export from the biofilm. A concentration of 2 mmol l-1 nitrate was shown to inhibit methanogenesis only in the upper layer of the biofilm, whereas a further addition of 2 mmol l-1 sulphate inhibited methanogenesis in the entire biofilm. The study demonstrated the power of the methane microsensor in the study of microhabitats with concurrent production and consumption of methane.

Microbiology of flooded rice paddies.

Flooded rice paddies are one of the major biogenic sources of atmospheric methane. Apart from this contribution to the 'greenhouse' effect, rice paddy soil represents a suitable model system to study fundamental aspects of microbial ecology, such as diversity, structure, and dynamics of microbial communities as well as structure-function relationships between microbial groups. Flooded rice paddy soil can be considered as a system with three compartments (oxic surface soil, anoxic bulk soil, and rhizosphere) characterized by different physio-chemical conditions. After flooding, oxygen is rapidly depleted in the bulk soil. Anaerobic microorganisms, such as fermentative bacteria and methanogenic archaea, predominate within the microbial community, and thus methane is the final product of anaerobic degradation of organic matter. In the surface soil and the rhizosphere well-defined microscale chemical gradients can be measured. The oxygen profile seems to govern gradients of other electron acceptors (e.g., nitrate, iron(III), and sulfate) and reduced compounds (e.g., ammonium, iron(II), and sulfide). These gradients provide information about the activity and spatial distribution of functional groups of microorganisms. This review presents the current knowledge about the highly complex microbiology of flooded rice paddies. In Section 2 we describe the predominant microbial groups and their function with particular regard to bacterial populations utilizing polysaccharides and simple sugars, and to the methanogenic archaea. Section 3 describes the spatial and temporal development of microscale chemical gradients measured in experimentally defined model systems, including gradients of oxygen and dissolved and solid-phase iron(III) and iron(II). In Section 4, the results of measurements of microscale gradients of oxygen, pH, nitrate-nitrite, and methane in natural rice fields and natural rice soil cores taken to the laboratory will be presented. Finally, perspectives of future research are discussed (Section 5).

Use of an oxygen-insensitive microscale biosensor for methane to measure methane concentration profiles in a rice paddy.

An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21 degrees C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 mum into the capillary, whereas atmospheric levels of oxygen were consumed within 50 mum. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 mum at 5 degrees C. The sensors could be manufactured with tip diameters as small as 25 mum, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.

Intrarenal oxygen tension measured by a modified clark electrode at normal and low blood pressure and after injection of x-ray contrast media.

The oxygen tension (pO2) in the rat kidney was studied using a Clark microelectrode with a guard cathode behind the sensing cathode. The mean (+/- SEM) outer tip diameter of the electrodes used was 5.5 +/- 1.9 microm. The zero-pO2 current amounted to 12.5 +/- 0.9 pA at 37 degrees C; at air saturation it was 252 +/- 22.9 pA. Rats with a systolic blood pressure (BP) above 80 mmHg (where 1 mmHg = 133 Pa) showed an average pO2 in the cortex of 45 +/- 2 mmHg and in the outer medulla of 31 +/-1 mmHg. In rats with a BP below 80 mmHg a paradoxically high outer medullary pO2 of 40 +/- 4 mmHg was found, while the pO2 in the cortex was 27 +/- 4 mmHg. Changes in pO2 were also noted in the renal cortex and outer medulla after intravenous injections of the x-ray contrast medium diatrizoate (370 mg iodine/ml). In rats with normal BP, injection of diatrizoate caused a slight fall in pO2 in the renal cortex, from 42 +/- 4 to 38 +/-4 mmHg. In the medulla pO2 decreased significantly from 34 +/- 6 to 20 +/-4 mmHg. Ringer's solution did not induce any changes.

Population structure and physiological changes within a hot spring microbial mat community following disturbance.

The influence of disturbance on a hot spring cyanobacterial mat community was investigated by physically removing the top 3.0 mm, which included the entire cyanobacterial layer. Changes in 16S rRNA-defined populations were monitored by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments. Some previously absent cyanobacterial populations colonized the disturbed areas, while some populations which were present before the disturbance remained absent for up to 40 days. Changes in physiological activity were measured by oxygen microelectrode analyses and by 14CO2 incorporation into cyanobacterial molecular components. These investigations indicated substantial differences between the disturbed and undisturbed mats, including an unexplained light-induced oxygen consumption in the freshly exposed mat, increased carbon partitioning by phototrophs into growth-related macromolecules, bimodal vertical photosynthesis profiles, and delayed recovery of respiration relative to photosynthesis.

Distinct differences in partial oxygen pressure at micrometer ranges in the rat hippocampal region.

A mapping at micrometer ranges of the partial oxygen pressure in the rat hippocampus was performed. The oxygen tension in the rat hippocampal region was measured using a glass oxygen microsensor in 30-microm steps along straight lines at a set of stereotactic coordinates. In the hippocampus the pattern of the oxygen tensions reflected the autometallographic zinc sulphide (AMG(ZnS)) pattern, i.e. the pattern of zinc enriched (ZEN) terminals. The highest levels of oxygen tension were recorded in the areas that are most heavily stained with the autometallographic zinc sulphide (AMG(ZnS)) method, like hilus fasciae dentatae. The zinc ions located in synaptic vesicles of the ZEN terminals can also be demonstrated by AMG silver amplification in brains from animals in vivo treated with sodium selenite. This method depends on the presence of a substantial reduction capacity of the tissues as selenite ions (SeO(2)(3)-) must to be reduced to selenide ions (Se2-) before the catalytic zinc selenide crystals can be formed. At some point, either during the transport from the infusion site to the actual target tissue or in the target tissue itself, selenium is reduced from Se(+ IV) to Se(- II). The importance of the reduction capacity of the target tissue in this process is demonstrated by the fact that areas found to have the highest concentration of zinc ions, e.g. hilus fasciae dentatae and the mossy fibres of CA3, are almost unstained after 1 h of i.p. Na2SeO3 exposure. An explanation of this phenomenon could be that the reduction process Se(+ IV) <==> Se(- II) leading to the formation of Se2- is moved to the left by the presence of oxygen, thus inhibiting the precipitation of ZnSe crystals. It is suggested that the subtle oxygen pressure pattern found in the rat hippocampus might also reflect essential biological zinc-related mechanisms vital to brain function.

Measurements of oxygen tension in the rat kidney after contrast media using an oxygen microelectrode with a guard cathode.

The oxygen tension (PO2) in the rat kidney was studied by modified Clark microelectrodes. Changes in PO2 were measured in the renal cortex and outer medulla after intravenous injections of the X-ray contrast medium (CM) diatrizoate, 370 mg iodine/mg body weight. Injection of diatrizoate caused a slight fall in PO2 in the renal cortex (from 42 +/- 4 to 38 +/- 4 mm Hg). In the medulla PO2 decreased significantly (from 34 +/- 6 to 20 +/- 4 mm Hg). Ringer's solution did not induce any changes.

A Microscale NO(3)(-) Biosensor for Environmental Applications.

A biosensor for NO(3)(-) containing immobilized dentrifying bacteria and a reservoir of liquid growth medium for the bacteria was constructed. The bacteria did not have a N(2)O reductase and therefore reduced NO(3)(-) to N(2)O, which was then subsequently quantified by a built-in electrochemical transducer for N(2)O. The only agents interfering with the determination of NO(3)(-) were NO(2)(-) and N(2)O. The sensitivity for NO(2)(-) was identical to the one for NO(3)(-) whereas the sensitivity for N(2)O was 2.4 times higher than for NO(3)(-). Diffusive supply of electron donors to the bacteria from the built-in reservoir of growth medium ensured that the biosensor could work for 2-4 days. The tip diameter was down to 20 µm, and the sensors exhibited perfectly linear responses to nitrate in both freshwater and seawater. The detection limit was ∼1 µM. The 90% response time to changes in NO(3)(-) concentration was from 15 to 60 s at room temperature and about twice that at 6 °C, which was the lowest temperature for successful operation. The new NO(3)(-) biosensor is a very useful tool for the study of nitrogen metabolism in nature.

A microscale biosensor for methane containing methanotrophic bacteria and an internal oxygen reservoir.

A microscale biosensor for continuous measurement of methane partial pressure based on a novel counterdiffusion principle is presented. Methane-oxidizing bacteria placed in the microsensor utilize oxygen from an internal oxygen reservoir when methane from the exterior diffuses through the tip membrane. The transducer is an internal oxygen microsensor with its tip positioned between the oxygen reservoir and the sensor tip membrane. The external partial pressure of methane determines the rate of bacterial oxygen consumption within the sensor, which in turn is reflected by the signal from the transducer. Tip diameters were down to 20 µm, enabling us to study methane distribution on a microscale. The microscale construction also results in a low stirring sensitivity and a 95% response time down to 20 s. By tailoring the geometry, sensors can be made to exhibit a linear response in the full range of 0-1 atm partial pressure of methane or, alternatively, to exhibit a linear response only at lower concentrations, improving the sensitivity to below 0.1 kPa, corresponding to ∼1 µM in aqueous solution. Temperature, oxygen, and H(2)S interfere with the signal; no interferences were detected from H(2), NH(3), CO(2), or acetate.

Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes.

Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system. By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions. Nitrification was restricted to a narrow zone of 50 microns on the very top of the film. In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy. Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters. Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm.

A microsensor for nitrate based on immobilized denitrifying bacteria.

A biosensor for NO(inf3)(sup-) was constructed by attaching a 30- to 70-(mu)m-wide capillary with immobilized denitrifying bacteria in front of an N(inf2)O microsensor. These bacteria reduced O(inf2) so that only bacteria in the very tip of the sensor were exposed to O(inf2) whereas bacteria at a greater depth could carry out the anaerobic process of denitrification. In the presence of acetylene, which inhibits nitrous oxide reductase, bacteria reduced NO(inf3)(sup-) (or NO(inf2)(sup-)) from the surrounding medium to N(inf2)O and the concentration sensed by the N(inf2)O microsensor was directly proportional to the concentration of NO(inf3)(sup-) in the medium. By applying a 250-(mu)m-long capillary in front of the N(inf2)O microsensor, the 90% response time of the biosensor was 50 s. Biosensors may also be made with nitrous oxide-deficient strains so that acetylene inhibition can be omitted.

Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Field Studies.

We investigated the microbial community that developed at an iron seep where anoxic groundwater containing up to 250 muM Fe flowed out of a rock wall and dense, mat-like aggregations of ferric hydroxides formed at the oxic-anoxic interface. In situ analysis with oxygen microelectrodes revealed that the oxygen concentrations in the mat were rarely more than 50% of air saturation and that the oxygen penetration depth was quite variable, ranging from <0.05 cm to several centimeters. The bulk pH of the mat ranged from 7.1 to 7.6. There appeared to be a correlation between the flow rates at different subsites of the mat and the morphotypes of the microorganisms and Fe oxides that developed. In subsites with low flow rates (<2 ml/s), the iron-encrusted sheaths of Leptothrix ochracea predominated. Miniature cores revealed that the top few millimeters of the mat consisted primarily of L. ochracea sheaths, only about 7% of which contained filaments of cells. Deeper in the mat, large particulate oxides developed, which were often heavily colonized by unicellular bacteria that were made visible by staining with acridine orange. Direct cell counts revealed that the number of bacteria increased from approximately 10 to 10 cells per cm and the total iron concentration increased from approximately 0.5 to 3 mmol/cm with depth in the mat. Primarily because of the growth of L. ochracea, the mat could accrete at rates of up to 3.1 mm/day at these subsites. The iron-encrusted stalks of Gallionella spp. prevailed in localized zones of the same low-flow-rate subsites, usually close to where the source water emanated from the wall. These latter zones had the lowest O(2) concentrations (<10% of the ambient concentration), confirming the microaerobic nature of Gallionella spp. In subsites with high flow rates (>6 ml/s) particulate Fe oxides were dominant; direct counts revealed that up to 10 cells of primarily unicellular bacteria per cm were associated with these particulate oxides. These zones exhibited little vertical stratification in either the number of cells or iron concentration. Finally, mat samples incubated anaerobically in the presence of acetate or succinate exhibited significant potential for iron reduction, suggesting the possibility that a localized iron cycle could occur within the mat community.

Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Laboratory Studies.

We constructed a small flow chamber in which suboxic medium containing 60 to 120 muM FeCl(2) flowed up through a sample well into an aerated reservoir, thereby creating an suboxic-oxic interface similar to the physicochemical conditions that exist in natural iron seeps. When microbial mat material from the Marselisborg iron seep that contained up to 10 bacterial cells per cm (D. Emerson and N. P. Revsbech, Appl. Environ. Microbiol. 60:4022-4031, 1994) was placed in the sample well of the chamber, essentially all of the Fe flowing through the sample well was oxidized at rates of up to 1,200 nmol of Fe oxidized per h per cm of mat material. The oxidation rates of samples of the mat that were pasteurized prior to inoculation were only about 20 to 50% of the oxidation rates of unpasteurized samples. Sodium azide also significantly inhibited oxidation. These results suggest that at least 50% and up to 80% of the Fe oxidation in the chamber were actively mediated by the microbes in the mat. It also appeared that Fe stimulated the growth of the community since chambers fed with FeCl(2) accumulated masses of either filamentous or particulate growth, both in the sample well and attached to the walls of the chamber. Control chambers that did not receive FeCl(2) showed no sign of such growth. Furthermore, after 4 to 5 days the chambers fed with FeCl(2) contained 35 to 75% more protein than chambers not supplemented with FeCl(2). Leptothrix ochracea and, to a lesser extent, Gallionella spp. were responsible for the filamentous growth, and the sheaths and stalks, respectively, of these two organisms harbored large numbers of Fe-encrusted, nonappendaged unicellular bacteria. In chambers where particulate growth predominated, the unicellular bacteria alone appeared to be the primary agents of iron oxidation. These results provide the first clear evidence that the "iron bacteria" commonly found associated with neutral-pH iron seeps are responsible for most of the iron oxidation and that the presence of ferrous iron appears to stimulate the growth of these organisms.

Microsensor Analysis of Oxygen in the Rhizosphere of the Aquatic Macrophyte Littorella uniflora (L.) Ascherson.

Oxygen released by the roots of submerged plants may oxidize organic compounds from the roots and reduced substances continuously supplied by diffusion from the surrounding anoxic hydrosoil. We provide here the first visualization of this gradient environment obtained by microsensor analysis of oxygen in the rhizosphere of the freshwater plant Littorella uniflora (L.) Ascherson. The plants were rooted in an agar medium, in which amorphous FeS provided the main oxygen sink. The oxygen concentration at the root surface ranged from 20 to 450 [mu]M (atmospheric saturation = 280 [mu]M) between darkness and saturating light, and the oxic shell surrounding the roots varied from about 0.5 to 5 mm in thickness. The oxygen flux from the roots was a saturating function of the incident light intensity on the leaves, and the oxygen released was consumed mainly at the fluctuating oxic/anoxic interface. The oxic zones around individual roots are under dynamic control by light, root morphology, root density, and sediment reducing capacity, and, therefore, oxygen concentrations should be subject to substantial diurnal fluctuations in dense Littorella populations in nutrient-poor sediments.

Estimation of nitrification and denitrification from microprofiles of oxygen and nitrate in model sediment systems.

The coupling between nitrification and denitrification and the regulation of these processes by oxygen were studied in freshwater sediment microcosms with O(2) and NO(3) microsensors. Depth profiles of nitrification (indicated as NO(3) production), denitrification (indicated as NO(3) consumption), and O(2) consumption activities within the sediment were calculated from the measured concentration profiles. From the concentration profiles, it was furthermore possible to distinguish between the rate of denitrification based on the diffusional supply of NO(3) from the overlying water and the rate based on NO(3) supplied by benthic nitrification (D(w) and D(n), respectively). An increase in O(2) concentration caused a deeper O(2) penetration while a decrease in D(w) and an increase in D(n) were observed. The relative importance for total denitrification of NO(3) produced by nitrification thus increased compared with NO(3) supplied from the water phase. The decrease in D(w) at high oxygen was due to an increase in diffusion path for NO(3) from the overlying water to the denitrifying layers in the anoxic sediment. At high O(2) concentrations, nitrifying activity was restricted to the lower part of the oxic zone where there was a continuous diffusional supply of NH(4) from deeper mineralization processes, and the long diffusion path from the nitrification zone to the overlying water compared with the path to the denitrifying layers led to a stimulation in D(n).

Limitation of primary production by heterotrophic assimilation and transformation of inorganic nutrients.

The planktonic environment is usually characterized by non-steady state conditions with events of phytoplankton blooms and sedimentation. Inorganic nutrients are stripped from the water column by sedimentation and end up in the sediments where they may be permanently deposited, or nitrogen may be liberated as nitrogen gas by denitrification. A major part of the denitrification activity is a coupled process of nitrification and denitrification which is dependent on a good supply of oxygen to the sediment. Urea may constitute a major part of the total outflux of dissolved N compounds from the sediment.

Microscale distribution of nitrification activity in sediment determined with a shielded microsensor for nitrate.

Microprofiles of O(2) and NO(3) were measured simultaneously in freshwater sediment with microsensors which were completely free from electrical interference because of coaxial designs. Depth profiles of nitrification (NO(3) production) and denitrification (NO(3) consumption) were subsequently determined by computer simulation of the measured microprofiles. The nitrifying bacterial community responded very quickly to changes in environmental conditions, and new steady-state microprofiles of O(2) and NO(3) were usually approached within a few hours after perturbation. Nitrification started quickly after introduction of O(2) in previously anoxic layers, suggesting prolonged survival of the nitrifiers during anaerobiosis. Changes in the availability of O(2) and NH(4) greatly affected the nitrification profile, and there was a high rate of coupled nitrification-denitrification under conditions in which nitrification occurred right above the oxic-anoxic interface. Addition of C(2)H(2) rapidly removed the NO(3) peaks, indicating that NO(3) production was due mainly to autotrophic nitrification.

Nitrification and denitrification in lake and estuarine sediments measured by the N dilution technique and isotope pairing.

The transformation of nitrogen compounds in lake and estuarine sediments incubated in the dark was analyzed in a continuous-flowthrough system. The inflowing water contained NO(3), and by determination of the isotopic composition of the N(2), NO(3), and NH(4) pools in the outflowing water, it was possible to quantify the following reactions: total NO(3) uptake, denitrification based on NO(3) from the overlying water, nitrification, coupled nitrification-denitrification, and N mineralization. In sediment cores from both lake and estuarine environments, benthic microphytes assimilated NO(3) and NH(4) for a period of 25 to 60 h after darkening. Under steady-state conditions in the dark, denitrification of NO(3) originating from the overlying water accounted for 91 to 171 mumol m h in the lake sediments and for 131 to 182 mumol m h in the estuarine sediments, corresponding to approximately 100% of the total NO(3) uptake for both sediments. It seems that high NO(3) uptake by benthic microphytes in the initial dark period may have been misinterpreted in earlier investigations as dissimilatory reduction to ammonium. The rates of coupled nitrification-denitrification within the sediments contributed to 10% of the total denitrification at steady state in the dark, and total nitrification was only twice as high as the coupled process.