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To permit CO 2 measurements behind stomatal pores of plant leaves a potentiometric CO 2 -biosensor was built with a tip diameter of 2 μm. The miniaturised sensor assembly consists of a H + carrier-based pH-microelectrode concentrically arranged within a sheathing micropipette. The tip of the latter is filled with carbonate buffer, the pH of which quickly responds to CO 2 concentration changes due to incorporated carbonic anhydrase. The CO 2 -microsensor shows a linear response to CO 2 in the physiologically relevant concentration range of 50–800 ppm (5.1–81.1 Pa). The 90% response time varied from sensor to sensor between 18 and 63 s (mean±S.E., 42±14 s; n =6). Sensor calibration and leaf experiments were performed in an open-flow tube-like minicuvette, allowing tangential air flow along the leaf surface with controlled gas mixtures and flow rates of choice. At 800 ppm external CO 2 concentration, CO 2 within the leaf was close to the external CO 2 concentration when stomatal pores were wide open. However, the concentration dropped to 350 ppm during stomatal closure due to CO 2 consumption by photosynthesis demonstrating distinct CO 2 -sensing of internal leaf CO 2 . Following ‘light-off’ internal CO 2 rapidly rose close to 700 ppm, a response which was completely reversed by ‘light-on’. We conclude that this sensor is a suitable tool for CO 2 monitoring in places too small to be accessible to conventional tools.

With microsensors, we measured the steady-state micro-profiles of O 2 , pH and Ca 2+ on the topside of young segments of Halimeda discoidea, as well as the surface dynamics upon light-dark shifts. The effect of several inhibitors was studied. The steady-state measurements showed that under high light intensity, calcium and protons were taken up, while O 2 was produced. In the dark, O 2 was consumed, the pH decreased to below seawater level and Ca 2+ uptake was reduced to 50%. At low light intensity (12 μmol photons m -2 s -1 ), Ca 2+ efflux was observed. Upon light-dark shifts, a complicated pattern of both the pH and calcium surface dynamics was observed. Illumination caused an initial pH decrease, followed by a gradual pH increase: this indicated that the surface pH of H. discoidea is determined by more than one light-induced process. When photosynthesis was inhibited by dichlorophenyl dimethyl urea (DCMU), a strong acidification was observed upon illumination. The nature and physiological function of this putative pump is not known. The calcium dynamics followed all pH dynamics closely, both in the presence and absence of DCMU. The Ca-channel blockers verapamil and nifedipine had no effect on the Ca 2+ dynamics and steady-state profiles. Thus, in H. discoidea, calcification is not regulated by the alga, but is a consequence of pH increase during photosynthesis. Acetazolamide had no effect on photosynthesis, whereas ethoxyzolamide inhibited photosynthesis at higher light intensities. Therefore, all carbonic anhydrase activity is intracellular. Carbonic anhydrase is required to alleviate the CO 2 limitation. Calcification cannot supply sufficient protons and CO 2 to sustain photosynthesis.

Stable associations of more than one species of symbiont within a single host cell or tissue are assumed to be rare in metazoans because competition for space and resources between symbionts can be detrimental to the host1. In animals with multiple endosymbionts, such as mussels from deep-sea hydrothermal vents2 and reef-building corals3, the costs of competition between the symbionts are outweighed by the ecological and physiological flexibility gained by the hosts. A further option for the coexistence of multiple symbionts within a host is if these benefit directly from one another, but such symbioses have not been previously described. Here we show that in the gutless marine oligochaete Olavius algarvensis, endosymbiotic sulphate-reducing bacteria produce sulphide that can serve as an energy source for sulphide-oxidizing symbionts of the host. Thus, these symbionts do not compete for resources but rather share a mutalistic relationship with each other in an endosymbiotic sulphur cycle, in addition to their symbiotic relationship with the oligochaete host.

Summary The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea-like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1‐2 orders of magnitude lower than those of N. europaea. Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic‐anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 μm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira -like bacteria in situ.

The synthesis of iodo{2,2′-[1,2-octadecanediylbis(nitrilomethylidyne)]diphenolato}cobalt is described. Liquid membrane microelectrodes based on this carrier exhibit Nernstian behaviour with a selectivity sequence according to the Hofmeister series: I– > NO3 – > NO2 – > Cl– > HCO3 – > AcO–. The selectivity coefficient of nitrate over nitrite and chloride amounts to –1.6 and –2.7, respectively. The detection limit for nitrate in water amounts to 10–5.2 mol/L. A nitrate profile measured in a nitrifying biofilm is presented as a practical application.

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10 8 SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O2, NH4+, NO2−, and NO3− and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift from an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 μm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Proteobacteria and a thus far unknown nitrite oxidising population.

The 0, dynamics at the base of biofilms was studied using planar optodes Biofllms were grown directly on the optodes and the 2-dimensional distnbution of O2 at the base of biofilms was resolved at a spatial resolution of 30 X 30 pm, using a CCD camera The average 0, saturahon at the base decreased and the heterogeneity increased as blofllms developed In mature blofilms heterogeneous 0, distributions were caused by clusters of high biomass which had low O2 saturations surrounded by 0,-nch voids and channels The O2 distribution at the base of biofilms was highly dependent on the free flow veloclty above the biofilm e g m a 400 pm thick biofllm the average 0, saturation increased from 0 to 23 1 % air saturation as the free flow velocity increased from 6 2 to 35 1 cm S ' Addition of glucose to a concentrat~on of 2 n1M in the water phase at maximum flow velocity caused the 0, consumption rate to increase and the base of the blofilm to go anoxlc The insertion of an O2 microelectrode into a biofdm caused the 0, saturation at the base of the biofiln~ to increase by approximately 25 pM This effect, presumably caused by hydrodynamic disturbances, typically extended several mm away from the position of the mcrosensor tip The presented data show for the first tune the true distnbution of O2 at the basis of heterogeneous biofilms and demonstrate the great potential of planar optodes for the study of solute dynamcs within biofllms at a very high spabal and temporal resolution

Microsensors used in microbial ecology are reviewed with emphasis on new sensor developments (NO3 -, NO2 -, NH4 +, CO2, H2, H2S and CH4 microsensors as well as fiberoptical microsensors for O2, temperature and pH). Examples of microsensor applications in biofilms and activated sludge flocs are presented, where sulfate reduction and denitrification were studied.

Two upflow anaerobic hybrid reactors treated lactose and a mixture of ethanol, propionate and butyrate, respectively, at a volumetric loading rate of 3.7 kg chemical oxygen demand (COD) m−3day−1, a hydraulic retention time of 5 days and a liquid upflow velocity of 0.01 m/h. Under steady-state conditions, the lactose-fed sludge had much higher (20%–100%) specific methanogenic conversion rates than the volatile-fatty acid␣(VFA)/ethanol-fed sludge for all substrates tested, including VFA. In both reactors, a flocculant sludge developed, although a much higher content of extracellular polysaccharide was measured in the lactose-fed sludge [1900 μg compared to 305 μg uronic acid/g volatile suspended solids (VSS)]. When the liquid upflow velocity of a third, VFA/ethanol-fed reactor was increased to 0.5 m/h, granulation of the sludge occurred, accompanied by a large increase (200%–500%) in the specific methanogenic conversion rates for the syntrophic and methanogenic substrates studied. Granulation reduced the susceptibility of the sludge to flotation. Glucose was degraded at a high rate (100 mg glucose gVSS−1h−1) by the sludge from the third reactor, despite not having been exposed to a sugar-containing influent for 563␣days.

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time-points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5-8.5. Filming the fluorescence decay by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations.

Quantitative studies of coral reefs are challenged by the three-dimensional hard structure of reefs and the high spatial variability and temporal dynamics of their metabolism. We used the non-invasive eddy correlation technique to examine respiration and photosynthesis rates, through O-2 fluxes, from reef crests and reef slopes in the Florida Keys, USA. We assessed how the photosynthesis and respiration of different reef habitats is controlled by light and hydrodynamics. Numerous fluxes (over a 0.25 h period) were as high as 4500 mmol O-2 m(-2) d(-1), which can only be explained by efficient light utilization by the phototrophic community and the complex canopy structure of the reef, having a many-fold larger surface area than its horizontal projection. Over diel cycles, the reef crest was net autotrophic, whereas on the reef slope oxygen production and respiration were balanced. The autotrophic nature of the shallow reef crests implies that the export of organics is an important source of primary production for the larger area. Net oxygen production on the reef crest was proportional to the light intensity, up to 1750 mu mol photons m(-2) s(-1) an d decreased thereafter as respiration was stimulated by high current velocities coincident with peak light levels. Nighttime respiration rates were also stimulated by the current velocity, through enhanced ventilation of the porous framework of the reef. Respiration rates were the highest directly after sunset, and then decreased during the night suggesting that highly labile photosynthates produced during the day fueled early-night respiration. The reef framework was also important to the acquisition of nutrients as the ambient nitrogen stock in the water had sufficient capacity to support these high production rates across the entire reef width. These direct measurements of complex reefs systems yielded high metabolic rates and dynamics that can only be determined through in situ, high temporal resolution measurements.

Convective mass transport in heterogeneous biofilms, consisting of cell clusters and voids, was investigated using oxygen microelectrodes. Oxygen concentration profiles were measured and contour plots constructed at different (average) flow velocities (Uavg). The profiles were used to determine the thickness of the mass transfer boundary layer (δh) above the voids and the cell clusters. The δh above the biofilm was inversely related to flow, as expected, and decreased exponentially with increasing flow velocity. However, the δh above the voids decreased more rapidly than the δh above the cell clusters resulting in two distinct situations; at low flow velocities the oxygen contours were parallel to the substratum but at high velocities were parallel to the irregular biofilm surface. It was concluded that at low flow velocities the biofilm could be modeled one-dimensionally, with fluxes perpendicular to the substratum and the exchange area being equal to the substratum area, but at higher velocities biofilm voids facilitate mass transport and a more complex, three-dimensional model would be more appropriate. In this latter case fluxes are multidirectional, and the exchange area is equal to that of the convoluted biofilm surface.

The structural heterogeneity of a microbial biofilm was demonstrated by scanning electron microscopy, confocal scanning laser microscopy (CSLM), and cryoembedding followed by sectioning and microscopic examination. Biofilm was composed of a binary population of Pseudomonas aeruginosa and Klebsiella pneumoniae grown in a continuous flow annular reactor. The three microscopic methods provided a consistent picture of the biofilm as a non-uniform structure characterized by variable thickness and variable local cell and polymer densities. Significant changes in these parameters occurred in the biofilm over distances of 10 μm or less. Though the biofilm was several hundred microns thick in places, areas of bare substratum were also observed on the same sample coupon. Cell-free pores and channels in the biofilm interior were evident. Specific staining of cellular nucleic acids with ethidium bromide and extracellular polymeric substances (EPS) with calcofluor showed that cell and EPS distributions did not always overlap. The ethidium bromide-stained region was contained within the larger region of calcofluor staining; thus, some cell-free areas actually were filled with EPS. CSLM and cryoembedding approaches are superior to SEM in their ability to image the biofilm interior and in their potential to provide quantitative information.

O2 and pH microprofiles were measured above corroding mild steel covered with a biofilm. The pH in the anodic areas (tubercles) ranged from 5 to 7 and was always 9.45 at the surface of the cathodic areas. After 1 month of biofilm development, O2 was depleted at the anodic area but could reach the cathodic surface where it was reduced. Consequently, differential O2 concentration cells were the driving force for corrosion. The O2 microprofiles indicated that O2 was consumed in the tubercles, probably by microbial activity, while O2 was reduced electrochemically in the cathodic areas. It was concluded that O2 transfer to the cathodic surface was the rate limiting step for the corrosion process.

In microbial consortia from rapid sand filters, manganese and nitrate transformations appear to be linked. Nitrate appears to have a beneficial effect on the removal of Mn. In aerated batch cultures derived from these consortia, the Mn removal rate was 38% higher in the presence of nitrate and was accompanied by nitrite accumulation. There was no increase in Mn removal when ammonium was added to the medium instead of nitrate. 15NO3 experiments with batch cultures of Mn removing mixed bacterial cultures showed denitrification. The nitrate profile inside bacterial flocs was determined by means of microelectrode measurements. Nitrate uptake inside flocs grown in the presence of nitrate occurred under both anoxic and aerated conditions. No or a very slight nitrate uptake occurred in a floc poisoned with 0.2% HgCl2 and a floc grown in the absence of nitrate, respectively. In microbial flocs originating from two different field sites incubated under anoxic conditions, the presence of Mn2+ increased the nitrate-N gradient in the floc by 35–130%. Nitrate also exerted a stabilizing effect on the removal of Mn: microbial consortia were prevented from reducing MnO2 to Mn2+ by the presence of nitrate

Glucose and pH microprofiles in UASB aggregates were interpreted with respect to the population distribution of fermentative and methanogenic bacteria. In order to map the activity distribution of these bacteria, microprofiles were measured with different substrates (glucose, propionate and acetate) and at varying buffer capacities of the medium. The measurements were compared to calculated microprofiles, in which the spatial location of the activities was varied. The results suggested an inhomogenic activity distribution in the aggregates investigated: fermentatives were located dominantly at the outer 200-300 μm whereas methanogens were homogeneously distributed over the aggregate.

[1] The use of multiphysics computational fluid dynamics (CFD) approaches to simulate surface–subsurface flow processes is evaluated by comparison with flume experiments on current-exposed permeable bed forms. The unique experimental data include measurements of the time-averaged surface water flow velocities, the pressure distribution at the sediment–water interface, and pore water flow paths. The modeling approach first simulates the time-averaged turbulent flow in the channel with CFD and then uses the predicted pressure distribution at the sediment–water interface to drive a flow and transport model for the sediment. The CFD-modeled velocity and pressure distribution and transient particle tracks within the sediment agree reasonably well with observations. Differences that exist between observations and simulations mainly concern the eddies in the wake zone downstream of the ripple crests that are slightly shorter than those predicted by the model. This deviation propagates from the surface to the subsurface domain, appearing in the pressure distribution along the bed and, consequently, the subsurface flow patterns. The good representation of general patterns and rates makes multiphysics CFD modeling a powerful and sufficiently accurate tool that can replace measurements for many studies of surface–subsurface processes involving current-exposed immobile bed forms. The approach can be used for predicting transport processes where they cannot easily be observed, such as in large rivers and coastal systems where boundary conditions such as mean currents and bed forms can be mapped.

Emission of the greenhouse gas nitrous oxide (N2 O) from freshwater and terrestrial invertebrates has exclusively been ascribed to N2 O production by ingested denitrifying bacteria in the anoxic gut of the animals. Our study of marine molluscs now shows that also microbial biofilms on shell surfaces are important sites of N2 O production. The shell biofilms of Mytilus edulis, Littorina littorea and Hinia reticulata contributed 18-94% to the total animal-associated N2 O emission. Nitrification and denitrification were equally important sources of N2 O in shell biofilms as revealed by (15) N-stable isotope experiments with dissected shells. Microsensor measurements confirmed that both nitrification and denitrification can occur in shell biofilms due to a heterogeneous oxygen distribution. Accordingly, ammonium, nitrite and nitrate were important drivers of N2 O production in the shell biofilm of the three mollusc species. Ammonium excretion by the animals was found to be sufficient to sustain N2 O production in the shell biofilm. Apparently, the animals provide a nutrient-enriched microenvironment that stimulates growth and N2 O production of the shell biofilm. This animal-induced stimulation was demonstrated in a long-term microcosm experiment with the snail H. reticulata, where shell biofilms exhibited the highest N2 O emission rates when the animal was still living inside the shell.

We investigated the mechanisms leading to rapid death of corals when exposed to runoff and resuspended sediments, postulating that the killing was microbially mediated. Microsensor measurements were conducted in mesocosm experiments and in naturally accumulated sediment on corals. In organic-rich, but not in organic-poor sediment, pH and oxygen started to decrease as soon as the sediment accumulated on the coral. Organic-rich sediments caused tissue degradation within 1 d, whereas organic-poor sediments had no effect after 6 d. In the harmful organic-rich sediment, hydrogen sulfide concentrations were low initially but increased progressively because of the degradation of coral mucus and dead tissue. Dark incubations of corals showed that separate exposures to darkness, anoxia, and low pH did not cause mortality within 4 d. However, the combination of anoxia and low pH led to colony death within 24 h. When hydrogen sulfide was added after 12 h of anoxia and low pH, colonies died after an additional 3 h. We suggest that sedimentation kills corals through microbial processes triggered by the organic matter in the sediments, namely respiration and presumably fermentation and desulfurylation of products from tissue degradation. First, increased microbial respiration results in reduced O 2 and pH, initiating tissue degradation. Subsequently, the hydrogen sulfide formed by bacterial decomposition of coral tissue and mucus diffuses to the neighboring tissues, accelerating the spread of colony mortality. Our data suggest that the organic enrichment of coastal sediments is a key process in the degradation of coral reefs exposed to terrestrial runoff.

Background: Coral reefs degrade globally at an alarming rate, with benthic algae often replacing corals. However, theextent to which benthic algae contribute to coral mortality, and the potential mechanisms involved, remain disputed.Recent laboratory studies suggested that algae kill corals by inducing hypoxia on the coral surface, through stimulatedmicrobial respiration.Methods/Findings: We examined the main premise of this hypothesis by measuring in situ oxygen microenvironments atthe contact interface between the massive coral Porites spp. and turf algae, and between Porites spp. and crustose corallinealgae (CCA). Oxygen levels at the interface were similar to healthy coral tissue and ranged between 300–400 mM during theday. At night, the interface was hypoxic (,70 mM) in coral-turf interactions and close to anoxic (,2 mM) in coral-CCAinteractions, but these values were not significantly different from healthy tissue. The diffusive boundary layer (DBL) wasabout three times thicker at the interface than above healthy tissue, due to a depression in the local topography. Anumerical model, developed to analyze the oxygen profiles above the irregular interface, revealed strongly reduced netphotosynthesis and dark respiration rates at the coral-algal interface compared to unaffected tissue during the day and atnight, respectively.Conclusions/Significance: Our results showed that hypoxia was not a consistent feature in the microenvironment of thecoral-algal interface under in situ conditions. Therefore, hypoxia alone is unlikely to be the cause of coral mortality. Due tothe modified topography, the interaction zone is distinguished by a thicker diffusive boundary layer, which limits the localmetabolic activity and likely promotes accumulation of potentially harmful metabolic products (e.g., allelochemicals andprotons). Our study highlights the importance of mass transfer phenomena and the need for direct in situ measurements ofmicroenvironmental conditions in studies on coral stress.

Based on combined microsensor measurements of irradiance, temperature and O(2) , we compared light energy budgets in photosynthetic microbial mats, with a special focus on the efficiency of light energy conservation by photosynthesis. The euphotic zones in the three studied mats differed in their phototrophic community structure, pigment concentrations and thickness. In all mats,1% of the absorbed light energy was conserved via photosynthesis at high incident irradiance, while the rest was dissipated as heat. Under light-limiting conditions, the photosynthetic efficiency reached a maximum, which varied among the studied mats between 4.5% and 16.2% and was significantly lower than the theoretical maximum of 27.7%. The maximum efficiency correlated linearly with the light attenuation coefficient and photopigment concentration in the euphotic zone. Higher photosynthetic efficiency was found in mats with a thinner and more densely populated euphotic zone. Microbial mats exhibit a lower photosynthetic efficiency compared with ecosystems with a more open canopy-like organization of photosynthetic elements, where light propagation is not hindered to the same extent by photosynthetically inactive components; such components contributed about 40-80% to light absorption in the investigated microbial mats, which is in a similar range as in oceanic planktonic systems.

While microbial communities of aerosols have been examined, little is known about their sources. Nutrient composition and microbial communities of potential dust sources, saline lake sediments (SLS) and adjacent biological soil crusts (BSC), from Southern Australia were determined and compared with a previously analyzed dust sample. Multivariate analyses of fingerprinting profiles indicated that the bacterial communities of SLS and BSC were different, and these differences were mainly explained by salinity. Nutrient concentrations varied among the sites but could not explain the differences in microbial diversity patterns. Comparison of microbial communities with dust samples showed that deflation selects against filamentous cyanobacteria, such as the Nostocales group. This could be attributed to the firm attachment of cyanobacterial filaments to soil particles and/or because deflation occurs mainly in disturbed BSC, where cyanobacterial diversity is often low. Other bacterial groups, such as Actinobacteria and the spore-forming Firmicutes , were found in both dust and its sources. While Firmicutes -related sequences were mostly detected in the SLS bacterial communities (10% of total sequences), the actinobacterial sequences were retrieved from both (11–13%). In conclusion, the potential dust sources examined here show highly diverse bacterial communities and contain nutrients that can be transported with aerosols. The obtained fingerprinting and sequencing data may enable back tracking of dust plumes and their microorganisms.

In this study, members of a specific group of thin (6-14 µm filament diameter), vacuolated Beggiatoa-like filaments from six different hypersaline microbial mats were morphologically and phylogenetically characterized. Therefore, enrichment cultures were established, filaments were stained with fluorochromes to show intracellular structures and 16S rRNA genes were sequenced. Morphological characteristics of Beggiatoa-like filaments, in particular the presence of intracellular vacuoles, and the distribution of nucleic acids were visualized. In the intracellular vacuole nitrate reached concentrations of up to 650 mM. Fifteen of the retrieved 16S rRNA gene sequences formed a monophyletic cluster and were phylogenetically closely related (≥ 94.4% sequence identity). Sequences of known filamentous sulfide-oxidizing genera Beggiatoa and Thioploca that comprise non-vacuolated and vacuolated filaments from diverse habitats clearly delineated from this cluster. The novel monophyletic cluster was furthermore divided into two sub-clusters: one contained sequences originating from Guerrero Negro (Mexico) microbial mats and the other comprised sequences from five distinct Spanish hypersaline microbial mats from Ibiza, Formentera and Lake Chiprana. Our data suggest that Beggiatoa-like filaments from hypersaline environments displaying a thin filament diameter contain nitrate-storing vacuoles and are phylogenetically separate from known Beggiatoa. Therefore, we propose a novel genus for these organisms, which we suggest to name 'Candidatus Allobeggiatoa'.

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length (ΔTL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold ΔTL of −4.2 kb was the best predictor for progression-free and overall survival. ΔTL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the ΔTL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the ΔTL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

Size distributions and glucose and pH profiles of aggregates of the d -(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.

Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in a fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a thickness of approximately 100 to 120 μm, of the aggregates. Under conditions in which ammonium and oxygen penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes alone would lead to erroneous results.

Photosynthetically driven calcification was investigated in diatom-dominated carbonate sediments from Bait Reef, Australia. Laboratory measurements conducted over complete diel cycles, using O 2 , pH, CO 2― 3 , and Ca 2+ microsensors, confirmed that photosynthesis and respiration drive calcification and calcium release via their respective effects on the local pH. However, the dark situation does not simply mirror the light situation. Profiles showed that calcification and calcium release are not necessarily tightly coupled to the light cycle and that mass transfer phenomena need to be considered in diel chemical dynamics. The magnitude and timing of pH and CO 2― 3 concentration changes did not simply follow the light cycle. The pH in the upper 3 mm of the sediment changed more rapidly upon illumination than upon darkening. Consequently, photosynthetically induced calcification began shortly (within I h) after illumination, but the pH remained elevated and calcification continued for ∼7 h after darkening. Thus, calcification in marine phototrophic sediments is not limited to light periods, but may continue for extended periods after darkening. This decoupling of light, photosynthesis, and calcification has profound consequences for estimates of daily calcification rates, which have previously been made from measurements assuming close to steady states and 12: 12 h light: dark calcification and decalcification. In Bait Reef sediments, such an assumption underestimates daily calcification rates by two- to threefold.

A mat-forming population of the giant sulfur bacterium Thiomargarita was discovered at the flank of the mud volcano Amon on the Nile Deep Sea Fan in the Eastern Mediterranean Sea. All cells were of a spherical and vacuolated phenotype and internally stored globules of elemental sulfur. With a diameter of 24-65 µm, Thiomargarita cells from the Eastern Mediterranean were substantially smaller than cells of previously described populations. A 16S rRNA gene fragment was amplified and could be assigned to the Thiomargarita-resembling cells by fluorescence in situ hybridization. This sequence is monophyletic with published Thiomargarita sequences but sequence similarities are only about 94%, indicating a distinct diversification. In the investigated habitat, highly dynamic conditions favour Thiomargarita species over other sulfur-oxidizing bacteria. In contrast to Thiomargarita namibiensis populations, which rely on periodic resuspension from sulfidic sediment into the oxygenated water column, Thiomargarita cells at the Amon mud volcano seem to remain stationary at the sediment surface while environmental conditions change around them due to periodic brine flow.

Udgivelsesdato: 2010 Several freshwater and terrestrial invertebratespecies emit the greenhouse gas nitrous oxide(N2O). The N2O production associated with these animalswas ascribed to incomplete denitrification byingested sediment or soil bacteria. The present studyshows that many marine invertebrates also emit N2O atsubstantial rates. A total of 19 invertebrate species collectedin the German Wadden Sea and in Aarhus Bay,Denmark, and 1 aquacultured shrimp species weretested for N2O emission. Potential N2O emission ratesranged from 0 to 1.354 nmol ind.–1 h–1, with an averagerate of 0.320 nmol ind.–1 h–1, excluding the aquaculturedshrimp Litopenaeus vannamei, which showedthe highest rate of N2O emission measured so far forany marine species (3.569 nmol ind.–1 h–1), probablydue to very high nitrate concentrations in the rearingtanks. The N2O emitted by L. vannamei was almostexclusively produced in its gut by incomplete denitrification.Statistical analysis revealed that body weight,habitat, and exoskeletal biofilms were important determinantsof animal-associated N2O production. Thesnail Hinia reticulata emitted about 3.5 times moreN2O with an intact exoskeletal biofilm on its shell thanwith an experimentally cleaned shell. Thus, the N2Oproduction associated with marine invertebrates isapparently not due to gut denitrification in every species,but may also result from microbial activity on theexternal surfaces of animals. The high abundance andpotential N2O emission rates of many marine invertebratespecies suggest significant contributions to overallN2O emissions from coastal marine environmentsand aquaculture facilities.

Dissimilatory NO 3 − reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO 3 − concentrations yields potential rather than actual activities of dissimilatory NO 3 − reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO 3 − consumption. The water column of the sediment core is amended with 15 NO 3 − at the in situ 14 NO 3 − concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH 4 + dissolved in the gel slices is chemically converted by hypobromite to N 2 in reaction vials. The isotopic composition of N 2 is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of 15 NH 4 + . The results were compared to the NH 4 + microsensor profiles measured in freshwater sediment and quartz sand and to the N 2 O microsensor profiles measured in acetylene-amended sediments to trace denitrification.

We show that the nitrate storing vacuole of the sulfide-oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellular energy conservation. Immunostaining by antibodies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gradient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These findings show consistently that nitrate respiration contributes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation.

The effect of NO(3)(-) addition on dissimilatory SO(4)(2-) reduction and sulfide conversion in organic-rich sludge from the digestion basin of a recirculating marine aquaculture system was studied. SO(4)(2-) reduction could only explain a minor fraction (up to 4-9%) of the observed total sulfide production (up to 35 mmol L(-1) day(-1)), indicating that the main source of sulfide in the sludge was not SO(4)(2-) reduction, but desulfuration during the decomposition of organic matter. Although NO(3)(-) inhibited SO(4)(2-) reduction, but not desulfuration, the primary NO(3)(-) mitigation effect was the onset of NO(3)(-)-mediated sulfide oxidation (up to 75 mmol L(-1) day(-1)), partially to elemental sulfur (S(0)). Above NO(3)(-) concentrations of 0.6 mM in the bulk water, the net sulfide production and oxidation zones were moved deeper into flocs and sludge cores, which effectively prevented sulfide from entering the water column. However, the sulfide efflux from the sludge instantly recovered after NO(3)(-) depletion. Thus, the NO(3)(-) level in the water column controls the zonation and magnitude of sulfur transformations in the sludge. The effect of NO(3)(-) relies therefore on its sustained presence in the water column, which in turn depends on a well-functioning nitrification in the mariculture system.

Here we present, to the best of our knowledge, the first balanced light energy budget for a benthic microbial mat ecosystem, and show how the budget and the spatial distribution of the local photosynthetic efficiencies within the euphotic zone depend on the absorbed irradiance (Jabs). Our approach uses microscale measurements of the rates of heat dissipation, gross photosynthesis and light absorption in the system, and a model describing light propagation and conversion in a scattering–absorbing medium. The energy budget was dominated by heat dissipation on the expense of photosynthesis: in light-limiting conditions, 95.5% of the absorbed light energy dissipated as heat and 4.5% was channeled into photosynthesis. This energy disproportionation changed in favor of heat dissipation at increasing irradiance, with >99% of the absorbed light energy being dissipated as heat and % used by photosynthesis at Jabs>700 μmol photon m−2 s−1 (>150 J m−2 s−1). Maximum photosynthetic efficiencies varied with depth in the euphotic zone between 0.014−0.047 O2 per photon. Owing to steep light gradients, photosynthetic efficiencies varied differently with increasing irradiances at different depths in the euphotic zone; for example, at Jabs>700 μmol photon m−2 s−1, they reached around 10% of the maximum values at depths 0−0.3 mm and progressively increased toward 100% below 0.3 mm. This study provides the base for addressing, in much more detail, the photobiology of densely populated photosynthetic systems with intense absorption and scattering. Furthermore, our analysis has promising applications in other areas of photosynthesis research, such as plant biology and biotechnology.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named ‘Candidatus Methylomirabilis oxyfera’, was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that ‘M. oxyfera’ bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis. A previously unknown pathway producing oxygen during anaerobic methane oxidation linked to nitrite and nitrate reduction has been found in microbes isolated from freshwater sediments in Dutch drainage ditches. The complete genome of the bacterium responsible for this reaction has been assembled, and found to contain genes for aerobic methane oxidation. The bacterium reduces nitrite via the recombination of two molecules of nitric oxide into nitrogen and oxygen, bypassing the familiar denitrification intermediate nitrous oxide. This discovery is relevant to nitrogen and methane cycling in the environment and, since nitrogen oxides arose early on Earth, raises the possibility that oxygen was available to microbes before the advent of oxygen-producing photosynthesis. In certain microbes, the anaerobic oxidation of methane can be linked to the reduction of nitrates and nitrites. Here it is shown that this occurs through the intermediate production of oxygen. This brings the number of known biological pathways for oxygen production to four, with implications for our understanding of life on the early Earth.

Due to the conversion of acetic acid into the weaker carbonic acid and methane, the pH inside methanogenic aggregates is higher than in the bulk liquid. The pH profiles in aggregates were measured with pH microelectrodes. These profiles strongly determine the macro-kinetics of the aggregate, by their influence on the values of the growth parameters Ks and μmax. Acetate transport resistances were not limiting for the conversion rate in methanogenic aggregates. Nevertheless, the effectiveness factor η did not approach unity, but amounted to 0.57–0.62 in the acetate concentration range relevant for most methanogenic reactors. The value of η is determined almost entirely by the pH profiles inside the aggregates. It was concluded that for the physical/mathematical description of the conversion in methanogenic aggregates, information on the pH gradients and the pH dependency of the growth parameters is indispensable. Batch experiments showed that acetate uptake by aggregates was not coupled directly to methanogenesis. Consumed acetate was not converted instantaneously to methane, suggesting the conversion to proceed via a pool of acetate or reserve material.

The role of microbial consortia on the removal of manganese (Mn) was examined on sand from three different Belgian rapid sand filters for the treatment of ground water. Microorganisms closely associated with deposits of Fe and amorphous Mn precipitates were observed by SEM and EDAX techniques on sand from the filters able to remove Mn efficiently. Bacterial counts were performed. Of the CFU enumerated on PYM-medium, 25-33% displayed Mn-oxidizing activity.Batch cultures were set up by inoculating a Mn-containing, low organic medium with sand from one of the filters. Microbial growth resulted in the formation of Mn-removing bacterial flocs and a pH increase. Suppression of microbial growth by addition of azide, kanamycin, or by autoclaving reduced removal of Mn(2+) from 0.5 mM/day to 0.05-0.11 mM/day. Buffering the pH of the medium at 7.5 (0.1 mM Hepes) decelerated the Mn removal but did not halt it, whereas microelectrode measurements revealed a clear pH drop of about 0.7 units inside bacterial flocs. In the absence of Mn(2+), the pH drop was only 0.4 units. The auto-catalytic removal of Mn by the Mn oxide coated filter sand was not sufficient to explain the Mn removal observed. Inactivated cells were not capable of a pronounced autocatalytic Mn removal. Experiments with enrichment cultures indicated that the Mn-removing capacity of the microbial sand filter consortia was not constitutive but was promoted by preadaptation and the presence of a substratum. These results clearly link Mn oxidation in rapid sand filters to microbial processes.

Product inhibition of the acidogenic population from an anaerobic wastewater treatment plant was studied in a carefully controlled fed-batch culture. Repeated feeding with a concentrated glucose solution allowed rapid determination of the critical product concentration at which metabolism ceased completely. From the course of the different products, butyrate could be identified as the main inhibitor. The critical concentration of 48 mmol/l butyric acid was in good agreement with extrapolated data from earlier continuous culture experiments. Since a good reproducibility was obtained as well, it seems that the experimental technique developed merits wider application for the identification of inhibitory products and the determination of kinetic parameters.

Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (> or =0.19 mmol N m(-2) h(-1)) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O(2) consumption overlapped. In slurry incubations conducted with (15)NO(3)(-) enrichment in gas-impermeable bags, denitrification assays revealed that N(2) production occurred at initial O(2) concentrations of up to approximately 90 microM. Initial denitrification rates were not substantially affected by O(2) in surficial (0-4 cm) sediments, whereas rates increased by twofold with O(2) depletion in the at 4-6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), (29)N(2) and (30)N(2) were produced and O(2) was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O(2) may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.

Diel cycles of dissolved cationic metal concentrations commonly occur in freshwater streams in apparent response to coincident cycles in water quality parameters (pH, O2, temperature). Hourly sampling of the Cd-contaminated Riou Mort (France) revealed large diel cycles in "total" dissolved Cd (232-357 nM;0.45 microm) and "truly" dissolved Cd (56-297 nM;0.02 microm) which were strongly correlated with changes in water pH. Using measured fluxes, a dissolved O2 model was constructed that indicated that benthic metabolic activities, respiration and photosynthesis, were responsible for the diel O2 (and thus, CO2 and pH) variation in the stream. However, microsensor measurements also showed that the pH changes occurred at the biofilm interface earlier than in the bulk water column. This difference in timing was reflected in the Cd dynamics, where pH-controlled sorption effects caused Cd partitioning from the truly dissolved pool onto the biofilm in the morning, and from the truly dissolved pool onto large colloids (0.02-0.45 microm) later in the day. Because this process causes large changes in the bioavailable Cd fraction, it has significant implications for Cd toxicity in freshwater streams. This study demonstrates the profound control of benthic microbiological processes on the cycling of heavy metals in aquatic systems.

Spectrally resolved imaging was applied to study the growth dynamics of phototrophic biofilms comprizing a mixture of one cyanobacterial and one diatom species. Linear spectral unmixing was combined with liquid chromatography to quantitatively discriminate the areal biomass densities of the two populations. The grown biofilms exhibited highly heterogeneous distribution with patches of 1–2mm in size, although the conditions provided for growth, including substrate roughness, illumination and flow of the overlying water, were homogeneous. The biomass was initially dominated by cyanobacteria, which exhibited an exponential-like growth phase during days 2–7. Their population declined during days 9–17, which coincided with the growth phase of the diatom population. By allowing non-invasive and real-time measurements and data evaluation, the spectral imaging approach constitutes a useful tool for microbial ecologists.

Ammonium microprofiles in two dominant types of lake sediment were measured using ion-selective microelectrodes. These profiles enabled establishment of local fluxes and convensions. Comparison of ammonium profiles obtained from the analysis of sediment slices with those measured with microelectrodes showed the superior spatial resolution of the latter method. Flux measurements with microelectrodes were confirmed by laboratory incubation experiments. In both sediments ammonium consumption was restricted to the oxic surface layer. In sandy sediments ammonium was only consumed, while in silty sediment both ammonium production and consumption were observed. In silty sediment ammonium production by anaerobic mineralization was not balanced by ammonium consumption in the oxic surface layer.

Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.

In contrast to other B-cell neoplasias, chronic lymphocytic leukaemia (CLL) is characterized by increased non-leukaemic T-cells. In order to assess their replicative history, telomere length was analyzed in subsets of leucocytes from CLL patients. Naive and memory T-cells from ZAP-70(+)/CD38(+) patients exhibited significantly shorter average telomere lengths than ZAP-70(-)/CD38(-) patients. Compared to the age-related percentiles of telomere length values from healthy individuals practically all values of the naive and memory T-cells from the ZAP-70(+)/CD38(+) CLL patients fell below the 50th percentile. This indicated an extensive expansion and a role for T-cells in ZAP-70(+)/CD38(+) CLL patients.