Lars Riis Damgaard

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.

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.

Mapping electron sources and sinks in a marine biogeobattery

Filamentous, multicellular bacteria of the Desulfobulbaceae family form a biogeobattery in marine sediments by mediating an electric coupling between sulfide oxidation in deeper anoxic layers and oxygen reduction at the sediment surface. The electric fields generated in such biogeobatteries were determined with microelectrodes and correlated well with geochemical indicators of microbial electrogenic activity in the top 2 cm of the sediment. The electric fields collapsed within less than a minute when oxygen was removed and reestablished when oxygen was reintroduced, demonstrating a direct coupling between aerobic microbial activity and presence of electric fields. Modeling of the electric potential distribution was used to quantify and localize electron sources and sinks. Most anodic oxidation was confined to the lower part of the anoxic zone with nondetectable free sulfide, suggesting that iron sulfide was the main sulfide source and had already been depleted in the upper part of the anoxic zone. The flow of electrons from anoxic sediment layers fuelled up to 80% of the aerobic activity of the sediment. Responses of the electric field to the presence and absence of nitrate, nitrite, or nitrous oxide in the overlying water readily demonstrated potentials for cathodic reduction of nitrite or nitric oxide and possibly nitrate but not nitrous oxide.

Cable Bacteria in Freshwater Sediments

ABSTRACT In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures and electric fields indicated electron transfer between vertically separated anodic and cathodic half-reactions. Fluorescence in situ hybridization revealed the presence of Desulfobulbaceae filaments. In addition, in situ measurements of oxygen, pH, and electric potential distributions in the waterlogged banks of Giber Å demonstrated the presence of distant electric redox coupling in naturally occurring freshwater sediment. At the same site, filamentous Desulfobulbaceae with cable bacterium morphology were found to be present. Their 16S rRNA gene sequence placed them as a distinct sister group to the known marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary origin of the cable phenotype within Desulfobulbaceae with subsequent diversification into a freshwater and a marine lineage.

Long-distance electron transfer by cable bacteria in aquifer sediments

The biodegradation of organic pollutants in aquifers is often restricted to the fringes of contaminant plumes where steep countergradients of electron donors and acceptors are separated by limited dispersive mixing. However, long-distance electron transfer (LDET) by filamentous ‘cable bacteria’ has recently been discovered in marine sediments to couple spatially separated redox half reactions over centimeter scales. Here we provide primary evidence that such sulfur-oxidizing cable bacteria can also be found at oxic–anoxic interfaces in aquifer sediments, where they provide a means for the direct recycling of sulfate by electron transfer over 1–2-cm distance. Sediments were taken from a hydrocarbon-contaminated aquifer, amended with iron sulfide and saturated with water, leaving the sediment surface exposed to air. Steep geochemical gradients developed in the upper 3 cm, showing a spatial separation of oxygen and sulfide by 9 mm together with a pH profile characteristic for sulfur oxidation by LDET. Bacterial filaments, which were highly abundant in the suboxic zone, were identified by sequencing of 16S rRNA genes and fluorescence in situ hybridization (FISH) as cable bacteria belonging to the Desulfobulbaceae. The detection of similar Desulfobulbaceae at the oxic–anoxic interface of fresh sediment cores taken at a contaminated aquifer suggests that LDET may indeed be active at the capillary fringe in situ.

Microscale biosensors for environmental monitoring

Abstract Microbiosensors for nitrate and methane have been constructed with diameters down to 30 μm. Both sensors are characterized by linear responses to their substrates and 90% response times from 10–120 s. The efficient diffusional transport at a micrometer scale makes it possible to utilize new concepts in the construction of biosensors.

EFFECT OF WATER VELOCITY AND BENTHIC DIATOM MORPHOLOGY ON THE WATER CHEMISTRY EXPERIENCED BY POSTLARVAL ABALONE

Abstract The water bathing postlarval abalone often lies within the diffusive boundary layer (DBL) so its chemistry is greatly influenced by the metabolism of the biofilm on which the abalone feed. This study used microelectrodes to investigate the influence of water velocity and diatom morphology on dissolved oxygen and pH in the DBL. Decreasing water velocity increased the thickness of the DBL, thereby increasing the amplitude of changes in oxygen concentration. Over a film of the prostrate diatom Nitzschia ovalis Arnot, DBL thickness averaged 71, 139, 177, and 406 μm at water velocities of 78, 15, 7, and 1 mm s−1 respectively. Corresponding oxygen concentrations at the biofilm surface under moderate light (75 μE m−2 s−1) and temperature (15°C) averaged 111%, 120%, 125%, and 151% of air saturation respectively, at the four velocities. The presence of a 1-mm tall diatom canopy (Achnanthes longipes Agardh) over a Nitzschia ovalis film thickened the DBL by 3-fold at 1 mm s−1 and 6-fold at ∼80 mm s−1. The thickened DBL and higher diatom biomass generated extreme conditions at the biofilm surface. Dissolved oxygen concentrations as high as 440% of air saturation, and pH as high as 9.8 were recorded beneath the canopy in moderate light (105 μE m−2 s−1) and temperature (15°C) at a water velocity of 1 mm s−1. Changes during darkness were less extreme, with 53% oxygen saturation and pH 7.7 the minima recorded. These measurements demonstrate the extreme water chemistry that can develop in the microhabitat of postlarval abalone. The changes will be amplified by the presence of filamentous diatoms, by increased light intensity, and by lack of water movement. Standard aeration will greatly reduce the extremes experienced by postlarvae by generating water movement sufficient to thin the DBL.

Electric potential microelectrode for studies of electrobiogeophysics

Spatially separated electron donors and acceptors in sediment can be exploited by the so-called “cable bacteria.” Electric potential microelectrodes (EPMs) were constructed to measure the electric fields that should appear when cable bacteria conduct electrons over centimeter distances. The EPMs were needle-shaped, shielded Ag/AgCl half-cells that were rendered insensitive to redox-active species in the environment. Tip diameters of 40 to 100 µm and signal resolution of approximately 10 μV were achieved. A test in marine sediments with active cable bacteria showed an electric potential increase by approximately 2 mV from the sediment-water interface to a depth of approximately 20 mm, in accordance with the location and direction of the electric currents estimated from oxygen, pH, and H2S microprofiles. The EPM also captured emergence and decay of electric diffusion potentials in the upper millimeters of artificial sediment in response to changes in ion concentrations in the overlying water. The results suggest that the EPM can be used to track electric current sources and sinks with submillimeter resolution in microbial, biogeochemical, and geophysical studies.

Motility of Electric Cable Bacteria

ABSTRACT Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces with a highly variable speed of 0.5 ± 0.3 μm s−1 (mean ± standard deviation) and time between reversals of 155 ± 108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment. IMPORTANCE This study reports on the motility of cable bacteria, capable of transmitting electrons over centimeter distances. It gives us a new insight into their behavior in sediments and explains previously puzzling findings. Cable bacteria greatly influence their environment, and this article adds significantly to the body of knowledge about this organism.

The effect of respiration on the phototactic behavior of the purple nonsulfur bacterium Rhodospirillum centenum

Abstract The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc1 complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion.