Microelectrodes in Microbial Ecology U
nderstanding the microenvironment of bacteria has presented many challenges for the microbial ecologist. Various high-resolution tech niques such as scanning and transmis sion electron microscopy and fluores cent staining, immunofluorescence, and other techniques for light micros copy have proved useful in reaching a high spatial resolution. However, the search for techniques to study the dy namics of the microenvironment has not been as successful until recently. The results obtained from most chemi cal and radiotracer techniques, other than those from the combined use of autoradiography and fluorescence mi croscopy, are so macroscopic in nature (1 cm-1 mm scale) that they cannot be directly related to the dynamics of the relevant microorganism. Simple intracellular capillary elec-
FOCUS trodes have been used in neurophysiol ogy since the 1950s to measure action potentials in ion transport over biologi cal membranes, and ion-selective elec trodes were developed soon thereafter for the determination of H + , Na + , K + , and Ca 2+ . However, these analytical techniques of obvious applicability to many problems in microbial ecology did not receive much attention until 1978, when Niels Peter Revsbech and Bo Barker Jjirgensen at the Institute of Ecology and Genetics, University of Aarhus, Denmark, began using oxygen microelectrodes in their studies of the ecology and biogeochemistry of marine
sediments and other microbial envi ronments. Today, Revsbech and J0rgensen use five types of microelec trodes, two types of oxygen microelec trodes, a combined microelectrode for nitrous oxide and oxygen, a sulfide mi croelectrode, and a pH microelectrode. The first three microelectrodes have diameters of about 10 μηι and the last two of about 50 μηι. Some of the elec trodes actually contain two or three cathodes plus a reference electrode, all situated behind a polymer membrane. The construction of these microelec trodes was described by Revsbech and J^rgensen in 1986 and by Revsbech and co-workers in 1988. These microelectrodes have been successfully used for field work, and data were obtained in Solar Lake, Si nai, in 1983 and in the hot springs of Yellowstone Park in 1984. In situ ex periments have been done for several years at a water depth of several me ters, where the micromanipulator is op erated by a diver. Recently measure ments were obtained in the deep sea with the microelectrodes mounted on a free-falling vehicle or operated from a submersible vessel. According to J0rgensen, this type of work may prove easier than field work in coastal sedi ments where snails, crabs, fish, and other creatures seem determined to ap proach and break the delicate micro electrodes. In 1986 Revsbech and J^rgensen de signed a data collection system in which the signal from the picoammeter is fed into a 12-bit analog-to-digital converter. The digital signals are fed into a data collection unit, and the pulses from a stepper motor on the mi cromanipulator are also counted by
this unit. In this way, coupled data points of electrode current and depth are received. The data are then trans mitted to a microcomputer that per forms calculations on the data with si multaneous collection of new data. Microelectrode applications in microbial ecology Of primary importance to the under standing of microbial environments is whether oxygen is present. According to J0rgensen, before the use of oxygen microelectrodes, assumptions were made based on visual interpretations. Surprisingly, oxygen measurements with the microelectrode showed an oxic zone of only 2 mm, whereas a positive redox potential down to a depth of 3.5 cm was found in typical sandy coastal sediments. The oxic-anoxic in terface often was not distinguishable on the redox profile. The sediments of ten contain large amounts of iron and manganese, which in their oxidized forms could be responsible for these positive redox potentials. However, it is difficult to understand how these of ten insoluble compounds are kept oxi dized without oxygen being present be cause the oxidized species are contin ually being reduced by metabolic activity of some bacteria as well as by the hydrogen sulfide formed by sulfate-reducing bacteria. It is probable, says Jjirgensen, that the reworking of sediment by infauna referred to as "bioturbation" occurs periodically and often keeps a thick layer oxidized. The surface of a microbial mat with tufts of unicellular algae (diatoms) and cyanobacteria. (3X magnification)
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FOCUS Diffusive boundary layers probably cover the entire sea floor and range in thickness from 0.1 to 0.2 mm at bulk water flow rates of 5-10 cm/s and up to approximately 1 mm in stagnant water. These diffusive boundary layers are actually thin films of water, the water of which does not participate in the general circulation and where the transport of solutes occurs by molecular diffusion. Oxygen microelectrodes are ideal for studying these diffusive boundary layers, giving information about thickness and stability. Microbial photosynthesis Oxygen microelectrodes are well-suited for studying photosynthesis in sediments with dense microalgae, cyanobacterial mats from saline environments, or in algal mats from hot springs because of high, rapidly changing concentrations of oxygen. Good oxygen and photosynthesis profiles can also be obtained in sediments having a relatively sparse population of diatoms that exhibit only modest photosynthetic activity. The steady-state oxygen concentration approached when the sediment is exposed to light for several minutes results from the equilibrium between oxygen-consuming and oxygen-producing processes at each depth. The processes removing oxygen within a layer are biological and chemical consumption of oxygen and molecular diffusion away from or into the layer. The only process producing oxygen is oxygenic photosynthesis, as carried out by the microalgae and cyanobacteria. If photosynthesis is stopped instantaneously by darkening, the oxygen-consuming processes continue while the oxygen-producing process of photosyn-
thesis stops. The oxygen concentration at each depth therefore will immediately start to decrease. Measuring the initial rate of decrease in oxygen concentration after darkening thus provides a method to quantitate photosynthesis. The advantages of the oxygen microelectrode method for measuring photosynthesis are numerous, according to J^rgensen. The method is nondestructive, repetitive measurements can be made on the same cluster of microalgae, the results of the measurement are known within seconds, and methodological errors are better controlled than in the two other methods used extensively to quantify the photosynthetic activity of benthic and epiphytic algae (the 14C method and the oxygenexchange method). J^rgensen indicates that the oxygen microelectrode method also has limitations. Fluctuations in light intensities caused by drifting clouds and movement of the substratum and low photosynthetic activity are but a few. Also, the presence of large mineral grains or carbonate shells may cause a "sudden death" for the fragile electrodes. Microbial sulfide oxidation In the oxic world (with oxygen), aerobic respiration dominates and metabolic processes are primarily oxidative. In the anoxic world (without oxygen), bacterial fermentation and anaerobic respiration dominate and metabolic processes are primarily reductive. The oxic-anoxic interface is an important boundary where reoxidation of these reduced products occurs. The only suitable microelectrode (at present) to detect these compounds is the sulfide electrode.
Solar Lake on the Red Sea coast. 426 A · ANALYTICAL CHEMISTRY, VOL. 61, NO. 6, MARCH 15, 1989
Certain microorganisms have adapted to life at the oxic-anoxic interface. Heterotrophic bacteria (i.e., bacteria that live on organic compounds) will often reveal the location of the interface by forming narrow bands in stagnant liquid cultures. Their adaptation to microoxic conditions is demonstrated with an oxygen electrode. Likewise, aerobic, heterotrophic bacteria, which through a chemotactic response aggregate in the microoxic zone, possibly enjoy this environment because it contains the optimal supply of organic substrates. Numerous chemoautotrophic bacteria (i.e., bacteria that live on inorganic compounds and assimilate CO2) adapt to this environment where H2, NH4, H2S, and Fe 2 + serve as electron donors for their energy metabolism. The specialized sulfur and iron bacteria, which have adapted to oxidize H 2 S and Fe 2 + , are in an unusual situation of having to compete with a simultaneous chemical oxidation of their energy substrates by oxygen. Referred to as gradient bacteria, they are intimately "bound" to the oxic-anoxic interface, where they live between the opposed gradients of their electron donor and acceptor and can readily be studied by microelectrodes. Life at the 0 2 -H 2 S interface Although most sulfide oxidation in nature takes place within sediments at depths of several centimeters from the surface, sediments with high organic turnover and thus high rates of oxygen consumption and sulfate reduction may push the O2-H2S interface up to the sediment-water interface. When this occurs, colorless sulfur bacteria such as the filamentous Beggiatoa spp. can sometimes be observed on the sedi. ment surface. Even though circulating, aerated seawater bathes the sediment, the bacteria live under microoxic or anoxic conditions. This is the result of both the presence of the diffusive boundary layer and the high oxygen uptake of the bacteria. The oxygen uptake is limited only by the diffusive flux through the boundary layer, and the potential oxygen uptake of the Beggiatoa mat is often much higher than the actual uptake. This phenomenon can explain why anaerobic bacteria, such as purple sulfur bacteria, can grow on mud surfaces that appear to be exposed to highoxygen concentrations. By using a gradient culture system of seawater partially solidified with agar and containing 4 mM H2S in the lower 2 cm only, a chemoautotropic strain of Beggiatoa could be grown as a 0.4-mmthiçk horizontal band just at the sharp O2-H2S interface. The oxidation of
H2S to sulfuric acid caused a sharp minimum in pH. The ratio of H2S/O2 consumption in the Beggiatoa mat was about 0.6 (if all the H 2 S were oxidized to H2SO4, the ratio would be 0.5) be cause 15% of the electrons from H 2 S were transferred to CO2. The position of the O2-H2S interface is dependent on light and photosynthetic activity. The diurnal light-dark variations create a cyclic pulse of oxy gen to which the gradient bacteria and photosynthetic organisms need to adapt. Revsbech and J0rgensen ana lyzed the dynamic distributions of O2, H 2 S, and pH in cyanobacterial mats from the hypersaline Solar Lake in Si nai in 1983. The three microelectrodes were glued together into one unit with the sensing tips less than 1 mm apart to obtain precise depth-time correlations among the three parameters during abrupt light-dark shifts and during the natural variations over a 24-h cycle. The organisms living at 0.5-mm depth were exposed to variations from anoxia to over 1 atm partial pressure of oxygen, depending on the light condi tions, and those living at 1.5 mm were exposed to 250 μΜ H 2 S in the dark and 500 μΜ 0 2 in the light. Most of the dominant mat organisms are motile and adjust to the changing conditions by moving up and down. Some, howev er, remain fixed and experience opti mum conditions for growth only during part of the light-dark cycle. Use of the appropriate microelectrode would probably also demonstrate similar fluctuations in NO^, N H j , H P O f , Fe2+, and Mn 2+ , as we'll as fluc tuations in organic compounds. Anoxygenic photosynthesis by pur ple or green sulfur bacteria or by cyanobacteria can also be studied using a combination of oxygen and sulfide mi croelectrodes. During anoxygenic pho tosynthesis, the bacteria use H 2 S in stead of H 2 0 to reduce CO2 into biomass. Denitrification The combined microsensor for nitrous oxide and oxygen can be used to mea sure naturally occurring nitrous oxide, which is formed as an intermediate in the bacterial reduction of nitrate to dinitrogen (denitrification). The emis sion of nitrous oxide from natural sources is of particular interest because nitrous oxide is one of the gases that catalyzes the degradation of ozone in the stratosphere. The reduction of ni trous oxide can be prevented by the addition of acetylene, and the accumu lation rate of nitrous oxide in such acety lene-inhibited samples is thus a mea sure of the denitrification rate. From microprofiles of nitrous oxide obtained
Microbial mat along the shore. Such mats are solid enough to walk on. by using the microsensor for nitrous oxide and oxygen, it is possible to quantify denitrification within sub strates such as soil, sediments, and waste water treatment biofilms. The results from sediments and biofilms show that denitrification occurs only at anoxic or near-anoxic conditions and that diffusion limitation of nitrate of ten restricts denitrification to a narrow layer immediately below the oxic sur face layer. With 250 μΜ nitrate in the water above a waste water treatment biofilm, this layer may only be 0.2 mm thick. Miscellaneous microenvironments The speed and high analytical capacity of the microelectrode lends it to wider and more general studies involving photosynthesis. In addition, because of the high spatial and temporal resolu tion of the microelectrode techniques, they are also ideally suited for in situ studies of the physiology of undis turbed microbial communities. Be cause of the small dimensions relevant to microbial environments, the impor tant transport process for gases, ions, and other dissolved molecules is pre dominantly molecular diffusion. A sim ple mapping of the concentration field around microbial communities may therefore allow the quantitative calcu lation of their metabolic rates. Microelectrode studies of soils, the rhizosphere around roots in soils and sediments, the Rhizobium nodules of legumes, microbial films from sewage treatment plants, and epiphytic com munities have led to a greater under standing of these microenvironments. These studies have demonstrated the existence of anoxic microzones in soil crumbs of otherwise well-aerated soils. Future developments As more ion-selective electrodes are de veloped and refined for measuring
NO^, NH4, and POij - at the macroelectrode level, it should eventually be pos sible to scale down these electrodes to the microelectrode level, thus making available new tools for the study of metabolic processes in natural micro bial communities. In addition to the appearance of these new sensors, more sophisticated methods of processing data are becom ing available. The use of microelec trodes in ecological research has led to the wish for additional microsensors to measure such environmental parame ters as light, temperature, and salinity at a spatial resolution sufficiently high to be relevant to the individual micro bial populations. A fiber-optic micro sensor with a sensing tip diameter of 15-20 μηι, recently developed by J0rgensen and Des Marais, was used to study the spectral light distribution in microbial mats and sediments. The microbial environments and ecological research applications of mi croelectrodes studied thus far are rath er limited. Microelectrodes appear to be ideal for measuring rapidly chang ing and often quite chemically differ ent bacterial microenvironments. Im portant future directions include the study of corrosion of off-shore steel constructions, oil degradation in the sea, the development of bacterial plaque on teeth, and the metabolism of immobilized bacterial films in biotech nology. Sharon Boots Suggested reading Revsbech, N. P.; Jdrgensen, Β. Β. In Ad vances in Microbial Ecology; Marshall, K. C, Ed.; Plenum Publishing Corp.: New York, 1986; pp. 293-352. Reimers, C. E.; Fischer, K. M.; Merewether, R.; Smith, K. L., Jr.; Jahnke, R. A. Nature 1986, 320, 741-44. Revsbech, N. P.; Nielsen, L. P.; Christensen, P. B.; Sorensen, J. Appl. Environ. Microbiol. 1988,54, 2245-49. J^rgensen, B. B.; Des Marais, D. J. Limnol. Oceanogr. 1988, 33, 99-113.
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