Microbial Ecology
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nderstanding the microenvironment of bacteria has presented many challenges for the microbial ecologist. Various high-resolution techniques such as scanning and transmission electron microscopy and fluorescent staining, immunofluorescence, and other techniques for light microscopy have proved useful in reaching a high spatial resolution. However, the search for techniques to study the dynamics of the microenvironment has not been as successful until recently. The results obtained from most chemical and radiotracer techniques, other than those from the combined use of autoradiography and fluorescence microscopy, are so macroscopic in nature (1cm-1 mm scale) that they cannot be directly related to the dynamics of the relevant microorganism. Simple intracellular capillary elec-
trodes have been used in neurophysiology since the 1950s to measure action potentials in ion transport over biological membranes, and ion-selective electrodes were developed soon thereafter for the determination of H+, Na+, KC, and CaZt. 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 Jfirgensena t 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 environments. Today, Revsbech and Jfirgensen use five types of microelectrodes, two types of oxygen microelectrodes, a combined microelectrode for nitrous oxide and oxygen, a sulfide microelectrode, and a pH microelectrode. The first three microelectrodes have diameters of about 10 pm and the last two of ahout 50 pm. Some of the electrodes actually contain two or three cathodes plus a reference electrode, all situated behind a polymer membrane. The construction of these microelectrodes was described by Revsbech and Jfirgensen 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, Sinai, in 1983 and in the hot springs of Yellowstone Park in 1984. In situ experiments have been done for several years a t a water depth of several meters, where the micromanipulator is operated by a diver. Recently measurements were obtained in the deep sea with the microelectrodes mounted on a free-falling vehicle or operated from a submersible vessel. According to Jfirgensen, this type of work may prove easier than field work in coastal sediments where snails, crabs, fish, and other creatures seem determined to approach and break the delicate microelectrodes. In 1986 Revsbech and Jfirgensen designed 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 micromanipulator are also counted by
this unit. In this way, coupled data points of electrode current and depth are received. The data are then transmitted to a microcomputer that performs calculations on the data with simultaneous collection of new data. Microelectrode applications in microbial ecology Of primary importance to the understanding of microbial environments is whether oxygen is present. According to J$rgensen, before the use of oxygen microelectrodes, assumptions were made based on visual interpretations. Surprisingly, oxygen measurements with the microelectrode showed anoxic zone of only 2 mm, whereas a positive redox potential down to a depth of 3.5 em was found in typical sandy coastal sediments. The oxic-anoxic interface often was not distinguishable on the redox profile. The sediments often 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 often insoluble compounds are kept oxidized without oxygen being present because the oxidized species are continually being reduced by metabolic activity of some bacteria as well as by the hydrogen sulfide formed by sulfate-reducing bacteria. It is probable, says Jfirgensen, 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 mot wirh rufrs of unicellular algae (diatoms)and ryanobacteria. (3X mognificafion)
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Diffusive boundary layers probably cover the entire sea floor and range in thickness from 0.1 to 0.2 mm a t bulk water flow rates of 5-10 cm/s and up to approximately 1mm 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.
MicroMal Photoarpnthesk 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 photmyntbetic 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-
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thesis stops. The oxygen concentration a t 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 14Cmethod and the oxygenexchange method). J#rgensen indicates that the oxygen microelectrodemethod 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.
Mlcmblal sulfide oxMatlon 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 comDounds is the sulfide electrode.
ANALYTICAL CMMISTRY. VOL. 61, NO. 6, MARCH 15, 1989
Certain microorganismshave adapted to life a t 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 COz) adapt to this environment where Hz, NH:, HzS, and Fez+ serve as electron donors for their energy metabolism. The specialized sulfur and iron bacteria, which have adapted to oxidize H2S and Fez+, 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.
Llfe at the @-HS lnteriace 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 OZ-HZSinterface up to the sedimeucwater interface. When this occurs, colorless sulfur baderia 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 ou 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 HzS in the lower 2 cm only, a chemoautotropic strain of Beggiatoa could he grown as a 0.4-mmthick horizontal band just a t the sharp OZ-H2S interface. The oxidation of
HzS to sulfuric acid caused a sharp minimum in pH. The ratio of HzS/Oz consumption in the Beggiatoa mat was about 0.6 (if all the HzS were oxidized to HzSOa, the ratio would be 0.5) because 15%of the electrons from HzS were transferred to Con. The position of the OZ-HZS interface is dependent on light and photosynthetic activity. The diurnal lighbdark variations create a cyclic pulse of oxygen to which the gradient bacteria and photosynthetic organisms need to adapt. Revshech and J#rgensen analyzed the dynamic distributions of 02, HzS, and pH in cyanohacterial mats from the hypersaline Solar Lake in Sinai in 1983. The three microelectrodes were glued together into one unit with the sensing tips less than 1mm apart to obtain precise depth-time correlations among the three parameters during abrupt lighedark shifts and during the natural variations over a 24-h cycle. The organisms living a t 0.5-mm depth were exposed to variations from anoxia to over 1atm partial pressure of oxygen, depending on the light conditions, and those living a t 1.5 mm were exposed to 250 fiM HzS in the dark and 500 pM Oz in the light. Most of the dominant mat organisms are motile and adjust to the changing conditions by moving up and down. Some, however, remain fixed and experience optimum conditions for growth only during part of the ligwdark cycle. Use of the appropriate microelectrode would probably also demonstrate similar fluctuations in NO;, NH:, HPOi-, Fez+,and MnZ+, as well as fluctuations in organic compounds. Anoxygenic photosynthesis by purple or green sulfur bacteria or by cyanobacteria can also he studied using a combination of oxygen and sulfide microelectrodes. During anoxygenic photosynthesis, the bacteria use HzS instead of HzO to reduce Cot into hiomass.
DeniMHcath The combined microsensor for nitrous oxide and oxygen can he used to measure naturally occurring nitrous oxide, which is formed as an intermediate in the bacterial reduction of nitrate to dinitrogen (denitrification). The emission 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 nitrous oxide can he prevented by the addition of acetylene, and the accumulation rate of nitrous oxide in such acetylene-inhibited samples is thus a measure of the denitrification rate. From microprofilesof nitrous oxide obtained
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hy using the microsensor for nitrous oxide and oxygen, it is possible to quantify denitrification within substrates such as soil, sediments, and waste water treatment biofilms. The results from sediments and hiofilms show that denitrification occurs only a t anoxic or near-anoxic conditions and that diffusion limitation of nitrate often restricts denitrification to a narrow layer immediately below the oxic surface layer. With 250 fiM nitrate in the water above a waste water treatment hiofilm, this layer may only he 0.2 mm thick. Miellam-v i-i 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 resolution of the microelectrode techniques, they are also ideally suited for in situ studies of the physiology of undiaturhed microbial communities. Because of the small dimensions relevant to microbial environments, the important transport process for gases, ions, and other dissolved molecules is predominantly molecular diffusion. A simple mapping of the concentration field around microbial communities may therefore allow the quantitative calculation 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 communities have led to a greater understanding of these microenvironments. These studies have demonstrated the existence of anoxic microzones in soil crumbs of otherwise well-aerated soils. Futuredevekpd \ As more ion-selective electrodes are developed and refined for measuring ~
N05, NH:, and PO!- a t the macroelectrode level, it should eventually he possible to scale down these electrodes to the microelectrode level, thus making available new tools for the study of metabolic processes in natural microbial communities. In addition to the appearance of these new sensors, more sophisticated methods of processingdata are hecoming available. The use of microelectrodes in ecological research has led to the wish for additional microsensors to measure such environmental parameters as light, temperature, and salinity at a spatial resolution sufficiently high to he relevant to the individual microbial populations. A fiber-optic microsensor with a sensing tip diameter of 1 5 2 0 pm, recently developed by J#rgensen and Des Marais, was used to study the spectral light distribution in microbial mats and sediments. The microbial environments and ecological research applications of microelectrodes studied thus far are rather limited. Microelectrodes appear to he ideal for measuring rapidly changing and often quite chemically different bacterial microenvironments. Important 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 hiotechnology. Sharon Boots
suseested~w Revsbech,,N. 9.; J rgenaen, B. B. In Advances m Mtcrofial Ecology; Marshall, K. C., Ed.; Plenum PublishingCorp.: New York, 1986, pp: 293-352. Reimers, C. E.;Fischer, K. M.; Merewether, R.;Smith, K. L.,Jr.; Jahnke,R. A. Nature 1986,320,74144.
Revsbech,N.P.;Nielsen,L. P.; Chriatensen, P. B.;Sorensen, J. Appl. Enuiron. Microbioi. 1988,54,2245-49. J$ ensen, B. B.; Des Marais, D. J. Limnol. 8ceeanogr. 1988,33, LXL113.
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