Distribution of Protozoa in Subsurface Sediments of a Pristine Groundwater Study Site in Oklahoma
Applied and Environmental Microbiology
Most-probable-number estimates showed that protozoan numbers declined steeply with depth in subsoil. Flagellates and amoebae dominated the protozoan population, which declined to a most probable number of 28 -g (dry weight)-1 in a clay loam layer at the bottom of the unsaturated zone. Samples from a texturally variable interface zone between 3 and 4 m down also were variable in their content of protozoa. Four contiguous clay loam samples in a single core from this zone contained variable
... of amoebae ranging from 0.2 to 44g (dry weight)-'. However, a sandy clay loam layer at the bottom of the core contained a mixture of flagellates and amoebae with a combined population density of 67. g (dry weight)-'. A slow-growing filose amoeba was isolated from interface zone samples and was tentatively classified in a new family in the order Aconchulinida. Protozoa were not detected in the saturated zone except in a very permeable gravelly, loamy sand layer at a depth of approximately 7.5 m. Low numbers (4 to 6g [dry weight]f1) of surface-type flagelates and amoebae, as well as the filose amoeba seen in the interface zone, were observed in this layer. Acid-treated and untreated samples contained equivalent numbers of protozoa, showing that the majority of protozoa in the layer at 7.5 m and the interface zone samples were encysted. Increased numbers of bacteria also were found in the layer at 7.5 m, indicating that it was biologically more active than other saturated-zone layers. Cyanobacteria grew in illuminated samples from this layer, suggesting that it may be connected hydrologically to a nearby river. Previous microbiological studies of oligotrophic sediments from unsaturated and saturated subsurface zones at sites in Oklahoma, Louisiana, and Texas (2, 10, 11, 27, 29, 30) have largely failed to demonstrate the presence of eucaryotic microorganisms or their characteristic long-chain polyenoic fatty acids (28). These negative results indicate that eucaryotic microorganisms are not dominant members of the subsurface microbiota; because the methods used may not have been sensitive enough to detect very low population densities, however, the results do not mean that eucaryotic microorganisms are absent from subsurface environments. Indeed, eucaryotic forms have been observed microscopically in some subsurface samples (4, 10), and recent work on the microbial community structure of subsurface soils (8) shows that eucaryotic fatty acids are present in most, if not all, subsoil horizons. Most-probable-number (MPN) procedures based on enrichment culture principles are more sensitive than other methods for detection and enumeration of microorganisms. Such methods have been particularly effective for estimating numbers of protozoa in soil (1, 7, 22), because a nearly universal and complete food source, the bacterial cell, is readily provided. However, MPN procedures for protozoa are subject to problems of underestimation and airborne contamination, which may significantly affect counts in samples containing few or no protozoa. Protozoan cysts, like fungal and bacterial spores, are ubiquitous airborne contaminants. On the average, air contains two protozoan cysts m-3 (23). Despite these problems, MPN and enrichment culture methods have been used successfully to demonstrate the presence of protozoa in subsurface samples. For example, * Corresponding author. groundwater (13), wastewater treatment wells (15), the waters and muds of caves (12, 16), unsaturated subsoils (19, 26), and subsurface clay sediment samples from as deep as 6.1 m (14) all have been found to contain viable protozoa. However, while these results reflect the ubiquity of protozoa, they do not reveal protozoan distribution in subsurface zones, nor do they indicate possible variations of protozoan population density in subsurface sedimentary layers. In the present work, we applied MPN counting methods to determine the distribution of protozoa in a depth profile at a groundwater microbiology study site near Lula, Okla. (2, 29, 30) . Aseptic procedures were used to ensure minimal airborne contamination of samples. Numbers of protozoa declined with depth, but amoebae or flagellates were present in all samples down to an interface zone just below the water table. Deeper in the profile, only samples from one layer of the saturated zone contained protozoa. The results emphasize the biological variability of deeper subsurface layers.