Hydrobiologia 482: 151–159, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
151
Culturable and non-culturable fractions of bacterial populations in sediments of a South Carolina stream Christopher J. McNamara1 , Michael J. Lemke∗ & Laura G. Leff Department of Biological Sciences and Water Resources Research Institute, Kent State University, Kent, OH 44242, U.S.A. (1 Author for correspondence. Current address: Laboratory of Microbial Ecology, Division of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, U.S.A. E-mail:
[email protected]) Received 2 March 2001; in revised form 2 May 2002; accepted 12 June 2002
Key words: stream, sediments, benthic, in situ hybridization, VBNC, bacteria
Abstract Population sizes of three bacterial species were examined in stream benthic habitats to assess differences in distribution and culturability among species. Population sizes were determined in sediments (both near bank and mid-channel) from three sites along Four Mile Creek (South Carolina, USA) using both culture-dependent (colony hybridization) and culture-independent (fluorescent in situ hybridization) techniques. The two methods used yielded different results. The numbers of colony forming units (CFU) of each species were similar in pattern to that found when the total number of CFU was enumerated (i.e., greater abundance in bank sediments and at downstream sites). In situ hybridization revealed a different distribution of these bacterial populations. Population sizes of the species were similar among sites. By using both the culture-based method and the culture-independent methods, the culturability of each species could be determined. The culturability of each species was at times much higher than the culturability of the overall assemblage. In spite of this higher culturability, viable but nonculturable cells commonly dominated the populations examined. These findings suggest that not only do bacterial species differ in population size and distribution, but also that cells within a population differ in their physiological state, or response to their environment, as reflected in differences in culturability.
Introduction Recent studies of planktonic bacteria have demonstrated that commonly measured features of the bacterial community, such as total number, may mask dynamic changes in the sizes of the underlying populations (Leff et al., 1998; Pernthaler et al., 1998; Lemke & Leff, 1999; Leff, 2000). Accordingly, community and population level measurements may provide different pictures of the abundance, distribution, and activity of planktonic microbial assemblages. Because physical and chemical factors that affect planktonic bacteria may also affect attached microorganisms, examination of the bacterial community of ∗ Current address: Biology Department, University of Illinois at Springfield, Springfield, IL 62794, U.S.A.
the benthos at a finer level of resolution than has been used in nearly all prior studies should be informative (Leff & Lemke, 1998). In stream benthos, differences among bacteria in nutritional and physical requirements (Nealson, 1997) and in attachment to various types of substrata (McEldowney & Fletcher, 1987; Brisou, 1995) may result in species-specific patterns of distribution and abundance that affect the role of these microorganisms in the ecosystem. However, few studies have quantified the abundance of different bacterial taxa in sediment microbial communities (Llobet-Brossa et al., 1998) because of limitations in methods of detection and enumeration (Leff, 1994). Thus, knowledge of stream sediment bacterial populations has largely been limited to organisms involved in specific, well-studied processes (e.g., nitrification and sulfur oxidation; Nealson, 1997).
152 The choice of methods used to enumerate bacteria may also affect the outcome of studies that examine bacterial distribution and abundance in the environment. Studies that enumerate colony forming units (CFU) are limited, in that nonculturable cells are undetectable (Jannasch and Jones, 1959; Joux and LeBaron 1997). Studies that utilize methods to enumerate cells without culturing (such as fluorescent in situ hybridization; FISH) could also be limited by the sensitivity of the detection system and dependency of detection on the physiological state or activity of the cells. Simultaneous usage of culture- and non-culture based-methods of enumeration provides information about the response of the bacterial community to environmental conditions that could not be gained when only one method is used. In this study, population sizes of three species of common, Gram-negative bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Pseudomonas putida) were examined using both a culture-based method (enumeration of CFU based on colony hybridization) and a culture-independent method (FISH). The three species were selected because they are common in streams (Lemke et al., 1997a; Leff et al., 1998; Lemke & Leff, 1999; Leff, 2000) and because they differ in their nutritional capabilities, which may affect patterns of distribution and abundance within benthic habitats. A. calcoaceticus is a non-motile, nonfastidious organism capable of utilizing a wide range of substances, such as aliphatic alcohols, unbranched hydrocarbons, and recalcitrant aromatic compounds (Towner, 1992). B. cepacia, originally described as a plant pathogen (Palleroni, 1992), has been well studied because of its unique degradative abilities (Stanier et al., 1966; Lessie & Gaffney, 1986) and because it is one of the few bacteria that contains multiple chromosomes (Cheng & Lessie, 1994). The third species, P. putida, is able to utilize a variety of low molecular weight compounds (Palleroni, 1992). Previous work has shown that these species of bacteria are common in the water of the study stream, Four Mile Creek (FOU), throughout the year (Lemke et al., 1997a; Lemke & Leff, 1999). These prior studies demonstrated that the distribution of the species in the water along FOU was not uniform and differences among sites could not be attributed solely to longitudinal changes in environmental conditions caused by natural and anthropogenic influences (Lemke et al., 1997a; Lemke & Leff, 1999). Because bacteria from benthic sources may contribute significantly to the assemblage found in water (Baker & Farr, 1977;
Edwards et al., 1990), differences in benthic bacterial populations may have contributed to the differences in distributions observed in the water of FOU. The purpose of this study was to use culturedependent (colony hybridization) and culture-independent (FISH) methods to compare population sizes of the three species of bacteria (A. calcoaceticus, B. cepacia, and P. putida) in benthic habitats of FOU to determine if spatial differences were similar to those found for planktonic bacteria (Lemke et al., 1997a; Lemke & Leff, 1999). Additionally, differences among populations were compared to community-level variables (i.e., numbers of colony forming units (CFU) and DAPI-stained cells). Colony and in situ hybridization quantify different fractions of a population (culturable versus active) and may provide different pictures of the abundance and distribution of bacterial species. Neither method, nor any other established technique, can enumerate all members of a population (Head et al., 1998). By using both methods, we were able to quantify the culturability of the three species and compare them to the overall culturability of bacteria at the study sites. Differences in distribution or culturability among bacterial species influence which methods can be appropriately used to study bacterial populations. Furthermore, variation in distribution and culturability demonstrate that bacterial species respond differently to their environment, and reflect the physiological state of the cells.
Methods Study site and sample collection Four Mile Creek (FOU) is a low gradient, blackwater stream located on the U.S. Department of Energy Savannah River Site (SRS) near Aiken, South Carolina, USA. FOU is one of five major drainages located on the SRS and traverses upland coastal plain soils with a mixture of uplands pine, mixed pine and hardwood forests (Workman & McLeod, 1990). The SRS was created and operated for the production of materials used in the construction of nuclear weapons, and portions of FOU have been impacted by a wide variety of radioactive (e.g., tritium, strontium-90, and cesium137), organic (e.g., oil, grease, and organic halogens), and inorganic (e.g., sodium hydroxide, arsenic, and cyanide) pollutants (Arnett et al., 1994; Chase, 1995). Stream sediments, consisting of sand and organic particles, were collected in November 1995 and Feb-
153 Table 1. Physical and chemical characteristics of study sites. Values indicate range across sample dates. Organic matter is percent of dry mass
Water temperature (◦ C)a Water velocity (m/s)a % Organic matter (bank) % Organic matter (mid-channel)
FOU1
FOU2
FOU3
12.7–23.3 0.29–0.53 0.72–6.74 0.62–2.56
15.3–24.1 0.11–0.31 19.89–25.48 1.28–4.25
13.0–24.5 0.29–0.59 1.50–19.66 0.19–1.22
a Data for November, February, and April from Lemke & Leff (1999).
ruary, April, and August 1996 from three sites at FOU. An unimpacted, reference site (FOU1) was located in the headwaters, while two anthropogenically impacted sites (FOU2 and FOU3) were located downstream. These sites were selected because they have been used in prior studies (Lemke et al., 1997a; Lemke & Leff, 1999). Differences among sites provided predictable environmental changes (e.g., decreased flow rate and increased organic content of the sediments at downstream sites) that facilitated comparison of bacterial populations among distinctive points along the stream (Table 1). Because bacterial abundance on a per g AFDM basis was strongly influenced by large differences in organic content of the sediments (Table 1), bacterial numbers are presented here on a per g dry mass basis (which was consistent among samples). Increased organic content at FOU2 probably resulted from deposition of seston caused by lower flow rates at that site. Bank and mid-channel samples (n = 3 − 5) were collected from the top 1–2 cm of sediment within 20 cm of the bank or in the middle of the channel with a scoop sampler. Bank samples were not collected in November 1995. Samples were transported on ice to the laboratory where 15 g of sediment was measured into sterile Whirlpaks (Nasco, Fort Atkinson, WI), 30 ml of 0.85% NaCl was added, and samples were sonicated (Branson 2210 Sonicator, Danbury, Connecticut) for 5 min to separate bacteria from the substrate. The remainder of the sample was frozen for determination of ash free dry mass (AFDM). One ml of the NaCl solution was then diluted 1:10 with additional 0.85% NaCl. Three modified Nutrient Agar (mNA, l−1 = 4 g nutrient broth, 15 g agar, 1.5 g NaCl, 0.03 g MgSO4 , 0.003 g CaCl2 , 2.1 g Na2 HPO4 , 0.9 g KH2 PO4 , 0.3 g NH4 Cl, 5 ml 2% cyclohexamide (in methanol), Leff & Meyer, 1991) plates were inoculated with 10 µl of the original 0.85% NaCl solution, and three with 10 µl from the dilution. In February and August, 5 ml from the original sample were preserved with 0.04
ml 37% formaldehyde, and 12 ml were preserved in 36 ml 4% paraformaldehyde, 0.5× phosphate buffered saline (1× PBS = 7.6 g NaCl, 1.9 g Na2 HPO4 , 0.7 g NaH2 PO4 per l, pH 7.2). To determine AFDM samples were dried at 103±2 ◦ C for 24 h (Tempcon Oven, Baxter, McGraw Park, IL), and then ashed for 12 h at 500±50 ◦ C (Thermolyne 1400 Furnace, Barnstead-Thermolyne, Dubuque, IA). Water velocity was determined using a FP 101/201 Global Flow Meter (Global Water Instruments, Fair Oaks, CA). Stream water temperature was determined using an Oakton WD-35615 pH/mV/Temperature Meter (Singapore). Plate counts and colony hybridization Plates were incubated at 23◦C for 7 days and the total number of colony forming units (CFU) was determined. To enumerate CFU of the bacterial species, colony hybridization was performed as previously described (Leff et al., 1995; Lemke et al., 1997a; Lemke & Leff, 1999). The speciesspecific probe sequences were: A. calcoaceticus, 5 AGCATCCTATCGCTAGGTA-3 (Braun-Howland et al., 1993); B. cepacia, 5 -CCCATCGCATCTAACAAT3 (Schleifer et al., 1992); and P. putida, 5 GCTGGCCTAACCTTC-3 (Schleifer et al., 1992). Controls used in each hybridization were: A. calcoaceticus (American Type Culture Collection #23055 [ATCC, Manassas, VA]), B. cepacia (ATCC #25416), P. putida (ATCC #12633), and Pseudomonas fluorescens (ATCC #13525). Enumeration of total bacteria Samples preserved with formaldehyde were stained for 3 min with 200 µl of 15 µg/ml 4 ,6-diamidino2-phenylindole (DAPI), concentrated by filtration (15 kPa vacuum) onto a 0.22 µm pore size black polycarbonate membrane (Poretics, Livermore, CA), and
154
Figure 1. Number of CFU per g dry weight of sediment (mean + SE). November and February, n = 3; April and August, n = 5. BA – bank; MC – mid-channel; and ND – no data.
rinsed with 1.0 ml deionized water (Porter & Feig, 1980). Bacteria were enumerated using epifluorescence microscopy (DAPI filter set 310000, Chroma Tech. Corp., Brattleboro, VT, exciter D360/40, dichroic 400DCLP, emitter D460/50645/75).
Figure 2. Total number of bacteria per g dry weight of sediment (mean + SE) as determined by epifluorescence microscopy after staining with DAPI. November n = 3; February, April, and August, n = 5. BA – bank; MC – mid-channel; and ND – no data.
probes for A. calcoaceticus, B. cepacia, and P. putida (same sequences as used in colony hybridization) were labeled at the 5 end with Texas Red and enumerated using epifluorescence microscopy (Texas Red filter set 41004, Chroma Tech. Corp., Brattleboro, VT, exciter HQ560/55, dichroic Q595LP, emitter HQ645/75).
In situ hybridization Calculation of percent culturable To enumerate bacteria of the three species without culturing, cells preserved with 4% paraformaldehyde, 0.5× PBS were subjected to in situ hybridization with fluorescently labeled oligonucleotides using the method of Lemke et al. (1997b). Species-specific
The percent of the bacterial community that was culturable was calculated by dividing the total number of CFU by the number of cells enumerated using DAPI and multiplying the quotient by 100. The culturabil-
155 Table 2. Mean CFU/g dry mass ± SE of each species (×103 ). November and February, n = 3, April; and August, n = 5. BA – Bank; MC – mid-channel; and ND – no data A. calcoaceticus November
FOU1 FOU2 FOU3
February
FOU1 FOU2 FOU3
April
FOU1 FOU2 FOU3
August
FOU1 FOU2 FOU3
BA MC BA MC BA MC
ND 0 ND 0 ND 0
B. cepacia
P. putida
ND 0 ND 0 ND 0.92±0.48
ND 0 ND 0 ND 0
BA MC BA MC BA MC
2.34±0.99 0.29±0.18 67.65±42.04 0.40±0.40 10.08±5.04 6.99±2.20
0 0 0 0 0 0
0 0 0 0 0 0
BA MC BA MC BA MC
0.18±0.11 0.15±0.15 47.09±14.17 1.68±0.98 49.06±15.89 5.28±1.23
0 0 27.44±24.29 0.45±0.45 28.83±18.19 1.14±0.97
0.10±0.10 0.13±0.07 21.26±15.77 4.34±1.52 10.32±4.91 2.39±1.01
BA MC BA MC BA MC
0.50±0.32 0 3.31±2.75 0.84±0.63 1.06±0.45 0.68±0.47
0.15±0.15 0 1.23±0.93 0.06±0.06 1.30±0.96 0.03±0.03
0.63±0.29 0.15±0.10 4.50±2.49 0.37±0.33 1.11±0.46 0
ity of individual species was calculated by dividing the abundance determined by colony hybridization by the abundance determined by in situ hybridization and multiplying the quotient by 100. Statistics Results were analyzed using Statview 4.01 (Abacus Concepts, Berkeley, CA). Differences among sites were determined by one-way ANOVA followed by Scheffe’s test (α = 0.05). Differences between habitats were determined using an unpaired sample t-test (α = 0.05) for each sampling date. Pearson’s correlation coefficient (α = 0.05) was determined for the proportion of sediments composed of organic matter (arcsin–square root transformed) and the number of CFU and cells in the total community and for each species.
Results The total number of CFU was higher at the downstream sites (FOU2 and FOU3) than at FOU1 in both bank and mid-channel habitats on some dates (Fig. 1). In November, there were significantly more total CFU in the mid-channel sediments at FOU2 (p = 0.017) and FOU3 (p = 0.017) than at FOU1. In April, there were significantly more CFU in the bank sediments at FOU2 than FOU1 (p = 0.026). At any given site during February, April, and August, there were more CFU in the bank than in the mid-channel sediment (Fig. 1). Statistically significant differences between the bank and mid-channel habitats were found in February at FOU1 (p = 0.027), April at FOU2 (p = 0.020) and FOU3 (p = 0.004), and August at FOU3 (p = 0.005). When detectable using colony hybridization, A. calcoaceticus CFU were often more abundant at the
156
Figure 3. Number of A. calcoaceticus, B. cepacia, and P. putida per g dry weight of sediment (mean + SE, n = 3), as determined by in situ hybridization. BA – bank; and MC – mid-channel.
downstream sites (FOU2 and FOU3) than at FOU1 (Table 2). In February, there were significantly more A. calcoaceticus CFU in the mid-channel at FOU3 than FOU1 (p = 0.030) or FOU2 (p = 0.032), and in April there were significantly more at FOU3 than FOU1 (p = 0.030) in the mid-channel. At any given site, A. calcoaceticus CFU were at times more abundant in the bank habitat than in the mid-channel habitat; statistically significant differences were detected in April at FOU2 (p = 0.026) and FOU3 (p = 0.025). Unlike A. calcoaceticus, no statistically significant differences among the three sites or between midchannel and bank sediments were found for B. cepacia CFU (Table 2). Differences in abundance of P. putida CFU were uncommon; only in August at one site (FOU3) were there significant differences in abundance between the bank and mid-channel sediments (p = 0.042). Overall, the total number of bacteria, based on the abundance of DAPI-stained cells, was relatively constant across sample dates (Fig. 2). However, there were differences among sites on some dates. Significant differences among sites were found in February in bank sediment (FOU2 > FOU1, p = 0.002) and mid-channel (FOU2 > FOU3, p = 0.048), and during April in both bank (FOU2 > FOU1, p = 0.011) and mid-channel (FOU2 > FOU1, p < 0.001 and FOU2 > FOU3, p = 0.002) sediment. More DAPIstained cells were at times found in the bank than in the mid-channel sediment (Fig. 2). Significant differences occurred between the two habitats in February at FOU1 (p = 0.004), FOU2 (p = 0.002), and FOU3 (p = 0.004), and in April at FOU2 (p = 0.012) and FOU3 (p = 0.004). When the culture-independent method (in situ hybridization) was used to quantify bacterial populations
of the three species, responses that differed from the other variables were detected. For A. calcoaceticus, there were no significant differences among the three sites (Fig. 3), but the abundance of A. calcoaceticus was at times higher in the bank than the mid-channel sediment. Significant differences between BA and MC habitats were found in February at FOU2 (p = 0.002) and FOU3 (p = 0.024) and in August at FOU1 (p = 0.003), FOU2 (p = 0.002), and FOU3 (p = 0.015). No significant differences were found in the abundance of B. cepacia among sites or between midchannel and bank sediments based on in situ hybridization results (Fig. 3). For P. putida, some significant differences were detected, but there were no consistent patterns among sites and between habitats. Significant differences in P. putida abundance were found among sites in the mid-channel during February, where numbers at FOU3 were greater than at FOU1 (p = 0.004) or FOU2 (p = 0.005). Also during February, significantly more P. putida cells were found in the mid-channel than the bank sediment at FOU3 (p = 0.012). The overall culturability of the bacterial community was generally less than 1% (except in August, at FOU2 bank (5.04%), and FOU3, bank and midchannel (3.87 and 1.03%, respectively)). In contrast, the percentage of culturable cells of two of the species, A. calcoaceticus and P. putida, were at times much higher than 1% (Fig. 4). B. cepacia had a lower percent culturable relative to A. calcoaceticus and P. putida, but its maximum culturability was greater than 1%.
Discussion In contrast to studies of planktonic bacteria in Four Mile Creek (FOU) (Lemke et al., 1997a; Lemke &
157
Figure 4. Percentage of culturable A. calcoaceticus, B. cepacia, and P. putida cells (mean + SE, February, n = 3; August, n = 5).
Leff, 1999), community-level variables (number of CFU and DAPI-stained cells) in the benthos were consistently different among the three sites. Generally, in this study, there were greater numbers of both CFU and DAPI-stained cells at the downstream sites, FOU2 and 3. The absence of such differences in the water column may be due to the transitory nature of the planktonic assemblage. Additionally, there are many possible sources of bacteria found in the water column. Each of these sources may contribute cells and confound the ability to detect patterns in the total planktonic community. Examination of attached (and plausibly spatially stable) communities, such as those in the sediments, may reveal patterns not evident in the water. When comparing the mid-channel and bank portions of the stream bed, greater numbers of both CFU and DAPI-stained cells were found in the bank habitat, likely because of differences in the organic content of the sediments. CFU and DAPI numbers were significantly correlated with the organic content of the sediments (CFU: r = 0.611, p < 0.001; DAPI: r = 0.721, p < 0.001). Other studies have also demonstrated a correlation between sediment bacterial abundance or biomass and sediment organic content (Bott & Kaplan, 1985; Fischer et al., 1996). Each of the three bacterial species exhibited responses to the environmental conditions among the sites and habitats that differed from the communitylevel responses. For A. calcoaceticus, the pattern of differences in abundance in the sediment was re-
markably similar to that reported for this species in previous studies of planktonic bacteria (Lemke et al., 1997a; Lemke & Leff, 1999). In both the water and the benthos, there were greater numbers of A. calcoaceticus CFU at the downstream sites and no apparent pattern of spatial differences in the number of cells determined using the culture-independent method (FISH). A. calcoaceticus is described as a soil bacterium (Towner, 1992) and did not grow effectively when incubated in the laboratory in flasks of stream water from the study sites (Lemke & Leff, 1999). Furthermore, A. calcoaceticus was at times much more culturable in the sediment than was reported for the water (Lemke & Leff, 1999). Together, these findings suggest that the sediments of FOU serve as a source of A. calcoaceticus for the water column, and that the spatial differences in the abundance of A. calcoaceticus in the water column could be attributable to differences in the distribution in the sediments. Patterns of abundance of culturable B. cepacia and P. putida in the sediments were also similar to those reported for the water column. In the water column, these two species often exhibited similar abundances at all sites (Lemke et al., 1997a; Lemke & Leff, 1999), and this same level of similarity was found in the benthos. In spite of differences in water chemistry and physical conditions, there were few significant differences among the three sites. The organic content of the sediments also seemed to influence certain species. The number of A. calcoaceticus CFU was correlated with the organic content
158 of the sediments (r = 0.614, p < 0.001), while B. cepacia and P. putida CFU numbers demonstrated weak, yet statistically significant, correlations (r = 0.349, p = 0.001 and r = 0.335, p = 0.002 respectively). Sediment organic content also appeared to be related to the numbers of A. calcoaceticus and B. cepacia determined by in situ hybridization (correlations were: r = 0.681, p < 0.001 and r = 0.541, p = 0.001 respectively). P. putida abundance determined by in situ hybridization was not significantly correlated with sediment organic content (r = −0.246, p = 0.147). The two methods used to determine population sizes, colony and in situ hybridization, yielded different results. The number of cells of each species determined by in situ hybridization was, in most cases, much greater than the numbers determined by colony hybridization. The difference between the numbers revealed by these two methods demonstrate that for each species there were a large number of cells that were active and viable (based on in situ hybridization) but that were not culturable. The occurrence of these viable, but non-culturable (VBNC) cells has been demonstrated for other species of bacteria (e.g., Vibrio cholerae) in marine environments (Colwell et al., 1984). However, most evidence previously reported was based on experimental manipulations rather than actual field observations (e.g., Amy & Morita, 1983). The presence in streams of VBNC cells of cultivatable species suggests that this is a widespread phenomenon (Leff & Lemke, 1998) and has ramifications for selection of methods for enumeration and for our understanding of the nature of bacteria in streams. In addition, measurement of culturability may reveal information about the physiological state of the cells in the population. The inability of viable cells to form colonies has been attributed to the effects of environmental conditions, such as low temperature and low nutrient concentrations (Grimes & Colwell, 1986; Oliver et al., 1995; Buswell et al., 1998) as well as radiation, osmolarity, aeration, desiccation, and the presence of organic and heavy metal biocides (Gauthier, 2000). The presence of VBNC cells of a normally culturable species may indicate stressful conditions caused by either normal environmental fluctuations or the presence of harmful substances. Culturability of specific populations, determined by comparing numbers from colony and in situ hybridization, revealed that on many occasions a given species was more culturable than the overall assemblage. Variation in culturability of the species among sites may indicate relative tolerance of the species to the
environmental conditions present at the different sites. Moreover, the results demonstrate that VBNC cells were a major component of the benthic populations of all three species examined. Thus, the pool of non-culturable bacteria found in streams includes species, such as those examined here, that have been cultivated and named and does not consist only of unknown and unculturable species. Similar conclusions have been reported in studies of marine sediments (Llobet-Brossa et al., 1998). The findings of this study support those of other studies, in that bacterial community-level responses differed from the population-level responses (Lemke et al., 1997a; Leff et al., 1998; Pernthaler et al., 1998) and demonstrate that benthic bacteria of different species differ in their population ecology. Differences observed among species in this study provide quantitative evidence for the conceptual model of Leff & Lemke (1998). Not only do bacterial species differ in population size and distribution, but also cells within a population differ in their physiological state, or response to their environment, as reflected in differences in culturability. Our findings and those from other studies (Lemke et al., 1997a; Lemke and Leff, 1999; Leff, 2000) demonstrate that bacteria of different species differ in their population ecology in streams. Such differences may affect the function of the bacterial community in the ecosystem.
Acknowledgements This research was supported by the U.S. Environmental Protection Agency Office of Exploratory Research (# R823749-01-0). Collection facilities were provided by a contract between the U.S. Department of Energy and the University of Georgia (DE-FL0996SR00819). Additional support was provided by a grant from the Water Resources Research Institute, Kent State University. We thank Derek Cody for assistance in sample collection.
References Amy, P. S. & R. Y. Morita, 1983. Starvation-survival patterns of sixteen freshly isolated open-ocean bacteria. Appl. environ. Microbiol. 45: 1109–1115. Arnett, M. W., A. R. Mamatey & D. Spitzer (eds), 1994. Savannah River Site environmental report for 1994. Publication WSRC-TR-95-077. USDOE Westinghouse Savannah River Co., Savannah River Site, Aiken, SC.
159 Baker, J. H. & I. S. Farr, 1977. Origins, characterization and dynamics of suspended bacteria in two chalk streams. Arch. Hydrobiol. 80: 308–326. Bott, T. L. & L. A. Kaplan, 1985. Bacterial biomass, metabolic state, and activity in stream sediments: relation to environmental variables and multiple assay comparisons. Appl. environ. Microbiol. 50: 508–522. Braun-Howland, E. B., P. A. Vescio & S. A. Nierzwicki-Bauer, 1993. Use of simplified cell blot technique and 16S rRNAdirected probes for identification of common environmental isolates. Appl. environ. Microbiol. 59: 3219–3224. Brisou, J., 1995. Biofilms: Methods for the enzymatic release of microorganisms. CRC Press, Boca Raton, FL. Buswell, C. M., Y. M. Herlihy, L. M. Lawrence, J. T. M. McGuiggan, P. D. Marsh, C. W. Keevil & S. A. Leach, 1998. Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescentantibody and -rRNA staining. Appl. environ. Microbiol. 64: 733–741. Chase, J., 1995. Mixed waste management facility FSS well data groundwater monitoring report. Fourth quarter 1994 and 1995. Publication WSRC-TR-94-0610 (NTIS No. DE95060118). USDOE Westinghouse Savannah River Co., Aiken, SC. Cheng, H. P. & T. G. Lessie, 1994. Multiple replicons constituting the genome of Pseudomonas cepacia 17616. J. Bacteriol. 176: 4034–4042. Colwell, R. R., P. A. West, D. Maneval, E. F. Remmers, E. L. Elliot & N. E. Carlson, 1984. Ecology of pathogenic vibrios in Chesapeake Bay. In Colwell, R. R. (ed.), Vibrios in the Environment. Wiley, Chichester. Edwards, R. T., J. L. Meyer & S. E. G. Findlay, 1990. The relative contribution of suspended bacteria to system biomass, production, and metabolism in a low-gradient blackwater river. J. n. am. Benthol. Soc. 9: 216–228. Fischer, H., M. Pusch & J. Schwoerbel, 1996. Spatial distribution and respiration of bacteria in stream-bed sediments. Arch. Hydrobiol. 137: 281–300. Gauthier, M. J., 2000. Environmental parameters associated with the viable but nonculturable state. In Colwell, R. R. & D. J. Grimes (eds), Nonculturable Microorganisms in the Environment. ASM Press, Washington, DC. Grimes, D. J. & R. R. Colwell, 1986. Viability and virulence of Escherichia coli suspended by membrane chamber in semitropical ocean water. FEMS Microbiol. Lett. 34: 161–165. Head, I. M., J. R. Saunders & R. W. Pickup, 1998. Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb. Ecol. 35: 1–21. Jannasch, H. W. & G. E. Jones, 1959. Bacterial populations in sea water determined by different methods of enumeration. Limnol. Oceanogr. 4: 128–139. Joux, F. & P. LeBaron, 1997. Ecological implications of an improved direct viable count method for aquatic bacteria. Appl. environ. Microbiol. 63: 3643–3647. Leff, L. G., 1994. Stream bacterial ecology: a neglected field? ASM News 60: 135–138. Leff, L. G., 2000. Longitudinal changes in microbial assemblages of the Ogeechee River. Freshwat. Biol. 43: 605–615. Leff, L. G., R. M. Kernan, J. V. McArthur & L. J. Shimkets, 1995. Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes. Appl. environ. Microbiol. 61: 1634–1636.
Leff, L. G., A. A. Leff & M. J. Lemke, 1998. Seasonal changes in planktonic bacterial assemblages of two Ohio streams. Freshwat. Biol. 39: 129–134. Leff, L. G. & M. J. Lemke, 1998. Ecology of aquatic bacterial populations: lessons from applied microbiology. J. n. am. Benthol. Soc. 17: 261–271. Leff, L. G. & J. L. Meyer, 1991. Biological availability of dissolved organic carbon to bacteria along the Ogeechee River continuum. Limnol. Oceanogr. 36: 315–323. Lemke, M. J., B. J. Brown & L. G. Leff, 1997a. The response of three bacterial populations to pollution in a stream. Microb. Ecol. 34: 224–231. Lemke, M. J. & L. G. Leff, 1999. Bacterial populations in an anthropogenically disturbed stream: comparison of different seasons. Microb. Ecol. 38: 234–243. Lemke, M. J., C. J. McNamara & L.G. Leff, 1997b. Comparison of methods for the concentration of bacterioplankton for in situ hybridization. J. microbiol. Methods. 29: 23–29. Lessie, T. G. & T. Gaffney, 1986. Catabolic potential of Pseudomonas cepacia. In Sokatch, J. R. & L. N. Ornston (eds), The Bacteria: A treatise on structure and function, Vol. X, The Biology of Pseudomonas. Academic Press, New York. Llobet-Brossa, E., R. Rossello-Mora & R. Amann, 1998. Microbial community composition of Wadden Sea sediments as revealed by fluorescence in situ hybridization. Appl. environ. Microbiol. 64: 2691–2696. McEldowney, S. & M. Fletcher, 1987. Adhesion of bacteria from mixed cell suspensions to solid surfaces. Arch. Microbiol. 148: 57–62. Nealson, K. H., 1997. Sediment bacteria: Who’s there, what are they doing, and what’s new? Annu. Rev. Earth Planet. Sci. 25: 403–434. Oliver, J. D., F. Hite, D. McDougald, N. L. Andon & L. M. Simpson., 1995. Entry into, and resuscitation from, the viable but non-culturable state by Vibrio vulnificus in an estuarine environment. Appl. environ. Microbiol. 61: 2624–2630. Palleroni, N. J., 1992. Human- and animal-pathogenic Pseudomonads. In Balows, A., H. G. Truper, M. Dworkin, W. Harder & K. H. Schleifer (eds), The Prokaryotes, Vol. 3. Springer, New York. Pernthaler, J., F. O. Glockner, S. Unterholzner, A. Alfreider, R. Psenner & R. Amman, 1998. Seasonal community and population dynamics of pelagic bacteria and archaea in a high mountain lake. Appl. environ. Microbiol. 64: 4299–4306. Porter, K. G. & Y. S. Feig, 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943–948. Schleifer, K. H., R. Amman, W. Ludwig, C. Rothemund, N. Springer & S. Dorn, 1992. Nucleic acid probes for the identification and in situ detection of Pseudomonas. In Galli, E., S. Silver & B. Witholt (eds), Pseudomonas: Molecular biology and biotechnology. American Society for Microbiology, Washington, DC. Stanier, R. Y., N. J. Palleroni & M. Doudoroff, 1966. The aerobic pseudomonads: a taxonomic study. J. gen. Microbiol. 43: 159– 271. Towner, K. J., 1992. The genus Acinetobacter. In Balows, A., H. G. Truper, M. Dworkin, W. Harder & K. H. Schleifer (eds), The Prokaryotes, Vol. 4. Springer, New York. Workman, S. W. & K. W. McLeod, 1990. Vegetation of the Savannah River Site: Major community types. Savannah River Site National Environmental Research Park Program, Aiken, SC.
