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Applied and Environmental Microbiology, May 2005, p. 2278-2287, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2278-2287.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Seasonal Response of Stream Biofilm Communities to Dissolved Organic Matter and Nutrient Enrichments

Ola A. Olapade* and Laura G. Leff

Department of Biological Sciences, Kent State University, Kent, Ohio 44240

Received 29 July 2004/ Accepted 24 November 2004


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ABSTRACT
 
Dissolved organic matter (DOM) and inorganic nutrients may affect microbial communities in streams, but little is known about the impact of these factors on specific taxa within bacterial assemblages in biofilms. In this study, nutrient diffusing artificial substrates were used to examine bacterial responses to DOM (i.e., glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate and phosphate singly and in combination). Artificial substrates were deployed for five seasons, from summer 2002 to summer 2003, in a northeastern Ohio stream. Differences were observed in the responses of bacterial taxa examined to various DOM and inorganic nutrient treatments, and the response patterns varied seasonally, indicating that resources that limit the bacterial communities change over time. Overall, the greatest responses were to labile, low-molecular-weight DOM (i.e., glucose) at times when chlorophyll a concentrations were low due to scouring during significant storm events. Different types of DOM and inorganic nutrients induced various responses among bacterial taxa in the biofilms examined, and these responses would not have been apparent if they were examined at the community level or if seasonal changes were not taken into account.


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INTRODUCTION
 
Biofilms play important roles in ecosystem processes in streams (35), and factors responsible for the abundance and distribution of microorganisms in such communities have often been studied (e.g., references 12, 16, 17, 33, 45, and 48). Inorganic nutrients (11, 18) and dissolved organic matter (DOM) (17, 23, 44, 45) have been shown specifically to influence microbial abundance in aquatic biofilms (31, 48, 51).

The source, quality, and type of DOM, as well as the quantity, may influence the abundance and distribution of bacteria in stream ecosystems (20, 21, 24). In addition, other aspects of microbial community function such as respiration, biomass, and extracellular enzyme activity may be limited by dissolved inorganic nutrients and organic matter in streams (49, 51). Also, epilithic bacterial populations can be affected indirectly by inorganic nutrients via the influence of nutrients on algal biomass (45, 50).

In spite of the number of studies that have looked at responses of microorganisms in streams to DOM and inorganic nutrients, little is known about the influence of nutrients and DOM on the composition and distribution of different bacterial taxa in stream biofilms. This is because earlier investigations were typically based on assemblage-level responses, such as examining total bacterial numbers (e.g., references 16, 24, 31, and 45), with few examining spatial and temporal changes in specific bacterial populations (4, 20, 32). In this study, we examined how different bacterial taxa in biofilms responded to DOM and inorganic nutrients by using nutrient diffusing artificial substrates (clay flowerpots), an approach commonly used to demonstrate nutrient limitation in streams (e.g., references 11 and 43). Responses to different treatments (leaf leachate [LL], glucose, algal exudates, and phosphate and nitrate singly and in combination) by different bacterial taxa and algae were compared among seasons.

The bacterial taxa examined were selected because they are common in streams (21, 25, 26) and known to use a wide array of organic compounds (28). The three species targeted (Burkholderia cepacia, Acinetobacter calcoaceticus, and Pseudomonas putida) are gram-negative bacteria that are abundant in water and sediment (22). Also, we examined three subgroups of proteobacteria ({alpha}, ß, and {gamma}), with diverse metabolic and physiological characteristics (42). The {alpha}-proteobacteria include species that thrive in oligotrophic conditions, whereas many species belonging to {gamma}-proteobacteria are opportunistic and dominate in nutrient-rich environments (39). We also examined the Cytophaga-Flavobacterium cluster, a group that has been reported to account for a high percentage of biofilm bacteria in aquatic benthos (38); these bacteria thrive in the presence of complex macromolecules (19).

We predicted that different bacterial taxa in biofilms would exhibit different responses to DOM and inorganic nutrient amendments, as a result of differences in their metabolic needs and abilities. Because of seasonal changes in bacterial community composition and environmental factors, we hypothesized that responses to the amendments would also vary seasonally.


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MATERIALS AND METHODS
 
Description of study site and experiment:.
The study was conducted in the West Branch of the Mahoning River in northeastern Ohio, a region with a temperate climate and located within latitude 20°41'N and longitude 51°80'W. The river lies within the Erie/Ontario Lake Plain ecoregion (37). The stream has dense shading caused by a riparian forest of maple, oak, sycamore, and poplar. The riffle section of the stream channel where artificial substrates were positioned is dominated by mid (50-cm2)- to large (100-cm2)-sized cobbles.

Experiments similar to those described by Fairchild et al. (11) and McCormick and Stevenson (33) were carried out in five consecutive seasons from June 2002 through August 2003. Replicate sets (n = 3) of clay flowerpots (8.8 cm in diameter and 8.0 cm in height) were filled with inorganic nutrients (nitrate, phosphate, or nitrate plus phosphate) or DOM (glucose, leaf leachate, or algal exudates) and solidified with 2.0% agar. Control pots contained only agar in artificial stream water (ASW; each liter contained 12 mg NaHCO3, 7.5 mg CaSO4 · 2H2O, 7.5 mg MgSO4, 0.5 mg KCl, 10 mg CaCO3, pH 6.4) (3). All the nutrients were sterilized at 121°C for 15 min, except for both leaf leachate and algal exudates, which were filter sterilized by passage through 0.2-µm-pore-size filters (Whatman, Maidstone, United Kingdom).

Inorganic nutrients were administered in ASW as K2HPO4 at 0.5 mol/liter and NaNO3 at 0.5 mol/liter, individually and in combination. DOM sources included glucose (administered at 1 mol/liter), algal exudates (EDOC), and LL. EDOC was extracted from Chlamydomonas and Synedra cultures grown in Bristol medium for 7 to 14 days (10 ml NaNO3, 10 ml CaCl2 · 2H2O, 10 ml K2HPO4, 10 ml KH2PO4, 10 ml MgSO4 · 7H2O, 10 ml NaCl in 940 ml dH2O; Ward's Natural Science, Rochester, NY). These algae were selected because they could be cultured axenically in medium that was free of added DOM. LL was extracted from maple leaves as described in the work of McNamara and Leff (34), and ASW was added to the EDOC and LL solutions to achieve a dissolved organic carbon (DOC) final concentration of 50 mg/liter. The pots were sealed with plastic petri plates using silicon gel and incubated in the stream for 3 weeks.

After incubation, the pots were retrieved, and a measured area (26.12 cm2) on each was carefully scraped with a sterile toothbrush (44). Scraped samples were diluted with ASW. Subsamples were removed to determine chlorophyll a concentration, types of algae present, and bacterial abundance.

Subsamples for bacterial enumeration were preserved using phosphate-buffered saline (1x phosphate-buffered saline = 7.6 g NaCl, 1.9 g Na2HPO4 · 7H2O, 0.7 g NaH2PO4 · 2H2O, pH 7.2) and 8% paraformaldehyde. The samples were sonicated (Branson Model 2210 ultrasonic bath; Ultrasonics Corporation, Danbury, CT) for 5 min in 0.1% tetrasodium pyrophosphate (Na4P2O7 · 10H2O). Bacteria were enumerated by staining with 4',6'-diamidino-2-phenylindole (DAPI) and fluorescent in situ hybridization (FISH).

Subsamples for determining algal biomass were filtered through 0.45-µm-pore-size filters (Whatman Corporation) and treated with 2 ml of 1% MgCO3 solution. Filters were then frozen at –25°C in the dark for 24 h. Chlorophyll a concentration was measured using the standard spectrophotometric method (3, 12). Subsamples for algal identification were preserved with Lugol's reagent (20 g KI and 10 g I in 200 ml deionized H2O containing 20 ml glacial acetic acid). Algae were identified according to the methods of Sobczak (48) and Prescott (41). Algal cells were enumerated using a Palmer Counter (Ward's Natural Science) and an inverted microscope (40x magnification).

Temperature and pH were measured using an Oakton meter (Singapore). Turbidity was measured using a Hach turbidity meter (model 2100P), and conductivity was determined using a Hach conductivity meter (model 44600). A colorimetric method was used to estimate nitrate/nitrite and soluble reactive phosphate (SRP) concentrations in triplicate water samples (Hach models 41100-13 and 41100-16, respectively). DOC concentration was determined using a TOC 5000 instrument (Shimadzu Scientific Instruments, Columbia, MD).

Bacterial enumeration.
Total bacterial number was determined by staining samples concentrated onto 0.2 µm-pore-size black polycarbonate filters (Poretics, Livermore, CA) with 15 µg/µl DAPI for 3 min (40).

The abundances of bacteria belonging to the domains Bacteria and Archaea; {alpha}-, ß-, and {gamma}-proteobacteria; Cytophaga-Flavobacterium cluster; gram-positive high-G+C bacteria; and B. cepacia, A. calcoaceticus, and P. putida were enumerated as in the work of Lemke et al. (27). Bacteria were concentrated on 0.2-µm-pore-size Anodisc filters (Whatman, Maidstone, United Kingdom) and treated with 1 ml 0.1% Nonidet P-40 (Sigma). Filters were placed in petri plates and treated with 40 µl Texas Red-labeled probe (Sigma Genosys; 5 ng/µl final concentration, in hybridization buffer [6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.02 M Trizma base (pH 7.0), 0.1% sodium dodecyl sulfate, and 0.01% poly(A)]) before incubation at appropriate temperatures for 4 h (Table 1).


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  		TABLE 1. Oligonucleotide sequences, targets, and hybridization conditions used for biofilm community analysisa

After incubation, cells were washed twice with 400 µl of wash buffer and incubated with 80 µl of wash buffer for 10 min at the hybridization temperature. Filters were rinsed twice with 400 µl water, and hybridized cells were enumerated using epifluorescence microscopy.

Statistical analyses.
Two-way analyses of variance were used to examine the influence of nutrient treatments and season on bacterial numbers and algal biomass. Differences among treatments were determined using Tukey's Studentized range (highest significant difference [HSD]). All tests were considered significant at the level {alpha} = 0.05, and values are given as means ± standard errors (SE). Biofilm variables are expressed per cm2 of surface area.


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RESULTS
 
Environmental variables.
The physical and chemical variables measured differed among seasons (Table 2). Water temperature and pH were highest throughout the summer and lowest during the winter. There were marked differences in conductivity among the seasons, with lowest levels during the summer months and substantially higher values in winter. Turbidity also varied and was lowest during spring and highest in summer. There were no drastic changes in nitrate, SRP, and DOC concentrations during the study period.


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  		TABLE 2. Mean values (± standard errors) of environmental variables measured during the 21 days of incubation of nutrient diffusing substrates at the study site

Bacterial abundance.
Total bacterial numbers based on DAPI staining differed significantly among seasons (F = 7.46, P < 0.0001) and treatments (F = 3.98, P < 0.05), and there was a significant season-treatment interaction (F = 2.47, P < 0.05). Total bacterial numbers were significantly higher on glucose-treated pots during fall, winter, and summer 2003 than on the controls (P < 0.05, Tukey's HSD; Fig. 1A). Also, in spring 2003, total bacterial numbers were higher on the leaf leachate-amended pots than on the controls.



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  		FIG. 1. Number of bacterial cells based on DAPI staining (A) and FISH with domain Bacteria (B) and Archaea (C) probes in biofilms on artificial substrates. Values are means of triplicate samples ± SE. An asterisk indicates that the bar was significantly different (P < 0.05) from the control. ND, not determined.

Likewise, numbers of cells hybridizing to the domain Bacteria probe were significantly different among seasons (F = 28.66, P < 0.0001), treatments (F = 6.20, P < 0.0001), and season-treatment combinations (F = 3.55, P < 0.0002). The glucose treatment elicited a greater response than the control did in winter and summer 2003 (P < 0.05, Tukey's HSD; Fig. 1B). Also in winter 2003, numbers were higher on the nitrate-treated pots than on the controls.

In contrast, the number of cells hybridizing to the domain Archaea probe differed significantly among seasons (F = 12.76, P < 0.0001) but not among treatments (F = 2.26, P > 0.05), and there was no significant season-treatment interaction (F = 1.44, P > 0.05). Numbers of archaea were similar among treatments and the control throughout the study (Fig. 1C).

Abundance of the {alpha}-proteobacteria differed significantly among seasons (F = 4.01, P < 0.05) and among treatments (F = 0.33, P < 0.05); also there was a significant season-treatment interaction (F = 0.5, P < 0.05). In summer 2003 and winter 2003, numbers were highest on the glucose-containing artificial substrates (Fig. 2A). During other seasons, there were no significant differences among treatments.



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  		FIG. 2. Numbers of {alpha} (A)-, ß (B)-, and {gamma} (C)-proteobacteria in biofilms on artificial substrates determined by FISH with taxon-specific oligonucleotide probes. Values are means of triplicate samples ± SE. An asterisk indicates that the bar was significantly different (P < 0.05) from the control. ND, not determined.

A similar response was observed for the ß-proteobacteria (Fig. 2B). The numbers of ß-proteobacteria differed significantly among seasons (F = 4.10, P < 0.05) and among treatments (F = 0.23, P < 0.05) and season-treatment combinations (F = 0.75, P < 0.05). Only in summer 2003 was there a significant difference among treatments. As with the {alpha}-proteobacteria, numbers were highest on the glucose-amended pots.

The numbers of {gamma}-proteobacteria in the biofilms differed among seasons (F = 19.97, P < 0.0001) and among treatments (F = 3.17, P < 0.05); there was a significant season-treatment interaction (F = 1.25, P < 0.05; Fig. 2C). In fall 2002 and winter 2003 (P < 0.05, Tukey's HSD), there were significantly more {gamma}-proteobacteria on the glucose pots than on the controls. In contrast to the {alpha}- and ß-proteobacteria, there were no significant differences in summer 2003.

The abundance of Cytophaga-Flavobacterium bacteria in the biofilms differed significantly among seasons (F = 26.26, P < 0.0001) and among treatments (F = 10.34, P < 0.0001), and there was a significant season-treatment interaction (F = 2.43, P < 0.01). As observed for the {alpha}-proteobacteria, numbers of Cytophaga-Flavobacterium bacteria on the glucose-treated pots were significantly higher than numbers on the controls in winter and summer 2003 (P < 0.05, Tukey's HSD; Fig. 3A). In contrast to the other taxa examined, there were significantly more Cytophaga-Flavobacterium bacteria on the glucose-, leaf leachate-, and algal-exudate-treated pots in fall 2002 than on the control.



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  		FIG. 3. Numbers of Cytophaga-Flavobacterium cluster (CF) (A) and gram-positive high-G+C (GPHGC) (B) bacteria determined by probes in biofilms on artificial substrates determined by FISH with taxon-specific oligonucleotide probes. Values are means of triplicate samples ± SE. An asterisk indicates that the bar was significantly different (P < 0.05) from the control. ND, not determined.

The abundance of gram-positive high-G+C cells in the biofilms was significantly different among seasons (F = 3.28, P < 0.05) but not among treatments (F = 1.15, P > 0.05), and the season-treatment interaction was not significant (F = 1.32, P > 0.05). Numbers on artificial substrates were similar among all treatments and the controls (Fig. 3B).

A. calcoaceticus populations in the biofilms differed significantly among seasons (F = 28.96, P < 0.0001), but responses to the different treatments were not significant (F = 0.94, P > 0.05), nor was the season-treatment interaction significant (F = 1.27, P > 0.05). None of the treatments significantly altered A. calcoaceticus numbers in comparison to the controls (Fig. 4A).



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  		FIG. 4. Numbers of A. calcoaceticus (A), B. cepacia (B), and P. putida (C) bacteria in biofilms on artificial substrates determined by FISH with species-specific oligonucleotide probes. Values are means of triplicate samples ± SE. An asterisk indicates that the bar was significantly different (P < 0.05) from the control. ND, not determined.

When B. cepacia was examined, numbers were significantly different among seasons (F = 159.94, P < 0.0001) and also among treatments (F = 3.12, P < 0.05). There was a significant interaction between season and treatment (F = 1.41, P < 0.05). In most seasons, there were no significant differences in B. cepacia numbers among treatments. However, in winter 2003, numbers from the nitrate and glucose treatments were significantly higher than on the control (Fig. 4B).

The numbers of P. putida bacteria were significantly different among seasons (F = 431.76, P < 0.0001) and among treatments (F = 7.31, P < 0.0001). There was a significant season-treatment interaction (F = 6.04, P < 0.05). Only in summer 2003 were there significant differences among treatments. Interestingly, in that season, numbers on all treatments were higher than on the controls (Fig. 4C).

The relative abundance of the different phylogenetic groups as fractions of the domain Bacteria varied among treatments and among seasons (Fig. 5). In summer 2002, winter 2003, and spring 2003, the relative abundance of the taxa examined appeared similar among treatments and between 30 and 80% of the domain was composed of taxa examined here. In fall 2002 and summer 2003, there was an obvious shift in the relative contributions of the various taxa in different treatments. In particular, the phosphate treatment in fall 2002 showed a marked difference from the control, and the contributions of all the taxa were greatly altered. In that season, only in the nitrate treatment was there no obvious community shift.



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  		FIG. 5. Percent abundance of different bacterial phylogenetic groups relative to the domain Bacteria in biofilms on artificial substrates in summer 2002, fall 2002, winter 2003, spring 2003, and summer 2003 assemblages. CF, Cytophaga-Flavobacterium; GPHGC, gram-positive high-G+C bacteria; {alpha}, {alpha}-proteobacteria; ß, ß-proteobacteria; {gamma}, {gamma}-proteobacteria; other, other phylogenetic groups. ND, not determined.

Algal biomass.
Chlorophyll a concentrations were significantly different among seasons (F = 3.71, P < 0.05) but not among treatments (F = 0.51, P > 0.05), and there was not a significant season-treatment interaction (F = 0.55, P > 0.05; Fig. 6). Chlorophyll a concentrations were the lowest during summer 2003 because of an unusual series of spates during this period that scoured much of the biofilm from the substrates.



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  		FIG. 6. Chlorophyll a concentration in biofilms on nutrient diffusing substrates. Values are means of triplicate samples ± SE. ND, not determined.

Diatoms accounted for the bulk of the algae in the biofilms. The dominant algal types were Achnanthes, Frustulia, Tabellaria, and Cymbella (Fig. 7). There were clear community shifts among seasons and treatments in the algal types. Frustulia species dominated in summer 2002, accounting for as much as 80% of the total number of algae. In fall 2002, Achnanthes and Frustulia constituted the bulk of the algae, while in winter 2003, the community shifted to Achnanthes, Tabellaria, and Cymbella. Frustulia and Cymbella contributions surged in spring and summer 2003, respectively, and together with Achnanthes dominated the algal community. The abundance of the algal types varied in response to the different treatments: for example in summer 2002, the contribution of Frustulia appeared to be affected by nitrate and glucose treatments, while in winter 2003, phosphate, N plus P, leaf leachate, and algal exudates appeared to alter Tabellaria relative abundance.



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  		FIG. 7. Percent abundance of different algal types in biofilms on nutrient diffusing substrates in summer 2002, fall 2002, winter 2003, spring 2003, and summer 2003 assemblages. ND, not determined.


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DISCUSSION
 
The initial prediction that different DOM and inorganic nutrient sources would elicit various responses among different bacterial populations in biofilms was supported by the results from this study. Generally, bacterial responses were most obvious to amendment with labile DOM (i.e., glucose), in contrast to leaf leachate, algal exudates, and inorganic nutrients. Other investigations in which glucose was used as a carbon source in enrichment experiments also reported similar results in comparison to other nutrient/DOM treatments (6, 14, 15). Glucose is a preferred organic source among several bacterial taxa because of its extreme lability (7) and its occurrence in temperate streams (52). Although DOM mixtures, such as leaf leachate, may serve as the largest single source of organic matter in many streams (20), not all bacterial species are able to use leaf leachate effectively as a carbon source (34).

Although there is prior evidence that inorganic nutrients are limiting to bacteria in aquatic systems (18), there were few cases in this study where abundance of any bacterial group examined was elevated on artificial substrates containing inorganic nutrients. In addition, chlorophyll a concentration did not differ between these treatments and the controls. These observations are consistent with a study by Tank and Dodds (50), who found that in a third of the streams they examined there was no response by epilithic biofilms to inorganic nutrient amendment. In contrast, phosphorus may be a primary limiting nutrient to bacterioplankton in lakes (9, 36) and tropical rivers (6).

Responses among the various bacterial taxa examined differed. Overall, the response of the total number of bacteria (based on DAPI staining) was dissimilar to the responses of particular taxa based on FISH. In turn, different taxa sometimes exhibited responses that differed from those of the other types of bacteria examined. For example, in fall, abundance of Cytophaga-Flavobacterium bacteria was high on all three DOM treatments, while at the same time, only one other taxon ({gamma}-proteobacteria) showed any significant differences between any treatment and the controls. Cytophaga-Flavobacterium is generally abundant in streams, at times accounting for up to 25% of bacteria in river epilithon (38) and 11.4% of bacteria in a stream after a storm (4). Abundance of Cytophaga-Flavobacterium in aquatic systems has been associated with high-molecular-weight carbon compounds (8, 19). For example, Cottrell and Kirchman (8) demonstrated that Cytophaga-Flavobacterium abundance is associated with chitin and protein concentrations in marine systems. In contrast, for ß-proteobacteria, which are also a dominant group of freshwater bacteria, often representing the highest proportion of the domain Bacteria (53), abundances are more closely related to concentrations of low-molecular-weight compounds (8). Along these same lines, in this study, the abundance of ß-proteobacteria was nearly always similar among the treatments, except in summer 2003, when it was highest in the glucose-treated biofilms.

The three bacterial species examined in this study also differed in their responses: A. calcoaceticus did not differ among treatments in any season, whereas B. cepacia numbers were high on nitrate- and glucose-treated pots in winter, and P. putida responded significantly to all treatments in summer 2003. The variations observed in response to the DOM and inorganic nutrient sources may be associated with differences in the species' metabolic capabilities. This trend is similar to those reported in earlier studies that reported differential responses among subpopulations within bacterial communities (13, 14, 34). For example, Flaten et al. (14), using denaturing gradient gel electrophoresis to examine carbon-enriched cultures, reported that only a few subpopulations of bacteria in a marine bacterial community responded to the enrichments. McNamara and Leff (34) found differences in the population sizes of these same species in response to leachates from maple leaves. They observed that, while populations of A. calcoaceticus were unaffected by leaf leachate, B. cepacia responded positively to leaf leachate treatments, while P. putida appeared to be inhibited by phenolic compounds in the leaf leachate.

Overall, the responses to the different nutrient treatments varied seasonally, suggesting that the resources that are limiting to bacterial community may change over time. Changes in the environment, such as type and concentration of DOM (6, 24), inorganic nutrients, temperature, discharge, etc., coupled with changes in the bacterial community taxonomic composition are likely components determining seasonal changes in resource limitation. In this study, during summer and spring 2002, there were few, if any, differences between controls and treatments, suggesting that some other factors were limiting epilithic bacterial communities at these times. In summer 2003, a large fraction of the taxa examined were enhanced by glucose treatment, while in winter and fall 2003 some were enhanced by glucose and others were enhanced by glucose plus other factors. Overall, the most readily apparent responses were observed in summer 2003, a time when chlorophyll concentrations were much lower than during other parts of the study. During summer 2003, the study stream experienced unusual flooding and scouring was elevated (in fact, the summer 2003 experiment was attempted three times, with two earlier attempts being washed away by floods). We suspect that the needs of the bacterial community for labile, low-molecular-weight DOM, which were met by the algal community in summer 2002, were not met during summer 2003 because scouring restricted the development of the epilithic algal community and thus many bacterial taxa responded to the glucose amendment. In addition, there were differences among seasons in the type of algae present, perhaps as the result of temperature changes (29) and scouring, which may have altered the amount and type of algal exudate. Differences in exudate production among algae may also explain why there was little response to the exudate treatment in the study, which used species of algae not found in the actual biofilms studied.

This study agrees with others that reported compositional shifts in bacterial assemblages in response to variations in DOM and inorganic nutrients (10, 13). We observed strong seasonal differences in the responses of bacterial taxa examined to the various treatments. The variations in the response patterns among the taxa to the DOM and inorganic nutrient sources are likely attributable to differences in their metabolic and functional capabilities. In conclusion, our results demonstrate that different types of DOM and inorganic nutrients induce various responses among bacterial taxa in stream biofilms, and these may not be apparent if they are examined at the community level or if seasonal differences are not taken into account.


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ACKNOWLEDGMENTS
 
This research was supported in part by the Kent State University Graduate Student Senate through a grant to O.A.O.

We are grateful to Ksenijia Dejanovic for assistance with DOC analysis. We thank Todd Royer for his critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Great Lakes WATER Institute, Wisconsin Aquatic Technology & Environmental Research, University of Wisconsin, Milwaukee, WI 53204. Phone: (414) 382-1700. Fax: (414) 382-1705. E-mail: olapade{at}uwm.edu. Back


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Applied and Environmental Microbiology, May 2005, p. 2278-2287, Vol. 71, No. 5
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.5.2278-2287.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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