Benthic microbial utilization of differential dissolved organic matter ...

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Benthic microbial utilization of differential dissolved organic matter sources in a forest headwater stream1 David P. Kreutzweiser and Scott S. Capell

Abstract: Streamside mesocosm experiments were conducted in a low-order forest watershed to directly examine responses by microbial communities on standardized substrates to different terrestrial and aquatic sources of dissolved organic matter (DOM). Community respiration (oxygen uptake), microbial density (colony-forming units on agar plates), leaf decomposition, and community metabolic profiles (metabolism patterns in sole carbon source utilization assays) were measured. Stream benthic microbial communities responded immediately and positively to increases in terrestrially derived DOM. Respiration activity and density estimates increased significantly, but there was no significant change in community metabolic profile. Responses were greater to DOM extracted from upper soil horizons than from deeper soils. Community respiration and bacterial abundance also increased in response to an aquatic DOM source, but were accompanied by a significant change in community metabolic profiles. Results provide direct experimental evidence that benthic microbial communities of forest headwater streams are able to rapidly utilize terrestrial DOM. Résumé : Des expériences sur le mésocosme en bordure d’un cours d’eau ont été réalisées dans un bassin versant boisé d’ordre inférieur pour étudier directement, sur des substrats standardisés, les réactions des communautés microbiennes à différentes sources aquatiques et terrestres de matière organique dissoute. La respiration des communautés (prélèvement d’oxygène), la densité microbienne (unités formatrices de colonies sur plaque de gélose), la décomposition des feuilles et les profils métaboliques des communautés (comportement du métabolisme lors d’essais d’utilisation avec une source unique de carbone) ont été mesurés. Les communautés microbiennes benthiques du cours d’eau ont réagi immédiatement et positivement à des augmentations de matière organique dissoute d’origine terrestre. Les estimations de l’activité respiratoire et la densité ont significativement augmenté mais il n’y a pas eu de changement significatif dans le profil métabolique des communautés. Les réactions étaient plus fortes en présence de matière organique dissoute extraite des horizons supérieurs que plus profondément dans le sol. La respiration des communautés et l’abondance des bactéries ont également augmenté en réponse à une source aquatique de matière organique dissoute mais étaient accompagnées de changements significatifs dans les profiles métaboliques des communautés. Les résultats fournissent une preuve expérimentale directe que les communautés microbiennes benthiques des cours d’eau de tête de bassin en forêt sont capables d’utiliser rapidement la matière organique dissoute d’origine terrestre. [Traduit par la Rédaction]

Kreutzweiser and Capell

Introduction Terrestrial organic matter inputs to forested headwater streams have been shown to be important drivers of aquatic microbial community dynamics (Hall and Meyer 1998; Suberkropp 1998). Dissolved organic matter (DOM) can be an important energy source for heterotrophic microbial communities in streams (Cummins et al. 1983; Bott and Kaplan 1985), and whereas most DOM in headwater streams is exported to larger rivers and lakes, even low levels of retention and assimilation of DOM fluxes are likely to be critical to stream food webs (Lock 1993). If DOM is a significant source of energy for aquatic microbial communities, then

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changes in the quantity and quality of DOM entering streams as a result of disturbances to watersheds from landuse practices (Findlay and Sinsabaugh 1999) or climate change (Carpenter et al. 1992), for example, could affect stream microbial community structure and function. This would be particularly true of forest headwater streams where ecological linkages between aquatic and terrestrial environments are strong (McDowell and Likens 1988), and where much of the DOM is derived from the surrounding terrestrial ecosystem (Aitkenhead-Peterson et al. 2003). However, studies have reported conflicting results with regard to the importance of DOM to attached microbial communities in streams. Meyer et al. (1987) showed that a

Received 11 June 2002. Accepted 29 January 2003. Published on the NRC Research Press Web site at http://cjfr.nrc.ca on 3 July 2003. D.P. Kreutzweiser2 and S.S. Capell. Canadian Forest Service, Natural Resources Canada, 1219 Queen Street East, Sault Ste. Marie, ON P6A 2E5, Canada. 1

This paper was presented at the symposium Small Stream Channels and Riparian Zones: Their Form, Function and Ecological Importance in a Watershed Context held 19–21 February 2002, The University of British Columbia, Vancouver, B.C., and has undergone the Journal’s usual peer review process. 2 Corresponding author (e-mail: [email protected]). Can. J. For. Res. 33: 1444–1451 (2003)

I:\cjfr\cjfr3308\X03-030.vp June 30, 2003 9:43:53 AM

doi: 10.1139/X03-030

© 2003 NRC Canada



proportion of DOM produced in floodplains of a lowgradient stream was directly metabolized by stream bacteria. Bott et al. (1984) demonstrated a clear association between DOM concentration and bacterial biomass in streams. Hill et al. (2000) also showed a significant correlation between benthic microbial respiration and dissolved organic carbon (DOC) concentrations in streams. On the other hand, neither Sobczak et al. (1998) nor Chafiq et al. (1999) found a significant correlation between bacterial productivity and DOC concentration in pore waters of streams. Hedin (1990) also reported no significant relationship between benthic microbial respiration and stream water DOM. McDowell (1985) experimentally added DOM from leaf leachate to a small forest stream and found little or no increase in microbial respiration. Given these variable results, we concur with Mulholland (1997), who suggested that there is a need to better understand the role of DOM, particularly terrestrially derived DOM, in supporting microbial metabolism and food webs in streams. In addition, Findlay and Sinsabaugh (1999) point out that studies on DOM utilization by lotic microbial communities have not made it clear whether changes in microbial function are always preceded by changes in community structure, or whether microbial metabolic plasticity is sufficient to allow the same community to function at different rates. In the present study, DOM additions were made to outdoor stream channels in a low-order forest watershed to directly measure effects on microbial community function (respiration, decomposition activity) and structure (metabolic profiles). Comparisons were made among communities in channels of ambient DOM concentrations, terrestrially derived DOM additions, and aquatic-derived DOM additions. This manipulative experiment was intended to directly address three questions: (i) is DOM derived from nearby forest soils readily utilized by microbial communities in a forest stream, (ii) is this terrestrially derived DOM more or less bioavailable to stream microbes than an aquatic-derived DOM source from the same watershed, and (iii) are the responses to DOM additions accompanied by changes in community structure or enzyme production as measured by their metabolic profiles in carbon-source utilization assays?

Materials and methods Stream mesocosms The experimental site was located adjacent to a secondorder forest stream about 50 km north of Sault Ste. Marie, Ontario, Canada (46°53′20′′ N, 84°07′45′′W). The stream has a mean bank-full width of 7.2 m, a mean base-flow depth of 42.4 cm over a 300-m reach near the mesocosms, and a channel slope of 3.7%, and it flows through a mixed forest consisting primarily of sugar maple (Acer saccharum Marsh.) and yellow birch (Betula alleghaniensis Britt.), with balsam fir (Abies balsamea (L.) Mill.), white spruce (Picea glauca (Moench) Voss), white birch (Betula papyrifera Marsh.), and eastern white-cedar (Thuja occidentalis L.) mixed in riparian areas. Stream water temperatures during the dosing and observation periods over the course of experimental period ranged from 13 to 16°C, with dissolved oxygen concentrations near saturation (8 to 10 mg·L–1), pH at 6.5 to 6.6, and conductivity near 19 ␮S·cm–1.

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Twelve stream channels were assembled on a raised platform about 15 m from the stream. Each channel was constructed of stainless steel, 10 cm wide by 3 m long, and filled with natural bottom substrate (fine and coarse gravel, organic debris) collected from the adjacent stream. Water was diverted from the stream through a plastic pipe and gravity fed to the top of the channels. Gate valves regulated the rates of flow through each channel. Test substrates were added to the channels before the DOM additions to facilitate colonization by microbial communities. These included wooden Popsicle sticks (1 × 11 cm, made of white birch) that were used as surrogates of small benthic woody debris and were placed in the channels 3 week before the applications. Leaf disks (22 mm diameter) cut from preleached yellow birch leaves collected immediately after leaf fall and stored dry were used as surrogates of benthic leaf material and were placed in 1-mm mesh containers in the stream channels 2 week before the applications. The 1-mm mesh containers were intended to exclude macroinvertebrates but allow good water flow through the containers and facilitate microbial colonization. Given the 1-mm mesh size, very small insects could have accessed the leaf disks, but no insects or microbenthos (e.g., cladocerans, copepods, oligochaetes, ostracods) were ever observed on the disks during sampling and handling procedures. Microbes on the wood pieces and leaf disks were not identified, but microbial communities were presumed to include bacteria, fungi, and protozoans. DOM additions DOM was extracted from three sources: (i) soil O horizon materials (about top 10 cm) collected from the forest floor adjacent to the stream, (ii) soil A horizon (about 10–20 cm deep) from the same area, and (iii) submerged aquatic vegetation (water milfoil, Myriophyllum sp.) from the same watershed. The materials were placed in large plastic containers outdoors, shielded from rain and direct sunlight, and soaked in distilled water for 3 days. Leachates were progressively sieved and filtered down to a 1.5-µm filter (Whatman 934AH glass-fibre filter), autoclaved, and stored at 2°C for about 4 days before the applications were made. Dissolved organic carbon (DOC) concentrations of the leachates were measured by a Technicon Autoanalyser IIC carbon analyzer, and adjustments were made by adding distilled water. Applications were made to stream channels by continuous delivery of DOM leachates from Mariotte dripper bottles constructed from 25-L plastic bottles. The treatments included four replicate channels of ambient stream water, four channels of terrestrially derived DOM (extracted from soil O horizon), and four channels of aquatic-derived DOM (extracted from submerged vegetation). The total application time was 72 h. Bottles were replaced and drip rates readjusted every 24 h. Water samples were periodically collected from the outflow of the channels for spot checks of test concentrations. The drip rates were calibrated to provide a 50% increase in DOC over ambient stream water concentrations, based on DOC concentrations in the leachates and water flow rates in each channel. The spot check samples indicated that ambient DOC concentrations ranged from 8.8 to 9.5 mg·L–1, and the DOM additions generally increased DOC to about 12.3 to 14.5 mg·L–1. © 2003 NRC Canada



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Response variables Community respiration was determined as dissolved oxygen uptake by leaf and woody material in sealed, darkened chambers. Respiration measurements were made at 1 h after the 3-day DOM additions, and again at 5 days after the additions. Twelve circular Plexiglas chambers (6.5 cm diameter, 7 cm high) were mounted in a Plexiglas tank that served as a water bath to maintain constant temperature in the circular chambers. Each circular chamber contained a magnetic stir bar in a screened cell at the bottom of the chamber to maintain constant water circulation. The chambers were filled with water from the same stream channels as the substrates and operated with the tops open and stir bars active to saturate the water with dissolved oxygen. Substrates were taken from the stream channels and added to the chambers, where initial dissolved oxygen concentrations were measured with Orion models 810 and 834 dissolved oxygen meters. The same meter was used for measurements in the same chambers at each time. For measurement of respiration on leaf disks, 10 disks were randomly selected from the screened containers in each channel and placed in the respiration chambers. After addition of the leaf disks, the chambers were sealed with a Plexiglas lid mounted on a rubber O-ring and fastened down with thumbscrews. A portal in the lid, sealed with a rubber stopper, provided access for the oxygen probe after incubation. The chambers were then darkened with black coverings and held at ambient temperature and in constant water circulation for 3 h. After the 3-h incubation period, the covers and rubber stoppers were removed, and final dissolved oxygen concentrations were measured. A similar procedure was used for the woody substrates. For each sample, four sticks were randomly taken from each stream channel, broken into 16 pieces in total (needle-nose pliers were carefully used to avoid handling the sticks during this process) and placed in each respiration chamber. Measurements were taken as for leaf disks. Because these chambers were sealed and darkened, microbial respiration was assumed to be directly proportional to oxygen consumption and was expressed as milligrams O2 consumed per litre per hour. Oxygen consumption was also monitored each time in a single blank chamber (ambient water only) to determine contributions of water column organisms to respiration measurements. These were determined to be negligible, and no corrections to measurements from chambers containing substrates were made. Two further short-term experiments were conducted to provide information on the timing of microbial responses to DOM additions, and on responses to differential sources of terrestrially derived DOM (soil O vs. A horizon). In these cases, DOM additions were made directly to recirculating respiration chambers containing preconditioned leaf disks from control stream channels, and responses in respiration activity were determined. In the timing experiment, comparisons of respiration rates were made among chambers with ambient DOM (water drawn from the stream channels), aquatic DOM, and terrestrial (soil O horizon) DOM. The DOM concentrations from leachates were adjusted to provide equal concentrations in amended chambers (aquatic and terrestrial DOM). Measurements were made at 3 and 5 h after the additions. In the soil comparison experiment, leachates were extracted from O- and A-horizon soils, adjusted to

Can. J. For. Res. Vol. 33, 2003

a common concentration, and comparisons made among ambient, O-horizon, and A-horizon DOM amended chambers. Decomposition activity of microbial communities was estimated from the mass of leaf material remaining in 1-mm mesh bags at 5 days after the treatments. Batches of 10 leaf disks were placed in each channel 12 days before the DOM additions to facilitate microbial colonization of the leaf material. At 5 days after the end of the 3-day additions, the leaf disks were removed from each channel, gently washed to remove accumulated sediment and biofilm, oven-dried at 25°C, and weighed. Mass of leaf material remaining in leaf packs was compared among treatments. The 8-day postDOM exposure period should have been sufficient to detect differences in decomposition of leaf material, based on previous experience with determining microbial decomposition of yellow birch leaf disks over a similar experimental period (Kreutzweiser et al. 1996). Bacterial density in fine sediments was estimated by extraction from the sediments and direct plate counts. Density estimates were made from samples collected 5 days before, 1 h after, and 5 days after the 3-day DOM additions. The inherent inefficiencies of plate counts for estimating bacterial abundance (Staley and Konopka 1985) are recognized. This was intended to provide relative measures of bacterial densities among replicate stream channels, not absolute densities of bacterial populations. Equal volumes of fine sediments were drawn from each channel with a large-bore syringe, placed in a sterile glass vial, and held overnight at 4°C. Bacterial cells were extracted by a procedure modified from England et al. (2001). This procedure was developed for extraction of bacteria from fine sediments and was not tested for the larger organic substrates (wood pieces, leaf disks) used for respiration measurements. Therefore, bacterial abundance was estimated in fine sediments only. The sediments were filtered onto a GF/F filter, and 1-g wet mass batches from each channel were placed in 125-mL Erlenmeyer flasks with 19 glass beads and 9.5 mL of 0.1% sodium pyrophosphate (NaPyro, pH 7), and placed on a shaker at 170 r/min for 45 min at 20°C. After shaking, the suspension was held for several minutes to allow the large particles to settle, and a 0.5-mL aliquot of the supernatant was extracted. A serial dilution was prepared to 106 and plated onto 1/10 strength LB agar (1/10 Luria-Burtani: 0.1% (m/v) tryptone, 0.05% (m/v) yeast extract, 0.05% (m/v) sodium chloride, 1.5% (m/v) agar). Duplicates were prepared for each dilution, and the number of colony-forming units was recorded after 48 h incubation at room temperature. The mean of two duplicates was taken as the density estimate for a given dilution. Microbial community metabolic profiles, or “metabolic fingerprints”, were determined by examining carbon source utilization patterns in 96-well Biolog microplates (Garland and Mills 1991). This approach is becoming widely used for classifying microbial communities of environmental samples (e.g., Lehman et al. 1997; Gamo and Shoji 1999), but has been criticized for inaccuracies in output interpretation (Haack et al. 1995) and in characterizing microbial diversity (Konopka et al. 1998). The use of Biolog plates in these experiments was intended to provide a measure of community structure as metabolic profiles or fingerprints for comparison among replicate stream channels over time. Differences in © 2003 NRC Canada




metabolic profiles among treatments were taken to infer different structural responses by microbial communities to the applications, either in terms of actual shifts in species assemblages or in physiological responses through differential enzyme production. The assays did not distinguish between these. Samples were obtained and processed as described for bacterial abundance, and for the same reasons (i.e., extraction methods had been developed for fine sediments), microbial community profiles were not determined for the leaf and woody material. To reduce the amount of extraneous organic material introduced into the Biolog plates, the samples extracted from the sediments were further processed through a cleanup procedure of differential centrifugations to discard organic particles and concentrate microbial cells. For each sample, a 500-µL aliquot was drawn from each flask on the shaker and centrifuged at 680 r/min for 10 min. The supernatant was decanted into a second tube, and the pellet was resuspended in 10 mL sterile 0.1% NaPyro and centrifuged at 500 r/min for 10 min. The supernatant was again collected, and the pellet was resuspended and centrifuged a second time at 500 r/min for 10 min. The final supernatant was decanted and collected, thoroughly mixed with the previous supernatant, and a 20-mL aliquot was centrifuged at 8000 r/min for 12 min. The supernatant was discarded, and the pellet was resuspended in 10 mL sterile 0.1% NaPyro and again centrifuged at 8000 r/min for 12 min. The pellet was resuspended in 25 mL sterile 0.1% NaPyro and poured into a sterile petri plate. A multi-tip micropipette was used to dispense 125 µL into each of the 96 wells, and the microplates were incubated in the dark at 14°C for 144 h. Optical densities at 630 nm were read on a Bio-Tek Instruments ELX808 plate reader at 24, 48, 72, 96, 120, and 144 h after inoculation of the Biolog plates. Optical densities were corrected for readings from the blank well, and values were normalized by dividing each cell optical density by the average well colour development for the plate (Garland and Mills 1991). The average well colour development, or average metabolic response (AMR), at 96 h was used as a measurement of the community’s average ability to utilize the 95 carbon sources (a metabolic “fingerprint”), for comparison across treatments. Patterns in carbon source utilization were examined by multivariate analyses as an indication of the community structure (species assemblages and (or) differential enzyme production) among treatments. Data for these analyses were further normalized for differences in average intensity between samples by using a threshold AMR of 0.75 (Garland 1996). In this case, readings from 96 or 120 h were at or above the AMR threshold. Data analysis Differences among treatments in respiration rates and in mass of leaf material remaining were examined by one-way ANOVA, followed by Tukey’s studentized range (HSD) tests, which corrected for experimentwise error rates. Residual plots were examined for homogeneity of variances, and log transformations were applied if necessary. Trends over time in bacterial abundance and in community AMR were analyzed by a repeated measures orthogonal polynomial coefficient analysis (Meredith and Stehman 1991). This ap-

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proach accounts for the correlation among samples drawn from the same experimental unit over time. It also focuses on the response curve, i.e., a comparison of changes over time among groups, and does not require the stringent assumptions of a split-plot analysis. The treatment × time interaction in the analysis was the primary term of interest for detecting significant treatment effects. Significant treatment × time interaction (p < 0.05) indicated that trends over time were not parallel among treatment groups. When this occurred, differences among groups at each time were investigated further by ANOVA and specified contrasts. Residual plots for all data sets were examined for homogeneity of variances and normality prior to analyses, and log transformations applied when necessary. The community carbon source utilization patterns among the 96-well Biolog plates were analyzed by principal components analysis (PCA). Two-dimensional plots of the first two PCA axes were constructed to detect patterns in carbon source utilization among treatments and over time. In these plots, each point represents the metabolic profile of the microbial community on sediments in a given stream channel at a given time. Channels in which the microbial metabolic profiles are similar are plotted close together; those in which microbial metabolic profiles are dissimilar are plotted far apart in ordination space. The metabolic profile data were then subjected to a multivariate analysis of similarity (ANOSIM) to determine whether differences in metabolic profiles among microcosms were statistically significant. This test was based on a nonparametric permutation procedure applied to a similarity matrix derived from a normalized Euclidean distance ordination (Clarke and Warwick 1994).

Results Microbial community respiration and decomposition activity on organic material At the end of the 3-day DOM additions, respiration on leaf disks was significantly higher in channels of both DOM additions than in controls (ANOVA, p = 0.0002), with a significantly greater response (HSD, p < 0.05) to terrestrial DOM than to aquatic DOM (Fig. 1A). By 5 days after the additions, there were no differences in respiration on leaf disks across treatments (ANOVA, p = 0.3590). Respiration on wood was also significantly different among treatments by 1 h after the 3-day additions (ANOVA, p = 0.0360), but although mean respiration was higher in both DOM additions, only the response to aquatic DOM was significantly different from control (HSD, p < 0.05) (Fig. 1B). There were no differences among treatments in respiration on wood by 5 days after the additions (ANOVA, p = 0.8825). In subsequent short-term experiments in which DOM additions were made directly to respiration chambers containing preconditioned leaf disks, the response by attached microbial communities to DOM was rapid. There were significant increases in respiration within 3 h after the additions of terrestrial and aquatic DOM on both leaf disks (ANOVA, p = 0.0030) and wood (ANOVA, p = 0.0022) (Fig. 2). Although there tended to be a greater response to terrestrial DOM than to aquatic DOM, there were no significant differences between the two DOM additions (HSD, p > 0.05). In a © 2003 NRC Canada



1448 Fig. 1. Mean (±1 SE, n = 4) microbial respiration on (A) leaf disks and (B) wood in stream channels at 1 h after and 5 days after the 3-day DOM additions. Means with different letters are significantly different (Tukey’s studentized range HSD test, p < 0.05).


tions from O-horizon soils, and 1.00 (±0.07) in DOM additions from A-horizon soils. Despite the significant increase in microbial respiration on leaf disks after the terrestrial and aquatic DOM additions to stream channels, there was no indication that this reflected increased decomposition of leaf material. There were no differences among treatments in the amount of leaf material remaining in fine mesh leaf packs 5 days after the end of the DOM additions (ANOVA, p = 0.8677). The mean (±1 SE) dry mass (mg) of leaf material remaining in leaf packs was 161.9 (±8.97) in controls, 153.9 (±11.3) in terrestrial DOM channels, and 159.5 (±11.8) in aquatic DOM channels. Bacterial density Bacterial (colony-forming units) density on plate counts increased immediately after both DOM additions (Fig. 3). The linear time model of the repeated measures orthogonal polynomial coefficient analysis was significant (p < 0.0001), and the time × treatment interaction was also significant (p = 0.0055), indicating that the trends over time were not parallel among treatments. The time trends were compared by specified contrasts, and both the terrestrial (p = 0.0201) and aquatic (p = 0.0403) treatments were significantly different from trends in control channels. Density in both treatments declined by 5 days after the DOM additions, but numbers remained significantly higher than in controls (ANOVA, p = 0.0012) with density in terrestrial DOM channels slightly higher than that in aquatic DOM channels.

Fig. 2. Mean (±1 SE, n = 4) microbial respiration on leaf and wood material in chambers at 3 h after DOM additions. Means with different letters are significantly different (Tukey’s studentized range HSD test, p < 0.05).

further experiment in which responses to differential sources of terrestrial DOM were determined, respiration was again significantly different among treatments (ANOVA, p = 0.0096). However, respiration in response to DOM extracted from the O-horizon soil was significantly higher (HSD, p < 0.05) than in control, whereas respiration in response to DOM extracted from deeper A-horizon soil was not (HSD, p > 0.05). Mean (±1 SE) respiration (mg O2 consumed·L–1·h–1) was 0.84 (±0.09) in the control, 1.30 (±0.08) in DOM addi-

Microbial community profiles Although there were significant responses in microbial activity and abundance to both DOM additions, there was a differential response in terms of community metabolic profiles. The average metabolic response (AMR), which was used as a metabolic fingerprint to describe community structure, differed between aquatic DOM channels and controls, whereas the AMRs of communities in terrestrial DOM channels were similar to those of controls (Fig. 4). The AMR varied over time (quadratic time trend, p = 0.0002), but there was also significant time × treatment interaction (p = 0.0267), indicating that trends over time were not parallel among treatments. The specified contrasts indicated that the trend in aquatic DOM channels was significantly different from that of the controls (p = 0.0232), but the trend in terrestrial DOM channels was not (p = 0.7730). The effect of aquatic DOM on microbial community profiles was only apparent immediately after the 3-day additions, with a return to a community profile similar to that of the control by 5 days after the exposure (Fig. 4). The carbon source utilization patterns (CSUP) in microplate assays gave further evidence of an effect of aquatic DOM on microbial community structure. Ordination of CSUP data by PCA revealed temporal shifts in community profiles over the experimental period (Fig. 5), and the ANOSIM tests detected significant differences in community profiles among treatments. Before the DOM additions, CSUP were clustered to the left on the PCA axis 1, and there were no differences among treatments (ANOSIM, p = 0.895). By the second sample time, 1 h after the 3-day additions, all CSUP among treatments shifted to the right along axis 1, indicating a temporal shift in community profiles, © 2003 NRC Canada



Kreutzweiser and Capell Fig. 3. Mean (±1 SE, n = 4) abundance of colony-forming units (CFU) in plate counts from sediments of stream channels before, 1 h after, and 5 days after the 3-day DOM additions.

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presumably from natural changes in community structure or enzyme production. However, the mean CSUP of aquatic DOM channels moved away from controls in ordination space, upward along PCA axis 2 (Fig. 5). This resulted in a significant difference in community CSUP among treatments (ANOSIM, p = 0.011). Pairwise comparisons at sample time 2 indicated a significant difference in CSUP between controls and aquatic DOM channels (ANOSIM, p = 0.029), but no difference between controls and terrestrial DOM channels (ANOSIM, p = 0.200). By 5 days after the 3-day additions, CSUP of all groups were again clustered closely in ordination space, and no differences among treatments were detected (ANOSIM, p = 0.564).

Discussion

Fig. 4. Mean (±1 SE, n = 4) of the average metabolic responses to sole carbon sources, as determined by optical density (OD) at 630 nm, in Biolog 96-well assays before, 1 h after, and 5 days after the 3-day DOM additions.

Fig. 5. Principal component analysis (PCA) ordination of average (n = 4) carbon source utilization patterns in control (C), terrestrial DOM channels (T), and aquatic DOM channels (A) at sample time 1 (pretreatment), time 2 (1 h after the 3-day DOM additions), and time 3 (5 days after the 3-day DOM additions).

These results provide direct, experimental evidence that benthic microbial communities of forest headwater streams can respond rapidly and positively to terrestrially derived DOM. It was clear that attached microbial communities on organic substrates were responding almost immediately with significant increases in respiration activity within 3 h of exposure to terrestrial DOM additions. This response was greater to DOM extracted from O-horizon soils than to DOM from A-horizon soils. The differential responses to the two sources of terrestrial DOM at the same concentration indicate that the quality and bioavailability of DOM from the uppermost forest soil layer is the most important soil-derived fraction for supporting benthic metabolism. DOM from deeper soil layers has generally undergone considerable degradation and transformation and is therefore less labile than DOM from organic-rich upper layers (Kaplan and Newbold 1993). The difference in microbial response to the two sources of terrestrial DOM in the present study supports the suggestion by Bott and Kaplin (1985) that the quality of DOM can be important in regulating benthic microbial respiration. The increase in attached microbial activity and density appeared to reflect direct mineralization of water column DOM and did not stimulate increased decomposition of particulate organic material. There were no differences among treatments in mass of leaf material at the end of the experimental period. Given that bacteria are the principal users of DOM, and that hyphomycete fungi are the predominant microbial decomposers of particulate matter (Bott et al. 1984; Suberkropp 1998), the significant increases in microbial activity detected in our experiments were probably bacterial responses to the DOM additions. The significant increases in bacterial density in plate counts further indicate a bacterial response to DOM. The rapid and positive bacterial responses to terrestrial DOM occurred without measurable changes in community structure, at least in terms of enzyme production and metabolic profiles as measured in the Biolog assays. Although there were significant increases in respiration activity and bacterial density following the terrestrial DOM additions, there were no significant differences between controls and terrestrial DOM additions in bacterial community metabolic profiles (average metabolic responses) or in sole carbon source utilization patterns in Biolog microplate assays. This indicates that there was no change in community structure, or in © 2003 NRC Canada



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the production of extracellular enzymes among microbes in these stream channels in response to the terrestrial DOM. Rather, the aquatic microbial communities were able to directly mineralize terrestrially derived DOM. This has been inferred from previous observations of microbial activity and composition among streams of differing DOM characteristics (Kaplan and Bott 1985; Foreman et al. 1998), but the present study provides experimental evidence to this effect. Most DOM in streams is made up of compounds that require extracellular hydrolysis for utilization by benthic bacteria (Findlay et al. 1997), and it appears that the aquatic microbial communities of forested headwater streams are enzymatically equipped to rapidly utilize terrestrial DOM. Microbial enzyme production and kinetics in aquatic systems can be induced by exposure to specific substrates (Arnosti 2003). This inducible microbial enzymatic system facilitates the use by benthic bacteria of short-term fluxes in terrestrial DOM associated with disturbance or storm events. The rapid response to terrestrial DOM without a shift in community structure indicates that the metabolic plasticity of microbial communities in these streams is sufficient to allow the same community to function at substantially different rates under differential DOM sources and concentrations. The microbial communities of these stream channels also responded positively to the aquatic DOM additions, but this appeared to be mediated by a shift in community structure or enzyme production. By the end of the 3-day additions, microbial communities in aquatic DOM channels exhibited significant increases in respiration activity and bacterial abundance and were significantly different from controls in community metabolic profiles. Whereas the microbial responses to terrestrial DOM appeared to be physiological (i.e., increased enzyme allocation and DOM utilization without a change in community structure or enzyme production), the responses to aquatic DOM were accompanied by a change in the underlying community structure or in the community enzyme production. It is possible that those species among the benthic bacterial assemblages that were capable of mineralizing aquatic DOM proliferated and overtook species less equipped to use the aquatic DOM, to the extent that there was a measurable shift in community structure. However, given the rapid response time, it is more likely that there was a substrate-induced enzymatic shift in the microbial cells present in response to the aquatic DOM. No such shift was evident in response to the terrestrial DOM. Given the singular source of aquatic DOM in our experiments (extracted from milfoil), it is unclear to what extent this response to aquatic DOM additions can be generalized to other aquatic DOM sources. Nevertheless, these results suggest that the microbial communities of forest headwater streams may be less enzymatically primed to utilize aquatic DOM, and they underscore the importance of terrestrially derived DOM in supporting microbial metabolism in streams. Given the role of terrestrial DOM in supporting microbial metabolism in forest streams, anthropogenic and natural disturbances to forested watersheds that alter the quantity, timing, and quality of DOM transported to receiving waters could affect microbial mineralization rates and carbon cycling. The short pulses of labile DOM from terrestrial inputs appear to be important nutrient and carbon sources for benthic microbial communities. Our results indicate that this is


particularly true of DOM from the uppermost organic-rich soil layers in forest areas. DOM from upper layers can be transported to receiving waters during periods of saturation in riparian areas (Schiff et al. 1997), or through preferential flow paths during storms (Hagedorn et al. 2000). Activities that disrupt or alter this transfer of terrestrial DOM to headwaters streams, such as soil disturbance or compaction by logging machinery, or changes in organic matter processing in DOM source areas through vegetation removal or disturbance, have the potential to alter aquatic microbial metabolism and processes. Microbial communities form the bases of food webs in streams and can directly transfer carbon to higher trophic levels through the microbial loop (Meyer 1994). The present study indicates that terrestrial waterborne carbon is readily utilized by aquatic microbial communities in forest streams. DOM exported from forest watersheds is not entirely lost to downstream areas but can be rapidly assimilated and potentially transferred to higher trophic levels. Terrestrial DOM appears to play an important role in organic matter budgets of forest streams and is therefore a useful response variable in streams for measuring impacts of land use on watersheds.

Acknowledgements The authors are grateful for the technical assistance of Melissa Murray, Armin Schatzler, Kevin Good, Robin Harding, and Jamie Broad in these experiments. Laura Hawdon provided the DOC analyses. The project was supported in part by the Climate Change Network of the Canadian Forest Service.

References Aitkenhead-Peterson, J.A., McDowell, W.H., and Neff, J.C. 2003. Sources, production, and regulation of allochthonous dissolved organic matter inputs to surface waters. In Aquatic ecosystems: interactivity of dissolved organic matter. Edited by S.E.G. Findlay and R.L. Sinsabaugh. Academic Press, Amsterdam. pp. 25–70. Arnosti, C. 2003. Microbial extracellular enzymes and their role in dissolved organic matter cycling. In Aquatic ecosystems: interactivity of dissolved organic matter. Edited by S.E.G. Findlay and R.L. Sinsabaugh. Academic Press, Amsterdam. pp. 315– 342. Bott, T.L., and Kaplan, L.A. 1985. Bacterial biomass, metabolic state, and activity in stream sediments: relation to environmental variables and multiple assay comparisons. Appl. Environ. Microbiol. 50: 508–522. Bott, T.L., Kaplan, L.A., and Kuserk, F.T. 1984. Benthic bacterial biomass supported by streamwater dissolved organic matter. Microb. Ecol. 10: 335–344. Carpenter, S.R., Fisher, S.G., Grimm, N.B., and Kitchell, J.F. 1992. Global change and freshwater ecosystems. Annu. Rev. Ecol. Syst. 23: 119–139. Chafiq, M., Gibert, J., and Claret, C. 1999. Interactions among sediments, organic matter, and microbial activity in the hyporheic zone of an intermittent stream. Can. J. Fish. Aquat. Sci. 56: 487–495. Clarke, K.R., and Warwick, R.M. 1994. Change in marine communities: an approach to statistical analysis and interpretation. Natural Environment Research Council, Plymouth, U.K. © 2003 NRC Canada



Kreutzweiser and Capell Cummins, K.W., Sedell, J.R., Swanson, F.J., Minshall, G.W., Fisher, S.J., Cushing, C.E., Petersen, R.C., and Vannote, R.L. 1983. Organic matter budgets for stream ecosystems: problems in their evaluation. In Stream ecology, application and testing of general ecological theory. Edited by J.R. Barnes and G.W. Minshall. Plenum, New York. pp. 299–353. England, L.S., Trevors, J.T., and Holmes, S.B. 2001. Extraction and detection of baculoviral DNA from lake water, detritus and forest litter. J. Appl. Microbiol. 90: 630–636. Findlay, S., and Sinsabaugh, R.L. 1999. Unravelling the sources and bioavailability of dissolved organic matter in lotic aquatic ecosystems. Mar. Freshw. Res. 50: 781–790. Findlay, S., Hickey, C.W., and Quinn, J.M. 1997. Microbial enzymatic response to catchment-scale variations in supply of dissolved organic carbon. N.Z. J. Mar. Freshw. Res. 31: 701–706. Foreman, C.M., Franchini, P., and Sinsabaugh, R.L. 1998. The trophic dynamics of riverine bacterioplankton: relationships among substrate availability, ectoenzyme kinetics, and growth. Limnol. Oceanogr. 43: 1344–1352. Gamo, M., and Shoji, T. 1999. A method of profiling microbial communities based on a most-probable-number assay that uses BIOLOG plates and multiple sole carbon sources. Appl. Environ. Microbiol. 65: 4419–4424. Garland, J.L. 1996. Analytical approaches to the characterization of samples of microbial communities using patterns of potential carbon source utilization. Soil Biol. Biochem. 28: 213–221. Garland, J.L., and Mills, A.L. 1991. Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization. Appl. Environ. Microbiol. 57: 2351–2359. Haack, S.K., Garchow, H., Klug, M.J., and Forney, L.J. 1995. Analysis of factors affecting the accuracy, reproducibility, and interpretation of microbial community carbon source utilization patterns. Appl. Environ. Microbiol. 61: 1458–1468. Hagedorn, F., Schleppi, P., Waldner, P., and Hannes, F. 2000. Export of dissolved organic carbon and nitrogen from Gleysol dominated catchments — the significance of water flow paths. Biogeochemistry, 50: 137–161. Hall, R.O., and Meyer, J.L. 1998. The trophic significance of bacteria in a detritus-based stream food web. Ecology, 79: 1995– 2012. Hedin, L.O. 1990. Factors controlling sediment community respiration in woodland stream ecosystems. Oikos, 57: 94–105. Hill, B.H., Hall, R.K., Husby, P., Herlihys, A.T., and Dunne, M. 2000. Interregional comparisons of sediment microbial respiration in streams. Freshw. Biol. 44: 213–222. Kaplan, L.A., and Bott, T.L. 1985. Acclimation of stream-bed heterotrophic microflora: metabolic responses to dissolved organic matter. Freshw. Biol. 15: 479–492.

1451 Kaplan, L.A., and Newbold, J.D. 1993. Biogeochemistry of dissolved organic carbon entering streams. In Aquatic microbiology: an ecological approach. Edited by T.E. Ford. Blackwell Scientific Publications, Oxford, U.K. pp. 139–165. Konopka, A., Oliver, L., and Turco, R.F. 1998. The use of carbon substrate utilization patterns in environmental and ecological microbiology. Microb. Ecol. 35: 103–115. Kreutzweiser, D.P., Gringorten, J.L., Thomas, D.R., and Butcher, J.T. 1996. Functional effects of the bacterial insecticide Bacillus thuringiensis var. kurstaki on aquatic microbial communities. Ecotoxicol. Environ. Saf. 33: 271–280. Lehman, R.M., Colwell, F.S., and Garland, J.L. 1997. Physiological profiling of indigenous aquatic microbial communities to determine toxic effects of metals. Environ. Toxicol. Chem. 16: 2232–2241. Lock, M.A. 1993. Attached microbial communities in rivers. In Aquatic microbiology: an ecological approach. Edited by T.E. Ford. Blackwell Scientific Publications, Oxford, U.K. pp. 113– 138. McDowell, W.H. 1985. Kinetics and mechanisms of dissolved organic carbon retention in a headwater stream. Biogeochemistry, 1: 329–352. McDowell, W.H., and Likens, G.E. 1988. Origin, composition and flux of dissolved organic carbon in the Hubbard Brook valley New Hampshire, USA. Ecol. Monogr. 58: 177–196. Meredith, M.P., and Stehman, S.V. 1991. Repeated measures experiments in forestry: focus on analysis of response curves. Can. J. For. Res. 21: 957–965. Meyer, J.L. 1994. The microbial loop in flowing waters. Microb. Ecol. 28: 195–199. Meyer, J.L., Edwards, R.T., and Risley, R. 1987. Bacterial growth on DOC from a blackwater river. Microb. Ecol. 13: 13–29. Mulholland, P.J. 1997. Dissolved organic matter concentration and flux in streams. J. North Am. Benthol. Soc. 16: 131–141. Schiff, S.L., Aravena, R., Trumbore, S.E., Hinton, M.J., Elgood, R., and Dillon, P.J. 1997. Export of DOC from forested catchments on the Precambrian Shield of central Ontario: clues from 13 C and 14C. Biogeochemistry, 36: 43–65. Sobczak, W.V., Hedin, L.O., and Klug, M.J. 1998. Relationships between bacterial productivity and organic carbon at a soil–stream interface. Hydrobiologia, 386: 45–53. Staley, J.T., and Konopka, A. 1985. Measurement of in situ activities of nonphotosynthetic micro-organisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39: 321–346. Suberkropp, K.F. 1998. Microorganisms and organic matter decomposition. In River ecology and management: lessons from the Pacific Coastal Ecoregion. Edited by R.J. Naiman and R.E. Bilby. Springer-Verlag, New York. pp. 120–143.

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