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Applied and Environmental Microbiology, February 2005, p. 609-620, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.609-620.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Complexity of Bacterial Communities in a River-Floodplain System (Danube, Austria)

Katharina Besemer,1 Markus M. Moeseneder,2 Jesus M. Arrieta,2 Gerhard J. Herndl,2 and Peter Peduzzi1*

Division of Limnology, Institute of Ecology and Conservation Biology, University of Vienna, Vienna, Austria,1 Department of Biological Oceanography, Royal Netherlands Institute for Sea Research, Texel, The Netherlands2

Received 21 March 2004/ Accepted 8 September 2004


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ABSTRACT
 
Natural floodplains play an essential role in the processing and decomposition of organic matter and in the self-purification ability of rivers, largely due to the activity of bacteria. Knowledge about the composition of bacterial communities and its impact on organic-matter cycling is crucial for the understanding of ecological processes in river-floodplain systems. Particle-associated and free-living bacterial assemblages from the Danube River and various floodplain pools with different hydrological characteristics were investigated using terminal restriction fragment length polymorphism analysis. The particle-associated bacterial community exhibited a higher number of operational taxonomic units (OTUs) and was more heterogeneous in time and space than the free-living community. The temporal dynamics of the community structure were generally higher in isolated floodplain pools. The community structures of the river and the various floodplain pools, as well as those of the particle-associated and free-living bacteria, differed significantly. The compositional dynamics of the planktonic bacterial communities were related to changes in the algal biomass, temperature, and concentrations of organic and inorganic nutrients. The OTU richness of the free-living community was correlated with the concentration and origin of organic matter and the concentration of inorganic nutrients, while no correlation with the OTU richness of the particle-associated assemblage was found. Our results demonstrate the importance of the river-floodplain interactions and the influence of damming and regulation on the bacterial-community composition.


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INTRODUCTION
 
Almost all large rivers in Europe are greatly affected by pollution, alterations in the catchment, and hydraulic engineering (30). The greatest impact of damming and regulation on the connectivity, thermal heterogeneity, and biodiversity of a river system becomes visible in the lowland region of a river. Side arms, dead side arms, separated meander segments, and expansive floodplains, as they naturally appear in the lowland reach of a river, create a broad range of lotic, lentic, and semiaquatic habitats, leading to hot spots of biodiversity (37). Natural floodplains serve as sinks, sources, or transformers of dissolved and particulate organic matter, inorganic nutrients, and biota and thus contribute greatly to the self-purification ability of a river. The driving force of exchange processes between the river and its floodplains is hydrological connectivity, via both surface and groundwater inflow (36).

The National Park of the River Danube (Austria) is one of the last remnants of a natural floodplain and one of the largest in Europe. It is located downstream of Vienna and extends to the Slovakian border. Due to regulation of the Danube River and the construction of hydropower dams upstream of Vienna, large parts of the floodplains have been drained or disconnected from the river, and the hydrology and limnology of the whole river-floodplain system has been changed. The free-flowing section of the Danube River downstream of Vienna, as well as its floodplains, was designated a national park in 1996. As the national park status demands improvement of the ecological situation, restoration programs are being carried out in order to raise the water level in the floodplain sections and thus to increase the surface connectivity of the floodplains with the river and stop terrestrialization processes (30).

Bacteria are the most important consumers of the organic carbon entrained in running waters and thus play a significant role in the aquatic carbon cycle. They metabolize not only detritus arising from the death of higher organisms, but also organic wastes from excretions, photosynthetic extracellular release, or sloppy feeding (2). As heterotrophic bacteria are a valuable nutritional resource for higher organisms, this pathway, called the microbial loop, profoundly increases the productivity of the whole system (3). Recent studies of marine environments indicate the major importance of the taxonomic composition of the bacterial assemblage for enzymatic activity and the biochemistry of organic-matter cycling (26, 28). Until recently, inadequate techniques limited our knowledge of the bacterial-community composition in aquatic systems. Conventional culture-dependent methods are severely limited by the facts that most bacteria from natural environments seem to be uncultivable and that cultivation may alter the community structure (12). Molecular-fingerprint techniques, such as terminal restriction fragment length polymorphism (T-RFLP) analysis, have been shown to be sensitive and powerful tools for characterizing complex bacterial communities and for rapid comparison of community structures (19, 22).

The study presented here compared planktonic bacterial communities of the Danube River and floodplain pools with different hydrological characteristics over a period of 1 year, based on T-RFLP. The first major goal was to obtain insight into the spatial heterogeneity and temporal dynamics of the community composition. While the results of several investigations of the bacterial assemblages of marine and estuarine environments have been published (1, 6, 22), the communities in the different habitats of a river-floodplain system have received less attention. Similarity between the bacterial assemblages of the various bodies of water was expected to depend on hydrological connectivity and related changes in biochemical parameters. Furthermore, the seasonal dynamics of temperature and phytoplankton were assumed to influence the community composition.

The second major goal of our study was to compare the particle-associated and free-living bacterial communities. Particle surfaces are important sites of microbial activity and material transformation in rivers. Differences in the species richness, compositions, and metabolic activities of particle-associated versus free-living bacterial assemblages have been demonstrated in marine environments (1, 6, 9). Tockner et al. (36) reported that in a floodplain segment of the National Park of the River Danube, a considerable amount of biota and dissolved organic material is exported from the floodplain to the river even at mean water levels, while the major proportion of the total annual transport of particulate matter is restricted to short intense flood pulses. Thus, differences between the free-living and the particle-associated bacterial fractions should contribute considerably to our understanding of the composition and exchange processes of the biota, organic matter, and nutrients appearing in a river-floodplain system.


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MATERIALS AND METHODS
 
Study sites.
The investigation was conducted in the National Park zone of the Danube River. Samples were taken from representative sites within the main river and four floodplain pools with different hydrological characteristics. The sampling site for the Danube River (sampling site D1) was located near the town of Haslau. As an example of a frequently connected floodplain pool, the downstream end of a former river channel near the town of Regelsbrunn (Regelsbrunn Traverse; site R2) was chosen. This backwater system was artificially cut off from the riverine channel network at its upstream end >100 years ago but still remained to some extent dynamically interlinked with the main channel. In 1997, a restoration project took place at this site, which led to an additional increase in surface connectivity with the river (connection 0.5 m below mean water) and consequently to lotic conditions in this floodplain area for >180 days of a hydrological average year (13, 30). The other three floodplain pool sampling sites, L3, L4, and L5, were located in a former river channel in the Lobau area within the district of Vienna. This water body is widely separated from the Danube River by a series of embankments with only one narrow downstream connection still existing. The channel is subdivided by weirs, and terrestrialization processes are far advanced. The potential for restoration of hydrological connectivity is low due to human requirements for flood control (30). Sampling point L3 was located at the downstream end of the channel, a water body that is connected to the river ~30 cm below mean water. Point L4 was located just below a weir (Gaenshaufentraverse). This site is separated from the main river downstream by another weir, the Schoenauer Traverse, and connected to the Danube at a water level of ~0.5 m above mean water. Sampling point L5 was located upstream of the weir Gaenshaufentraverse. This backwater is connected to the river at 1.7 m above mean water; thus, hydrological connectivity to the river is established solely via groundwater inflow and during high-flood events. Water level recordings of the Danube were taken from the hydrograph at Orth provided by the Federal Waterway Agency.

Sample collection.
Samples from sites D1, R2, and L5 were taken on 17 dates from June 2000 to June 2001. From March to June 2001, sites L3 and L4 were sampled in addition (on eight dates). Samples were collected at 0 to 0.5 m depths at intervals of 7 to 21 days, depending on the changes of water level, with a longer interruption from December 2000 to March 2001. Due to methodological constraints, D1-R2 and L3-L4-L5 were not sampled on the same dates but within 2 days. Water samples were collected in acid-rinsed polyethylene bottles or high-temperature-treated glass bottles. Samples were brought to the laboratory within 2 h and were processed immediately.

Water analysis.
The water temperature at the sampling sites was recorded in the field. Depending on the respective seston contents, 300 to 1,500 ml of water was filtered through preashed glass fiber filters (Millipore APF/F filters). Suspended-solid (SS) and organic-suspended-solid (OSS) concentrations were measured by dry weight and ash-free dry weight determinations. The organic content of SS was calculated as the ratio of OSS to SS (in percent). To estimate the concentration of chlorophyll (Chl) a, 350 to 900 ml of water was filtered through APF/C filters (Millipore). Chl a was measured by the protocol of Talling and Driver (34). APF/F-prefiltered water was used to analyze the concentration and selected optical properties of dissolved organic material. Dissolved organic carbon (DOC) concentrations were measured using the high-temperature combustion method with a Shimadzu TOC 5000 C analyzer according to the method of Benner and Strom (5). The optical properties (fluorescence index and ratio of absorption) of the water samples were investigated to gain information on the origin and molecular size distribution of the dissolved organic material. Fluorescence at an excitation wavelength of 370 nm was determined in a Shimadzu RF-1501 spectrofluorometer, and the ratio of the fluorescence intensity at 450 nm to that at 500 nm (FI) was calculated. An FI of ~1.2 corresponds to allochthonous terrestrial organic matter, and an index of ~1.9 corresponds to autochthonous microbially derived organic matter (20). Absorption of the water samples under UV light was measured in a Hitachi U-2000 spectrophotometer, and the ratio of absorption at 250 to absorption at 365 nm (RA) was determined (18). Small molecules have been suggested to absorb at shorter wavelengths than large molecules (33). Analyses of nitrate (NO3), nitrite (NO2), ammonium (NH4), soluble and total Kjeldahl nitrogen, orthophosphate (PO4), and soluble and total phosphorus were carried out using standard methods (32). Concentrations of dissolved organic nitrogen (DON), particulate organic nitrogen (PON), dissolved organic phosphorus (DOP), and particulate phosphorus (PP) were calculated by using the following equations: DON = soluble Kjeldahl nitrogen – NH4; PON = total Kjeldahl nitrogen – soluble Kjeldahl nitrogen; DOP = soluble phosphorus – PO4; PP = total phosphorus – soluble phosphorus.

Bacterial abundance.
For total abundance of bacteria, 1 to 3 ml of formaldehyde-fixed sample was stained with 4',6-diamidino-2'-phenylindole-dihydrochloride, and bacteria were counted in 30 random selected fields under an epifluorescence microscope (Nikon E800) according to the method of Porter and Feig (27).

Concentration of bacteria and extraction of DNA.
Water was prefiltered through a net with a 20-µm mesh size. To concentrate the particle-associated bacterial community, 150 to 1,300 ml of water was filtered through a 3.0-µm-pore-size filter (Millipore TSTP). This prefiltered water was then filtered through a 0.22-µm-pore-size filter (Millipore GSWP) to obtain the free-living bacterial assemblage. The DNA of the bacterial community was extracted and purified with the UltraCleanTM Soil DNA Isolation kit from Mo Bio (Carlsbad, Calif.), following the manufacturer's recommendations. To improve the efficiency of extraction from the filters, they were cut into small pieces using ethanol-flamed tweezers and scissors. Empty filters served as negative controls. DNA concentrations were determined using a Bio-Rad SmartSpec 3000 spectrophotometer according to the manufacturer's instructions and ranged from 17 to 85 µg/ml.

PCR.
The primers used for PCR were 27F-FAM and 1492R-JOE, which give a 1,503-bp product of the 16S rRNA genes (17). 27F-FAM was 5'-labeled with phosphoramidite fluorochrome 5-carboxy-fluorescein by Eurogentec (Searing, Belgium). Each 50-µl PCR mixture contained 1 µl of sample, 12.5 pM both primers, 12.5 nM (each) deoxynucleoside triphosphate, 2.5 U of Taq polymerase, and the recommended PCR buffer (all from Amersham Pharmacia). Samples were amplified according to the protocol used by Moeseneder et al. (22). The PCR mixtures were further processed following the protocol of Moeseneder et al. (21). Then, the PCR mixtures were dissolved in 5 µl of agarose gel loading buffer (Perkin-Elmer Applied Biosystems; included with the TAMRA 2500 size marker) after precipitation and washing and were submitted to electrophoresis in a 1% agarose gel at 75 V for 50 min. The PCR products were visualized with a UV transilluminator, and bands were excised and purified with the QIAquick Gel Extraction kit (QIAGEN). The fluorescently labeled primers were removed from the PCR products most efficiently by this gel electrophoresis step (21).

Restriction digestion.
Fluorescently labeled PCR products were digested with the restriction enzyme HhaI. This enzyme has been shown to be suitable for T-RFLP analysis (19). Each 100-µl restriction digest mixture contained 10 U of enzyme and the recommended buffer (both from Amersham Pharmacia). Samples were digested at 37°C for 6 h. The restriction digest reaction mixtures were precipitated, washed, and resuspended in 2 µl of ultrapure water (Sigma), as described by Moeseneder et al. (21).

T-RFLP analysis: separation and detection of restriction fragments.
TAMRA 2500 size marker (0.5 µl; Perkin Elmer) was added to the samples, and DNA fragments were denatured in the presence of 7.5 µl of deionized formamide at 94°C for 3 min. DNA fragments were separated and detected with a Prism 310 capillary sequencer (Perkin-Elmer Applied Biosystems) run under GeneScan mode, as described previously (22). Samples were injected electrokinetically at 15 kV for 5 s and run at 15 kV for 45 min. Fluorescently labeled fragments were detected by laser-induced fluorescence detection using the virtual filter set A of the 310 acquisition software.

Each DNA extract was submitted to PCR and restriction digestion twice, and each restriction digest mixture was injected and separated twice to check for mistakes that might occur during PCR or injection. No differences between replicates could be observed. The possibility of preferential amplification of sequences cannot be excluded, but as we analyzed T-RFLP patterns, not quantitatively, this should not influence the interpretation of our data. Despite the high resolution of the T-RFLP analysis, two different species might have the same restriction site in the 16S rRNA genes and thus produce identical peaks (19). On the other hand, one species can have more than one copy of the 16S rRNA genes (11) and therefore produce more than one peak. Thus, the term operational taxonomic unit (OTU) is used instead of species.

Size determination and transformation in a binary matrix.
The sizes of the fluorescently labeled fragments were determined by comparison with the internal TAMRA 2500 size standards using the local southern size calling method of GeneScan version 3.1. Peak drifts for larger-size standard fragments can cause artifacts (22); therefore, all peaks were checked manually for correct size. Peak profiles were then transformed into a binary matrix according to the presence (represented by 1) or absence (represented by 0) of a peak. This matrix was used for further analyses.

Statistical analyses.
The software package SPSS for Windows version 10.0 was used for principal-component analysis (PCA) of environmental variables and calculation of significant differences between groups of values and correlation coefficients (parametric and nonparametric procedures, after testing for normal distribution and homogeneity of variances). The similarity of samples was determined by evaluating Jaccard coefficients [(number of OTUs shared/number of OTUs in sample A or sample B) x 100]. We used the software package Canoco for Windows version 4.0 (35) to compare OTUs, according to the environmental conditions under which they appear, and bacterial-community compositions. A correspondence analysis (CA) that assumes a unimodal response of OTUs on environmental conditions was performed. Multivariate statistical methods, such as CA and PCA, which summarize data in scatter plots and relate patterns to explaining variables, are now commonly used to deal with large data sets (8, 23, 29).


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RESULTS
 
Characterization of abiotic and biotic conditions.
From June to October 2000. the water level of the Danube River fluctuated around mean water (145.68 m above sea level), with a short spate at the beginning of August. From October 2000 to March 2001, it remained below mean water level most of the time (lowest water level, 144.20 m above sea level in January). After a high-flood event in March 2001 (highest water level, 148.32 m above sea level), the water level remained constantly above the mean water level, with another smaller spate in June 2001. The floodplain pool's extent of connection to the river was expressed by subtracting the water level at which surface connectivity of the respective floodplain pools to the river was established from the actual water level.

A PCA was performed on the biochemical parameters to get an overview of the biotic and abiotic conditions in the investigated water bodies and to relate observed patterns with changes in the composition of the bacterial communities. We restricted this analysis to those environmental variables that correlated strongly with the three most relevant components (Table 1). Component 1 was positively correlated with inorganic nutrients (NO3, NO2, NH4, and PO4), component 2 represented particulate organic material (OSS, PON, and PP) and algae, and component 3 was determined by dissolved organic material (DOC and DON). Samples were separated according to component 1, depending on the influence of river water. D1 had significantly higher values than L4 and L5 (analysis of variance [ANOVA], P < 0.001; Scheffe's test, P < 0.01); R2 had significantly higher values than L5 (ANOVA, P < 0.001; Scheffe's test, P < 0.05). The highest values at all sampling sites were observed during the high spate in March 2001. No difference between sampling sites was observed with respect to component 2. For the third component, L5 was clearly separated from the other sampling sites; the differences between L5 and sites D1, R2, and L4 were significant (ANOVA, P < 0.001; Scheffe's test, P < 0.001). The samples taken during the flood event in spring 2001 had higher values than the other samples in D1, R2, and L3, but not in L4 and L5 (Fig. 1).


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  		TABLE 1. PCA: explained variation in percent and correlations of environmental variables with the first three componentsa



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  		FIG. 1. Principal-component analysis ordination plot of environmental variables (components 1, representing inorganic nutrients, and 3, representing dissolved organic material). Samples are separated according to their sampling sites. Samples marked by arrows were taken during the flood event in March 2001.

In accordance with the PCA, inorganic nutrients (NO3, NO2, NH4, and PO4) were positively correlated and dissolved-organic-matter parameters (DOC, DON, and DOP) were negatively correlated with the floodplain's extent of connection to the river (data not shown). For particulate matter, SS, OSS, and PP were positively correlated with the extent of connection. The OSS/SS ratio was negatively correlated with the extent of connection. PON showed no correlation to hydrology but to Chl a, indicating that a substantial part of it was contributed by algal material. The FI varied between 1.20 and 1.59, and the RA varied between 3.79 and 7.92. Temperatures in the river and in the floodplains ranged from ~5°C in December to ~25°C in August. The temperature in the floodplain pools was usually some degrees higher than in the river (data not shown). Inorganic nutrients (NO3, NO2, and PO4) were negatively correlated with the temperature.

Numbers of OTUs and similarity between samples.
Since the different sampling periods of the water bodies limited the comparability of L3 and L4 with the other sampling sites, the samples from the year 2001 were also analyzed separately. However, the calculated mean values were very similar, and the results and conclusions were not altered by this reduction of data. Therefore, only the results obtained by analyzing all samples are presented below.

The mean numbers of OTUs per sample in the sampling sites ranged from 28 to 35; however, most differences between the mean values were not significant. Comparing all samples from the respective communities, the mean number of OTUs per sample was significantly higher in the particle-associated (35 OTUs) than in the free-living (29 OTUs) fraction (Student's t test; P < 0.001). The relative percentages of the particle-associated and the free-living fractions differed among the sampling sites. At D1, L3, and L4, the complexity of the particulate fraction was only slightly higher than that of the free-living fraction, while the difference at R2 and L5 was significant (Student's t test; P < 0.05), with 26 and 40% more particle-associated OTUs, respectively (Fig. 2).



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  		FIG. 2. Mean numbers and standard deviations of free-living and particle-associated OTUs in the investigated water bodies. In R2 and L5, the differences between the two fractions are significant (Student's t test; P = 0.019 and 0.001, respectively).

At all sampling sites, the absolute number of OTUs detected was higher in the particle-associated bacterial fraction than in the free-living fraction. Comparing all samples, 84 out of 124 OTUs detected were found in both fractions, 38 were found only in the particulate fraction, and 2 were found only in the free-living fraction (Fig. 3). Thirteen OTUs were restricted to the water body R2, and another 13 were restricted to L5, whereas only one OTU occurred only in D1 and none occurred only in L3 or L4. Nine of the R2-restricted OTUs and all 13 L5-restricted OTUs were only found associated with particles.



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  		FIG. 3. Absolute number of OTUs found. Notice that about one-third of the OTUs occurred only in the particulate fraction, whereas hardly any were detected only free living.

The mean similarity of the samples from one site and habitat (free or on particles) was estimated from the Jaccard coefficients, derived by comparison of the samples from all dates. Thus, mean similarity values included only temporal heterogeneity, both between consecutive dates and over the whole sampling period. The mean similarities among the samples of the free-living communities were significantly higher than those among the samples of the particle-associated communities in all water bodies, with the exception of the samples from L3 (Student's t test; P < 0.01). The mean Jaccard coefficients of the free-living and, less definitely, of the particle-associated assemblages decreased with increasing distance from the river. The mean values and significant differences among the sampling sites are summarized in Table 2.


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  		TABLE 2. Mean Jaccard coefficients comparing particle-associated and free-living communities from each site

Correspondence analysis.
A CA was performed, with downweighting of rare OTUs and biplot scaling focused on intersample distances. OTUs that occurred in less than five samples were excluded from calculation and added as passive OTUs to the resulting ordination diagrams. The cumulative percentage of variance of OTU data, as well as the cumulative percentage of explainable variance of OTU-environment relationships of the first three CA axes, is given in Table 3. The correlation matrix between CA axes and environmental variables, produced by the Canoco software, is given in Table 4. Only correlations which exceed 0.2 will be discussed for all environmental parameters, except for the FI, which showed no correlation coefficient of >0.2 with any of the three axes. The first CA axis showed a lower relation to environmental variables than both the second and the third CA axes. It was positively correlated with Chl a, PON, and the FI, among which Chl a and the FI showed higher correlation coefficients with the first axis than with the second and third. The second axis was correlated positively with temperature, DOP, and the OSS/SS ratio and negatively with PP, OSS, and inorganic nutrient parameters. The third CA axis showed positive correlations with DOC, DON, RA, the OSS/SS ratio, and PON and negative correlations with SS and inorganic nutrients.


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  		TABLE 3. CA: cumulative percentage of variance of OTU data and of explainable OTU-environment correlation explained by first three axes


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  		TABLE 4. Correlations of environmental variables with CA axesa

Samples representing community compositions and their relationship to environmental variables were plotted into graphs with the calculated CA axes. The samples were marked according to their origins and habitats (free living or on particles). The resulting graphs demonstrated that particle-associated and free-living bacterial-community compositions formed two clearly separated, though overlapping, clusters along axis 1 (Fig. 4). When the samples' coordinates along axis 1 were compared, the particle-associated communities had significantly higher values than the free-living ones (Table 5). As the two bacterial fractions investigated originated from the same water samples, a separation of samples along environmental gradients, which correlated with the first axis (i.e., Chl a), obviously occurred within the two clusters.



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  		FIG. 4. CA ordination plot of samples (axes 1 and 3). The arrows indicate correlation coefficients between environmental variables and the given ordination axes. Note the different scaling of the ordination plot and the arrows. temp., temperature.


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  		TABLE 5. Significant differences between values of CA axes of different groups of samples

Samples were separated along axes 2 and 3 according to the water bodies from which they originated (Fig. 5). Significant differences along the second CA axis existed between the values of L5 samples and those of D1, L3, and L4 samples, as well as between the coordinates of R2 and L4 samples. At the third CA axis, community compositions from D1 and R2 had significantly lower values than those from L3, L4, and L5 (Table 5). The environmental variables representing organic matter pointed in the direction where the L5 samples were found, while the inorganic-nutrient variables pointed to the cluster of D1 and R2 samples. However, the separation of samples according to their origins was overlapped by a separation along temperature, correlated with the second axis.



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  		FIG. 5. CA ordination plot of samples (axes 2 and 3). Samples are separated according to their sampling sites. The arrows indicate correlation coefficients between environmental variables and the given ordination axes. Note the different scaling of the ordination plot and the arrows. temp., temperature.

OTUs were plotted in graphs according to their appearance to investigate differences between particular groups of OTUs and their relation to explaining variables. We compared the OTUs that appeared only on particles with those that appeared both free and on particle surfaces (Table 6). OTUs which were only found free living were included in the latter group because of their small numbers. OTUs restricted to particle surfaces were plotted in the area to which arrows representing organic matter variables pointed and separated significantly from the others along the first two axes (Fig. 6).


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  		TABLE 6. Significant differences between values of CA axes of different groups of OTUs



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  		FIG. 6. CA ordination plot of OTUs (axes 1 and 2). The white circles include OTUs which were found only free living (not separately marked because of small numbers [2 OTUs; see the text]). The arrows indicate correlation coefficients between environmental variables and the given ordination axes. Note the different scaling of the ordination plot and the arrows. temp., temperature.

OTUs that appeared only in R2 or L5 were marked and compared with all OTUs that appeared in more than one water body (including the one OTU which appeared only in D1). L5- and R2-associated OTUs were plotted separately from the other OTUs, as well as from each other (Table 6). The coordinates of L5-associated OTUs had higher values than those of the unrestricted OTUs along the first and second axes and higher than the R2-associated OTUs along the third axis. OTUs from R2 showed higher values than unrestricted OTUs along axis 2 and lower values along axis 3. The distribution of OTUs according to the first two axes is shown in Fig. 7.



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  		FIG. 7. CA ordination plot of OTUs (axes 1 and 2). The black diamonds include OTUs that occurred only in D1 (not separately marked because of small number [1 OTU; see the text]). The numbers below the circles indicate that two or three OTUs, as indicated, were plotted at the same coordinates. The arrows indicate correlation coefficients between environmental variables and the given ordination axes. Note the different scaling of the ordination plot and the arrows. temp., temperature.

Correlations of OTU richness with biochemical parameters.
Correlation coefficients between the number of OTUs and biochemical data were calculated. The number of free-living OTUs correlated positively with soluble inorganic nutrients (PO4, NO3, NO2, and NH4) and with the FI, whereas it correlated negatively with DOC and DON (Table 7). A negative correlation was found between the number of free-living OTUs and bacterial abundance. No significant correlations were found for the number of particle-associated OTUs.


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  		TABLE 7. Correlations of number of free-living OTUs with environmental variables and bacterial abundance


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DISCUSSION
 
Characterization of hydrological and biochemical conditions.
Hydrological connectivity proved to be the determining factor for the biochemical conditions in the backwaters. The influx of water from the Danube River resulted in high levels of inorganic nutrients, and separation from the river resulted in increased levels of dissolved organic matter. It had been reported that the Danube supplies inorganic nutrients and suspended solids to the floodplains and that the floodplain pools, on the other hand, serve as a source of organic matter for the river (15, 36). In this study, the significance of flood events for the biochemical conditions in the river and its floodplains was evident from the PCA coordinates of the samples taken during the spate in spring 2001. The Danube carried high concentrations of inorganic nutrients into all water bodies, and simultaneously, in the river and the frequently connected floodplain pools, the amount of dissolved organic matter rose, probably due to an input from the previously disconnected pools and to an increased terrestrial inflow. In addition to hydrology, seasonal changes influenced the concentrations of inorganic nutrients. Concentrations were higher during the cold season, when decomposition exceeded production and inflow of organic matter. As indicated by the FI, the source of DOC was predominantly allochthonous; autochthonous production was of minor importance. This agrees with the results of Kirschner and Velimirov (16), who reported that for the major part of the year, the bacterial-plankton community of a water body of the Lobau backwater was dependent on carbon sources other than phytoplankton production.

Total suspended solids, as well as organic suspended solids, decreased with decreasing connectivity to the river. However, the organic percentage of the particulate matter rose, most probably because of sedimentation of inorganic particles under lentic conditions and autochthonous production of particulate organic matter (36). Particulate phosphorus was also positively correlated with the extent of connection to the river; however, it is quite possible that it consisted of phosphate adsorbed to inorganic particles rather than of organic matter. Particulate organic nitrogen showed no relation to hydrology, nor did Chla. Algal blooms in floodplain pools have been shown to depend on nutrient supply by floods and on grazing pressure (13, 15).

OTU richness and temporal dynamics.
The most pronounced differences in OTU richness, as well as in temporal dynamics, were found between the free-living and the particle-associated communities. The relative proportions of free-living and particle-associated bacteria differed considerably between the river and the various floodplain pools. In R2 and L5, the particle-associated fraction exhibited a significantly higher OTU richness than the free-living fraction, which was not the case in D1, L3, and L4. The higher absolute number of OTUs in these two water bodies was also due to a higher number of particle-associated OTUs, while the numbers of free-living OTUs were rather similar for all sites. Assuming a relationship between bacterial taxonomic composition and cycling of organic matter, this might lead to differences in the transformation and decomposition processes on particle surfaces in R2 and L5 compared to the other sampling sites. It was found that R2 and L5 also differed from the other water bodies in that a significant part of all detected OTUs were restricted to each of these two sites. The importance of the particle-associated OTUs for the OTU richness in these two sampling sites is underlined by the fact that most of the R2- and all of the L5-restricted bacteria were found only on particles. The high proportion of particle-associated bacteria among the site-restricted bacteria also indicates that they are less homogeneously distributed in a floodplain system than are the free-living bacteria.

Differences between particle-associated and free-living assemblages were described previously. Similar free-living bacterial-community compositions were identified in the Columbia River, its estuary, and the adjacent coastal ocean, while a uniquely adapted estuarine community existed attached to particles (9). In marine and estuarine environments, higher species richness and greater spatial heterogeneity were found for the free-living bacterial community in several studies; however, specialization for the microenvironment offered by particle surfaces was also reported for marine bacteria (1, 6, 22). The higher percentage and variability of the particle-associated bacterial community in limnetic environments can be explained by the differences in the concentration and quality of particulate matter. A lowland river and its floodplains receive large amounts of allochthonously derived organic matter, which is rapidly transformed and decomposed within the river, leading to diverse microhabitats on particles. In several studies, a very small percentage of the total organic matter was found to be particulate in marine environments (4, 22).

The temporal dynamics of the bacterial assemblages were clearly dependent on the influence of the river (Table 2). As for the bacterial-community composition, the Danube River represented a rather stable system; the seasonal changes were small compared with those of the communities in the floodplains. When surface connectivity was established, this bacterial community was washed into the floodplain pools, while the backwater's bacterial assemblages were washed out or mixed with the Danube's bacteria. Thus, the frequently connected floodplain pools were strongly influenced by the river's bacterial assemblage, and the community composition probably fluctuated around an average composition. With decreasing influence of the river, seasonal dynamics became more important, leading to the lower mean similarities between the sampling dates of a given disconnected floodplain pool. Seasonal changes in bacterial compositions have previously been reported for lakes (10, 25). The importance of seasonal changes was also evident from the strong correlation of temperature with the second CA axis. In addition, occasional high-flood events may alter the composition of the bacterial community of an isolated water body to a higher degree than that of a frequently connected water body, leading to lower similarity indices between consecutive dates in the former. It has been reported that chemical conditions were more severely influenced by spates in isolated water bodies than in frequently connected floodplain pools of the Danube (15).

The particle-associated bacterial assemblage showed higher temporal dynamics than the free-living community in all water bodies investigated. The particle-associated bacterial fraction was probably more strongly influenced by hydrological and seasonal changes, such as inflow of terrestrial material during flood events, leaf fall, or algal blooms. All of these would eventually also influence the free-living bacteria; however, the fraction of bacteria that is restricted to the microenvironment offered by particle surfaces might be more dependent on the organic matter entrained in the river-floodplain system than the group which can live on particles as well as free. Another reason could be an inflow of bacteria attached to particles from the terrestrial environment during periods of high water levels, which might survive in the aquatic environment for some time.

Community composition.
The CA revealed that the greatest differences occurred between the particle-associated and the free-living communities, as was also shown by the comparison of OTU richness. The clear separation of the two groups of samples along the first CA axis explains the low percentage of OTU-environment relationship, because every pair of free-living and particle-associated communities was taken from the same water sample and thus under the same biochemical conditions. The environmental parameters that the first CA axis was correlated to were parameters related to phytoplankton. The algal biomass was represented by Chl a and PON. The positive correlation with the FI indicated a higher percentage of autochthonously produced carbon, originating from algal exudates. A separation along these factors obviously occurred within both clusters of samples; however, the particle-associated communities were distributed over a larger area and might thus be more strongly influenced by algal blooms.

Particle-restricted OTUs seemed to have their optimum under environmental conditions that were in some respects extreme and differed in some way from the conditions preferred by most unrestricted bacteria (Fig. 6). Along axis 1, the particle-restricted OTUs were plotted in an area with high values for alga-related parameters and thus obviously appeared mainly at times of algal blooms. This underlines the assumption that the particle-associated bacterial community was more strongly influenced by phytoplankton dynamics than the free-living community. L5-restricted OTUs differed from the unrestricted OTUs along the first axis, which was not the case for R2-restricted bacteria. Thus, phytoplankton dynamics could have some influence on the L5-restricted OTUs but not on the R2-restricted OTUs. We conclude that in the rather isolated L5 pool, interactions between groups of organisms led to a biologically controlled system, while in the hydrologically controlled R2 system, frequent disturbances prevented a strong coupling between plankton compartments and the establishment of seasonal cycles. Correlations between phytoplankton dynamics and bacterial composition have been reported previously (10, 38). Riemann et al. (28) concluded that bacteria specialized in particle colonization proliferated during the death phase of a diatom bloom.

Along the second and the third CA axes, samples were separated according to their origins. However, hydrology was obviously not the determining factor, because the observed separation of samples resembled the pattern observed for OTU richness, in which R2 and L5 varied from the other sampling sites. The environmental variable that correlated most strongly with the second axis was temperature. This indicates a strong seasonality of the bacterial community rather than explaining the detected separation of the sampling sites, since the variations in temperature between the different water bodies were small compared with the seasonal changes. The several organic and inorganic variables, which were strongly correlated with the second CA axis, were all related to hydrology; however, inorganic nutrients were also related to temperature, so the calculated gradients might reflect seasonal rather than hydrological changes. The higher percentages of particle-associated communities in R2 and L5, and the groups of OTUs that were found only in these two water bodies, might drive the separation of their community compositions from the other bacterial assemblages. This was also indicated by the separation of the particle- or site-restricted OTUs from the unrestricted OTUs along axis 2. The appearance of these OTUs might have been favored by high temperatures and related seasonal changes in biochemical variables.

The separation of samples along axis 3 and its correlation with environmental parameters was clearly determined by hydrology. The Danube River (D1) and the frequently connected backwater at Regelsbrunn (R2) were found on the negative side of the axis, while the three Lobau sites (L3, L4, and L5) plotted on the positive side. Environmental variables pointing to D1 and R2 were parameters characteristic of the Danube and its influence on backwaters. The organic-matter variables, correlated positively with axis 3, also fit well with the observed biochemical conditions in the water bodies. Concentrations of DOC, DON, and OSS-SS increased with decreasing influence of the river. The arrow representing RA indicated a higher percentage of small molecules among the organic matter in disconnected pools. This low-molecular-weight organic matter might include refractory humic material, but also compounds easily degradable by microorganisms, like sugars. The correlation of PON to the third axis indicates a relationship of phytoplankton dynamics with this hydrology-dependent axis.

The R2-restricted OTUs were found on the inorganic-nutrient-determined side of the third CA axis. This indicates that these OTUs were found mainly in situations of high connectivity to the river, which are common in this frequently connected floodplain pool. We conclude that hydrological connectivity influenced the structures of the bacterial assemblages by determining the concentrations of organic and inorganic nutrients in the water bodies and by mixing communities. The combined effects of succession of bacterial communities, due to changed environmental conditions and inflow of allochthonous bacteria, have been reported for lakes (10) and estuaries (7).

OTU richness and bacterial abundance.
The correlations of the number of free-living OTUs with bacterial abundance, organic matter, and inorganic nutrients revealed two possible situations for the free-living bacterial community. In situations where there were high loads of organic matter and low concentrations of inorganic nutrients, and thus, in situations with low river influence, bacteria could reach high densities, but only strong competitors among the free-living bacteria would survive, leading to a low OTU richness. Situations in which there were high concentrations of inorganic nutrients and small amounts of organic matter, on the other hand, as found in the Danube and during periods of high river influence, would limit the bacterial abundance but allow a higher OTU richness, perhaps because of the survival of weak competitors. Species able to use inorganic nutrients as sources of nitrogen and phosphorus were probably favored under these conditions. The facts that the correlation coefficients were rather low and that the bacterial abundance refers to all bacteria, free and particle associated, certainly must be taken into account. The situations outlined have to be considered extremes. The positive correlation of the number of free-living OTUs with the FI indicates that high autochthonous productivity allowed the growth of bacteria that were not present in the bacterial community when mainly terrestrially derived carbon was available (10, 28, 38). The absence of significant correlations with the particle-associated bacteria could be explained by a higher dependence on the quality of particles, which was not further investigated, rather than on the overall trophic conditions.

Conclusions.
The most remarkable differences were found between the particle-associated and the free-living communities, with respect to community composition, OTU richness, and temporal dynamics. We conclude that the particle-associated community was more heterogeneous and more dependent on changes in the environmental conditions, such as algal blooms or inflow of terrestrial organic matter, than the free-living bacterial community.

The clear separation of the samples according to their origins indicated a significant influence of damming and regulation on the floodplains of a river even at the microbial level. Hydrology-dependent concentrations of organic matter and inorganic nutrients, phytoplankton dynamics, and seasonal changes were important but were not the only determinants of the bacterial composition. In the frequently connected floodplain pool, R2, and the most separated floodplain pool, L5, site-restricted and particle-associated bacteria constituted a major part of the community, which was not the case in the other water bodies. The reasons for this may be connectivity of the water bodies within the two backwaters. In the disconnected floodplain pool L5, a particular bacterial community could develop due to river-independent biocoenoses and isolation (30). As a consequence of the weirs within the Lobau channel, the bacterial communities are exchanged between L5 and the other Lobau pools only during periods of high water levels. The side channel at the sampling site R2, on the other hand, is connected to the whole related floodplain segment with its various water bodies for the better part of the year (30). Part of the R2-restricted bacteria could thus originate from bacterial communities of more separated floodplain pools within the Regelsbrunn channel network that were washed into the main channel of the side arm. In general, another controlling factor might have been grazing by protozoa or viral lysis (24, 31). Significant transport of the specified particle-associated bacterial communities to the river will occur in situations of high connectivity, and thus, in L5 only during high spates, while the free-living bacteria may also be exchanged during periods in which there is an intermediate extent of connection, as shown for dissolved and particulate organic matter (14, 36).

For eukaryotic organisms, it is well documented that severe river regulation results in a loss of biodiversity and a decline in and endangerment of the original biota (30). In this study, we were able to demonstrate that even the bacterial community composition in a river-floodplain system is affected by changes in the complex mixture of habitat properties and hydrology-related parameters. The ecological consequences of these findings have to be investigated in more detail in light of the functional role of bacteria in the cycling of energy and material.


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ACKNOWLEDGMENTS
 
We thank our colleagues from the Department of Limnology (University of Vienna) and the Department of Biological Oceanography (Netherlands Institute of Sea Research), especially C. Winter, J. Schelest, B. Luef, T. Hein, S. Glaser, I. Kolar, I. Zweimüller, W. Reckendorfer, C. Barany, and H. Kraill, for their help and support.

This work was supported by grants from the National Park Authority (grant no. Life98Nat/A/005422), the Hochschuljubiläumsstiftung der Stadt Wien (grant no. H57/2000), and the Austrian Science Fund (FWFgrant no. 14721-BOT) to P. Peduzzi.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Limnology, Institute of Ecology and Conservation Biology, Althanstr. 14, A-1090 Vienna, Austria. Phone: 43-1-4277-54342. Fax: 43-1-4277-9542. E-mail: peter.peduzzi{at}univie.ac.at. Back


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Applied and Environmental Microbiology, February 2005, p. 609-620, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.609-620.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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