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Applied and Environmental Microbiology, April 2004, p. 2072-2078, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2072-2078.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effect of Trophic Status on the Culturability and Activity of Bacteria from a Range of Lakes in the English Lake District

Jonathan Porter,^* Samantha A. Morris, and Roger W. Pickup

Centre for Ecology and Hydrology, Windermere Laboratory, The Ferry House, Far Sawrey, Ambleside, Cumbria LA22 0LP, United Kingdom

Received 14 July 2003/ Accepted 2 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The lakes in the English Lake District are classified according to their size, catchment, and trophic status, and they range from oligotrophic to eutrophic (11, 22, 36). Pearsall's original series arranged many of the major lakes in a series according to a wide range of criteria, which generally reflects biological production (22). Other lakes have been fitted into the Pearsall series, with Priest Pot (PP) being the most recent addition and the most productive (hypereutrophic) water body of the English Lake District (8). The relationship between lake trophic status, microbial numbers, biomass, and activity has been studied with respect to the benthic bacterial communities (11). That work suggested that microbiologically, the benthic communities of the more productive lakes could be considered to be similar but that they were different from those of the lakes of lower nutrient status (11). Freshwater bacterial assemblages often show between-site physiological differences, which may arise from differences in community composition or from differential responses of similar communities to environmental conditions. Between-site variability in bacterioplankton activity has been suggested to be a function of differences in community compositions rather than a conditional response by the same community (14). However, few data exist on whether the overall activity of bacterial communities in lakes is affected by trophic status. In one shallow eutrophic lake (Zwischenahner Meer, Germany), approximately 8% of the total cells reduced 5-cyano-2,3-ditoyltetrazolium chloride (CTC) and culturability determined by the most probable number method never exceeded 7%, although approximately 58% of cells were shown to divide on a membrane incubated on the most probable number test medium (3). Thus, conclusions concerning viability and/or activity may vary with the method of assessment (12). Activity is affected by seasonal change (7) and species composition (31). However, no direct comparison between lakes that differ in nutrient status has been made.

Molecular techniques, although rarely giving an insight into the physiological status of individual cells, have increased the reliability of detecting bacterial populations, species, or specific organisms. However, an assessment of their viability remains problematic (12, 26). Posch et al. (30) posed two questions which exemplify the goal of any ecological bacterial-activity assessment: (i) what is the total activity of the bacterial community and (ii) which members make a significant contribution to the overall activity? The former can be approached by taking gross activity measurements which can be enhanced if the in situ conditions are maintained during sampling and subsequent measurements (10, 23). Assigning activity to individual cells can be achieved with a range of dyes (3, 25), but each dye presents its own unique interpretational challenge (12, 26). Nucleic acid-specific fluorescent dyes, used in conjunction with flow cytometry cell sorting and subsequent leucine incorporation assays, have shown that bacteria with high apparent nucleic acid contents may be the most active and that activity can vary by a factor of 7 (16). Further work using a similar approach showed that the growth rate (activity) of the larger cells (>0.25-µm³ biovolume) was higher than that of the smaller-volume cells in seawater samples, demonstrating that cell size is an issue (34). This issue has also been raised for soil bacteria (2).

This study utilized the series of lakes in the English Lake District, including the hypereutrophic PP, to assess whether the prevailing lake conditions influenced culturability and activity among different size classes of bacteria.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sampling.
Water samples were taken above the deepest point of the following lakes in the English lake District, each representing decreasing degrees of eutrophication as described by Pearsall (22): PP (grid reference [GR] NY 357978; hypereutrophic), Windermere South Basin (WSB; GR NY 383904; eutrophic), Esthwaite Water (EW; GR NY 363957; eutrophic), Windermere North Basin (WNB; GR NY 381015; mesotrophic or oligotrophic), and Wastwater (WW; GR NY 165065; oligotrophic). Samples of up to 100-liter volumes were pumped from a 2.5-m depth and collected in 25-liter containers, except at PP, where samples were collected from a 1-m depth. Particles that were >0.22 µm in diameter were concentrated on site by tangential flow filtration (TFF) with a Pellicon tangential flow filtration unit (Millipore Ltd., Watford, United Kingdom) fitted with three 0.22-µm-pore-size Pellicon filters (Millipore). The concentrate (TFF retentate representing up to a 200-fold concentration) was returned to the laboratory within 2 h. The concentrate was then subjected to a number of tests, which were all completed within 7 h of collection. Cell and colony counts were converted to cells and numbers of CFU per milliliter of original lake water.

Size fractionation of samples.
Samples of TFF-concentrated lake water from the different water bodies were individually filtered through a polycarbonate filter with a 5.0-µm pore size (Millipore) and cellulose acetate filters with pore sizes of 3.0, 1.0, 0.8, 0.65, and 0.45 µm (Millipore). Where necessary, prefiltration was carried out (filter pore size, 80 µm; Millipore). An unfiltered sample was retained for comparative purposes. The filtrates from each sample were then treated as described in the following sections.

Bacterial culture.
Numbers of CFU were determined by plating after serial dilution of samples in filter-sterilized lake water (filter pore size, 0.22 µm) from the lake under study onto R2A (32) for 7 days at 18°C.

Direct counting and DVC.
Total direct counting using DAPI (4',6'-diamidino-2-phenylindole; Sigma, Poole, United Kingdom) was performed as described previously (29). Direct viable counts (DVC) were obtained with incubation of the samples at 18°C in the presence of nalidixic, piromidic, and pipemidic acids for 6 to 24 h prior to fixation in formaldehyde (final concentration, 4% [vol/vol]) and storage at 4°C for up to 3 days (13). The samples were then stained with DAPI prior to cells being counted. All cells were collected on 0.22-µm-pore-size black membrane filters (Millipore Costar, High Wycombe, United Kingdom) and counted on a Leitz Orthoplan fluorescence microscope under x1,000 magnification. At least 400 cells were counted on replicated filters for each sample (9).

Activity assessment.
Respiration activity was assessed by incubating the samples for 2 h at 18°C in the presence of 3.5 mM CTC (33). Intracellular enzyme activity was assayed using Chemchrome B (ChemB; Chemunex, Cambridge, United Kingdom) as described previously (26). Control samples were prepared by heat treating cells at 80°C for 10 min prior to staining them. Cells which labeled red were deemed CTC positive and counted by epifluorescence microscopy. Samples for the ChemB assay were analyzed by flow cytometry.

Flow cytometry.
Flow cytometry was performed on a FACStar Plus flow cytometer (Becton Dickinson, Oxford, United Kingdom) set to trigger on forward scatter at a threshold of 50 (arbitrary units). Three parameters were measured: forward light scatter, 90^o side scatter, and fluorescence at 525 nm. The laser power at a wavelength of 488 nm was set at 25 mW, and all recordings were taken on a 4-decade log scale. The photomultiplier voltages were set at 450 V for side scatter and 500 V for the fluorescence detector. The cytometer was aligned with 0.5-µm-diameter Fluoresbrite YG fluorescent latex beads (Polysciences Inc., Warrington, Pa.). The nozzle diameter was 70 µm. For each sample, 5,000 events were recorded. The FACStar Plus software package was used to determine the proportion of cells that were fluorescently labeled after assessment of background fluorescence levels from unstained and control samples.

Data analysis.
Duplicate or triplicate samples were processed for each assay from each of two sampling visits to each lake. Bacteria labeled with ChemB were assessed by flow cytometry, and data were expressed as a percentage of the total number of bacterial events determined by light scatter characteristics with the Becton Dickinson Lysis version 1.0 software. Bacteria labeled with CTC or responding to the DVC assay were counted microscopically, and data were expressed as a proportion of the total DAPI count. Statistical analysis was performed on these data using MINITAB version 12.1 (Minitab, Warrington, Pa.). Total microscope counts were log₁₀ transformed prior to analysis of variance (ANOVA), with the ANOVA assumptions of ensuring homogeneity of variance and normality of distribution being confirmed with published MINITAB macros (9). Means were compared by calculating minimum significant differences determined by the Tukey-Kramer method as discussed by Fry (9). Where data transformations did not fully ensure homogeneity at the 5% probability level, the nonparametric Kruskal-Wallis test was performed to confirm the ANOVA results.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterioplankton in eutrophic and oligotrophic water bodies.
Water samples were taken from WW, WNB, EW, WSB, and PP. Direct DAPI counts from untreated lake water samples and from the independent size-fractionated lake water samples are presented in Fig. 1. It was established that passing the samples through a prefilter had no statistically significant effect overall on the total number of bacteria counted by epifluorescence microscopy after DAPI staining (Fig. 1). Some samples required prefiltration prior to size fractionation, especially at the smaller pore sizes, due to the presence of particulate matter. In the prefiltered and nonfiltered samples, DAPI direct counts showed significantly higher numbers of bacteria in the eutrophic water bodies (PP, WSB, and EW) than in the oligotrophic systems (WW and WNB) (Fig. 1). Moreover, there were significantly fewer bacteria in WW than in WNB (Fig. 1).

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FIG. 1. Total numbers of bacterial cells from five freshwater lakes remaining after passage through different-pore-size filters. MSD, minimum significant difference, by the Tukey-Kramer method, at the 95% confidence level.

Among lakes, no significant differences in cell numbers observed for each size fraction down to 0.8 µm were found. After passage through 0.65- and 0.45-µm-pore-size filters, significantly fewer cells were observed (Fig. 1).

The effects of size fractionation within lakes was also analyzed, and the proportion of cells remaining after each step is shown in Table 1. Differences in cell size are apparent in the proportions of cells remaining during fractionation through progressively smaller filters, with higher numbers passing through the larger-pore-size filters for WW and WNB than for EW, WSB, and PP (Table 1). The size cutoff at which a significant fraction of cells was withheld varied from the oligotrophic to the eutrophic lakes. There were greater proportions of small cells in the lower-nutrient-status waters (WW and WNB; with a <0.45-µm-pore-size filter, 10 and 7.4%, respectively) than in the higher-nutrient-status waters (PP and WSB; 0.9 and 1.3%, respectively).

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TABLE 1. Percentages of bacterial cells remaining after passage through different-pore-size filters

Culturability.
Subsamples from each size fraction were assayed for culturability by spread plating onto R2A. Culturable counts (expressed as a percentage of the total DAPI direct count for that fraction) are presented in Fig. 2. For unfiltered lake water, EW showed significantly higher culturability than the other lakes (Fig. 2). No significant difference was detected between the percentages of cells cultured from PP and WSB (Fig. 2). However, the numbers of culturable cells from EW, PP, and WSB were significantly higher than those from WNB and WW. Therefore, the eutrophic lakes showed higher proportions of culturable bacteria. Apart from WW, cells from all lakes showed significantly less culturability below the 5.0-µm size fraction, with recovery rates of <0.4%. WW showed no significant change across the size fractions, with culturability ranging between 0.4 and 0.1%. The nutrient statuses of the lakes could not be clearly distinguished on the basis of culturability.

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FIG. 2. Proportion of culturable bacteria from five freshwater lakes remaining after passage through different-pore-size filters. MSD, minimum significant difference, by the Tukey-Kramer method, at the 95% confidence level.

DVC.
The DVC was ineffective in the eutrophic waters of PP, WSB, and EW (Table 2; Fig. 3). Where a response was detected, it was judged to be insignificant, as it comprised 0.1% of the total cell numbers. Increasing the incubation times from 6 to 24 h did not increase the numbers of responsive cells for these lakes (data not shown). In contrast, WNB and WW showed between 5.5 and 1.5% DVC-positive cells after 6 h of incubation in samples with and without prefiltration and in size fractions that were 5.0 to 3.0 µm (Table 2; Fig. 3). After 1.0 (WW)- and 0.8 (WNB)-µm-pore-size filtration, no DVC-responsive cells were detected. Oligotrophic lakes could be distinguished from eutrophic lakes in the DVC assay by the significantly higher numbers of responsive cells in size fractions of >3.0 µm (Table 2; Fig. 3).

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TABLE 2. Activity estimates of bacteria from different-size fractions across five freshwater lakes

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FIG. 3. Proportion of viable or active bacteria from five freshwater lakes. MSD, minimum significant difference, by the Tukey-Kramer method, at the 95% confidence level.

Activity by CTC and ChemB labeling.
Activity, independent of culture by plating, was assessed with the dyes CTC and ChemB. Different-size fractions from the range of lakes that differed in trophic status were assayed with the two tests.

The levels of effectiveness of the activity assays were initially compared by using unfiltered lake water from each of the lakes. For all lakes, ChemB labeling produced a higher proportion of responsive cells than the CTC, DVC, and culture methods. A clear distinction was evident between the eutrophic and oligotrophic systems (>40 to 35% compared to <13% labeling) (Fig. 3). CTC labeled <20% of the total cells and did not distinguish between the trophic statuses of the water bodies, although cells from WSB showed a significantly greater CTC-positive count than cells from the other waters (Table 2). ChemB and CTC provided similar responses for samples from the oligotrophic WNB (Fig. 3). Activities after labeling with these dyes were compared across size fractions between lakes. For the unfiltered and prefiltered fractions, labeling with ChemB clearly distinguished the higher-nutrient-status waters (PP, WSB, and EW) from WNB and WW (Table 2), whereas CTC distinguished only WSB from PP and PP and WSB from EW and WNB. Bacteria in EW were less responsive in the CTC assay than in the esterase assay, leading to two different (oligotrophic and eutrophic, respectively) lake classifications. This pattern was observed for the 5.0-µm fraction with both ChemB and CTC. After filtration through pore sizes of less than 5.0 µm, organisms from WSB and PP (eutrophic) and WW and WNB (oligotrophic) were distinguishable with ChemB. Only EW showed significant activity after filtration through pore sizes below 5.0 µm. Despite the fact that activity decreased with the smaller fractions, activity in EW water was significantly higher than the activities in the other water bodies down to the 0.45-µm fraction. For size fractions between 3.0 and 0.8 µm, CTC was able to distinguish eutrophic from oligotrophic waters: WSB generated the highest response in the eutrophic group. For the size fractions of 0.65 and 0.45 µm, PP (highly eutrophic) and WNB activities were indistinguishable with CTC, yet significantly greater numbers of responsive cells were detected in EW and WSB.

Activities after labeling with ChemB and CTC were then compared across size fractions within lakes. Results are presented in Table 3. After labeling with ChemB, significant differences were observed between size fractions, but no overall pattern emerged with respect to trophic status (Table 3). For instance, the eutrophic water of PP showed two activity classes (5.0 and 3.0 µm), whereas for WSB, three classes were observed (unfiltered, filtered through 5.0-µm-pore-size filters, and filtered through 3.0-µm-pore-size filters). Data from bacteria from EW could be divided into four classes (Table 3). In the oligotrophic group, WW and WNB also differed in response to labeling with ChemB, and WNB was thus more similar to the eutrophic lakes with respect to subdivision into size classes.

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TABLE 3. Activity estimates of size fractions of bacteria within five freshwater lakes as determined by esterase activity or tetrazolium salt reduction assay

Size classes of CTC activity also showed no overall pattern within the lakes. EW showed no significant difference between size fractions, and WNB was subdivided into CTC-responsive cells (diameters, 5.0 µm) and nonresponsive cells (diameters, 3.0 µm). WSB divided into three subclasses (diameters, 3.0, 1.0, and 0.80 µm). PP, like WNB, divided into CTC-responsive and nonresponsive cells, which separated around the 0.65-µm fractionation point.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study attempted to characterize by size and activity the heterogeneous bacterial populations found in lakes that differed in trophic status in the English Lake District. Analyses based on planktonic variables (chlorophyll and ATP chloroplast determinations) show a steady transition from oligotrophic to eutrophic. Previous work assessing microbiological parameters of sediments from these lakes has suggested that the order in Pearsall's original series be modified (11). However, data from this study did not reveal differences that would support altering the series order as suggested.

Direct counting of total bacteria with DAPI showed significant differences between the oligotrophic lakes and the rest and between the two oligotrophic lakes, yet it maintained their series order. WNB is acknowledged to have moved toward mesotrophic status due to anthropogenic influences, hence its lack of parity with WW (G. H. Hall, personal communication). Direct counts of size class did not discriminate between the eutrophic and hypereutrophic levels with PP, although other data have shown elevated seasonal numbers that were up 10⁸ cells ml^–1 at 3- to 3.5-m depths, which would have distinguished PP from the other eutrophic lakes (8). This seasonal variation is typical for PP, which represents a unique microbial environment within the English Lake District (8), but has not been observed for the other lakes (8, 11).

Trophic status can affect bacterioplankton community composition (17, 18). Given that the trophic status of the lake influences total bacterial numbers (11), we assessed whether it also affected bacterial size. Significant differences in total direct counts generally occurred after 0.45-µm size fractionation, showing the majority of bacteria in each lake to be above 0.5 µm in diameter. Fractionation above this size did not significantly change total numbers. Although further fractionation below 0.45 µm may have been more discriminatory, this size boundary appears not to be related to trophic status.

Culturability has proven controversial for assaying viability due to the selectivity of media and the large fraction of bacteria that remain unculturable (1). Culturability in nearly all environments varies between 0.1 and 10% (1) but can be increased with improvements in cultivation methods (5, 6). In this study, similar variation was observed, with culturability levels between 0.5 and 4% in the nonfractionated samples. However, culturability was not able to clearly discriminate the water bodies according to trophic status. Although we were able to culture cells in all size fractions, the effect of fractionation for each lake was most apparent with organisms with diameters below 3.0 µm, at which size culturability was reduced in all samples. However, both EW and WNB provided levels of culturability higher than those of the other lakes within this size fraction. These results reflect culturability on a commonly used medium designed to maximize the culture of bacteria from potable water (32). This medium has been demonstrated to work effectively for lake water samples of this type (27).

Linked to culturability, the DVC assay was applied to the samples to assess the responses of individual cells. Cell enlargement is a subjective criterion (39); however, a careful comparison was made between untreated DAPI-stained cell samples and DVC-responsive samples prior to the acquisition of data. It is implicit within the DVC assay that cells which can respond by elongating as opposed to simply showing activity are probably capable of division and thus viable. In eutrophic waters, the DVC assay was not as representative of viability as was culture by plating for the majority of samples and size fractions, as no change in the proportion of substrate-responsive cells was detected. However, for the oligotrophic lakes, significantly higher numbers of DVC-responsive cells were detected in the larger-size fractions than in the equivalent samples subjected to culture. This greater prevalence was presumably a function of trophic status and cell size within the samples, as equivalent cell sizes in the eutrophic samples did not respond to the assay. Therefore, trophic status is reflected by physiological differences in different lakes and is distinguishable by the DVC assay with a defined size range.

In order to test the physiological aspects of the activities of the cell fractions, samples were assayed with two commonly used activity dyes, CTC and ChemB (27). The data show that ChemB labeled a higher proportion of cells in all samples and in all size fractions than CTC. It clearly distinguished the larger-size fractions from the eutrophic lakes from the equivalent fractions in both WW and WNB but could not resolve fraction sizes in the eutrophic lakes within the Pearsall series. The response with CTC was significantly lower than, but very similar to, that of the ChemB assay. CTC activity indicated WSB as the water body with the most-active bacteria, a feature not reflected by the other assays. However, the eutrophic status of WSB and PP was detectable by the CTC assay, as were the higher activities of all the larger-size classes apart from EW (WW not determined). Significant differences were noted in the proportions of bacteria labeled with ChemB and CTC, suggesting that there is selectivity, possibly due to cell physiology, in the assays, although uptake of the dyes may influence labeling. Other workers have also noted that bacteria may be killed due to CTC reduction (34), which may limit labeling and detection through this assay. This lack of correlation between the two methods confirms the results of other studies suggesting that there is no universal dye for such measurements (see, for examples, references 27 and 37).

The use of microscopy in such studies has been criticized, as it may introduce errors due to subjectivity or induced by tedium. Alternatives such as image processing (30) and flow cytometry (15, 28, 35) are both more rapid and more objective. However, in this case, the use of two different enumeration methods (flow cytometry and microscopy) is unlikely to influence the data, as the results of both counting methods strongly correlate (24).

The data with respect to size fraction showed that for the unfiltered, prefiltered, and 5.0-µm-pore-size-filtered fractions, EW and PP were distinguishable from WSB and from WW and WNB as separate clusters. However, as the larger cells (diameters, 3.0 to 0.65 µm) were removed, the remaining cells in EW samples were more active than those from other lakes, all of which showed equal activities. Only after passage through a 0.45-µm-pore-size filter did the activities of EW, WSB, PP, and WW reach parity. Other studies compared a range of dyes based on fluorogenic esters and CTC, demonstrating that ChemB showed higher activity than CTC and that 7% of active cells could be found in WNB (27). This is consistent with the values found in this study. However, in microbiologically contaminated sewage-receiving river water, the esterase and DVC assays matched culturability (27). In a study of polluted water, numbers of bacteria which showed esterase activity were higher than those detected by the CTC assay (37). In addition, higher levels of esterase activity were observed in polluted sites than in nonpolluted sites, with approximately 2% of the bacteria being culturable. In this study, direct pollution is analogous to increasing eutrophication, and the bacterial community responded in similar manners to both fluorogenic compounds used with respect to their activities under the prevailing conditions. Both ChemB and CTC were able to distinguish the eutrophic from oligotrophic environments but not always unanimously. This lack of unanimity again raises the question of which method may serve as a definitive measure of activity. As in the case of many techniques applied to environmental samples, universal applicability of any one technique is rare. When they are used in conjunction with each other, some confidence may be gained in the assessment. However, in all cases the assays distinguished the larger cell fractions from the smaller; for these assays, the only variation was the particular size fraction at which the cutoff occurred.

A previous attempt based on microbial enzyme activity failed to distinguish lakes that differed in trophic status in the English Lake District (20). It was found that there was no relationship between the range of enzymes found and trophic status. Thus, the discriminatory potential of enzyme activity was low (20). This study suggests that culture, direct counting of bacteria, and activity determination enable a series of lakes to be distinguished by trophic status. The resolution is such that oligotrophic waters are distinguishable from eutrophic waters (by direct counting, culture, DVC, and ChemB assays). With culture and direct counting, mesotrophic status (WNB) was distinguishable as an intermediate trophic state. All of the assays failed to distinguish between the hypereutrophic and eutrophic states, although EW appears from these data to have a status comparable to that of the hypereutrophic PP. This may be due to their close proximity within their catchment. These findings further reinforce other observations that productive lakes are microbiologically very similar, certainly at a process level, and are different from those of lower trophic status (11). However, despite similarities at the process level, genetic diversity has been demonstrated in other lakes (17). The bacterioplankton community in two shallow eutrophic lakes showed distinct seasonal within-lake patterns, and contrasting between-lake patterns were determined largely by the presence or absence of macrophytes (38). Bacterial-community structure can also be influenced by the import of allochthonous bacteria, pH, humic acids, and grazing (17-19). Clearly, the next steps are to examine whether these similarities and differences occur at the genetic level by use of diversity profiling methods such as temperature gradient gel electrophoresis and denaturing gradient gel electrophoresis (21) and to associate diversity (including cell size) with productivity (4), taking into account that a significant contribution may come from cells not shown to be active with the more common dyes (12, 34).

 
We thank the Natural Environment Research Council (Swindon, United Kingdom) for funding through the fellowship scheme (J.P.; grant reference GT5/96/7/FS) and CEH for providing support and facilities.

 
* Corresponding author. Present address: Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter EX4 4PS, United Kingdom. Phone: 44 1392 264607. Fax: 44 1392 263700. E-mail: J.D.Porter{at}exeter.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, April 2004, p. 2072-2078, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2072-2078.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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