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Depth distribution of active bacteria and bacterial activity in lake sediment
FEMS Microbiology Ecology 46 (2003) 31^38
www.fems-microbiology.org
Depth distribution of active bacteria and bacterial activity in lake sediment Ann-Louise Haglund b
a;b;
, Peter Lantz b , Erik To«rnblom b , Lars Tranvik
b
a Department of Public Technology, Ma«lardalen University, Box 883, SE-721 23 Va«sterafis, Sweden Department of Limnology, Evolutionary Biology Centre, Uppsala University, Norbyva«gen 20, SE-752 36 Uppsala, Sweden
Received 16 April 2003; received in revised form 10 June 2003; accepted 26 June 2003 First published online 12 August 2003
Abstract The bacterial activity in sediments is often low considering the generally high bacterial abundance. Still, a large fraction of bacteria have been found active even in deep sediments. These findings suggest that sediment bacteria have comparatively low cell-specific production. We studied bacterial activity and the active fraction of bacteria in a lake sediment profile. Bacterial production and metabolism were measured by thymidine and leucine incorporation and by microcalorimetry. In addition to counts of total bacteria, we estimated the nucleoid-containing fraction of the bacteria by adding a destaining step to the DAPI staining method, and the live fraction using the Live/ Dead Baclight bacterial viability kit. The bacterial activity and abundance decreased with sediment depth, while the proportion of active bacteria remained similar at all depths. Between 57 and 63% of the bacteria were scored viable, and 13^52% were scored as nucleoidcontaining cells. Consequently, there was no accumulation of dead bacterial cells in deeper sediments. Cell-specific production of sediment bacteria may be severely underestimated if the active fraction of the sediment bacterial community is not considered during enumeration. 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Bacterium ; Sediment ; Production; Metabolically active bacterium ; Cell-speci¢c production
1. Introduction Sediment bacteria constitute an important component of the benthic food web as well as of nutrient cycling and decomposition of organic matter. Bacterial activity and biomass are generally highest near the sediment surface and decrease with depth (e.g. [1]). Considering the generally high abundance of bacteria in sediments, the bacterial activity therein is often low. This indicates that the cell-speci¢c activity of sediment bacteria is low, or that only a small fraction of the bacterial community is metabolically active [2]. Several studies have reported relatively large proportions of active bacteria in sediments (e.g. [3]), which favors the hypothesis of low cell-speci¢c productivity. Even in deep sediments, there are viable bacteria (e.g. [4,5]), however, the metabolic state of sediment bacteria in depth gradients is not well known.
* Corresponding author. Tel. : +46 (18) 471 27 18; Fax : +46 (18) 53 11 34. E-mail address : [email protected] (A.-L. Haglund).
Fluorochromes such as acridine orange and 4P,6P-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) may overestimate the number of bacteria due to staining of dead cells and non-bacterial particles [2]. In addition, all cells are not actively participating in production and growth, which may lead to underestimates of cell-speci¢c growth rates using the traditional enumeration methods. There are several methods to distinguish live cells from dead cells but they have been used mostly on bacterioplankton. In this study, we employed two di¡erent methods to determine the number of viable bacteria, neither of which has been used on freshwater sediment bacteria before. We used Zweifel and Hagstro«m’s [6] method to detect cells containing nucleoids and the Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, OR, USA) to separates live cells from dead cells. Zweifel and Hagstro«m [6] added a destaining step to the traditional DAPI staining procedure to avoid unspeci¢c staining of cells lacking nucleoids. The empty cells were referred to as ‘ghosts’ incapable of growth while the nucleoid-containing cells were considered viable and capable of metabolic activity.
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00190-9
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The Live/Dead BacLight bacterial viability kit is a twocolor £uorescence assay. Cells with intact membranes stain £uorescent green and are considered to be live while cells with damaged membranes stain £uorescent red. Combining several methods for bacterial activity and abundance, we examined the changes in the distribution of viable bacteria as well as the total and cell-speci¢c metabolic activity in depth pro¢le of a sediment.
2. Materials and methods 2.1. Sampling Seven sediment cores were taken in June 1999 at 12 m depth at an accumulation bottom in Lake Erken. Lake Erken is a relatively large mesotrophic lake situated 60 km north of Stockholm, Sweden (59‡51PN, 18‡35PE). The sediment cores were transported to the laboratory in darkness and stored at 4‡C until sliced. The cores were sliced at 6-mm intervals. Eight layers were saved for further analysis, taken at depths below the sediment surface of 0^6, 12^18, 24^30, 48^54, 78^84, 102^108, 162^168, and 222^228 mm. The sampling site is in an area with a sediment accumulation rate of 6 mm year31 (G. Weyhenmeyer, personal communication). Consequently, a core represents sediment accumulated over approximately the last 60 years, taking the compaction of the sediment into account. The sediments were muddy and had an organic content of approximately 18% [7]. After slicing, the sediment was analyzed within 24 h. Samples for DAPI staining were taken immediately after slicing and ¢xed with 4% formaldehyde. The rest of the staining and analyses was performed on live sediment. Water content was determined and content of carbon and nitrogen were measured using elemental microanalysis (LECO, CHNS 932) on all the layers in seven cores. 2.2. Abundance of live and dead bacteria We counted live and dead bacteria in the sediment using the Live/Dead BacLight bacterial viability Kit, which contains a mixture of SYTO0 9 and propidium iodide. The SYTO0 9 stain is a nucleic acid stain with green £uorescence, which labels all cells with intact cell membranes. Propidium iodide penetrates those cells that have damaged cell membranes, where it causes a red £uorescence and a reduction in the SYTO0 9 £uorescence when both dyes are present. Thus, when excited with blue light, the live cells, with intact cell membranes, will appear green. Dead cells, with damaged cell membranes, will appear red. Portions of 0.5 g of live sediment were transferred to glass vials and mixed with 4.5 ml of ¢lter-sterilized (0.2 Wm) water. The mixture was diluted 1000 times, and a 2.5ml subsample was mixed with 2.5 ml of water and soni-
cated for 90 s at 30 W using an ultrasonic cell disrupter (Microson1). Filter-sterilized (0.2 Wm) water was used in all dilutions. A 1:1 mixture of SYTO0 9 and propidium iodide was added (7.5 Wl), and the sample was incubated for 15 min. Thereafter, it was ¢ltered onto a 0.2-Wm black polycarbonate ¢lter (Micron Separations). The ¢lter was dried and placed on a slide in mounting oil (Molecular Probes) together with the 0.2-Wm cellulose acetate ¢lter (Lida), used as a drain disc during ¢ltration. Once mounted, ¢lters were immediately counted using epi£uorescence microscopy. A minimum of 400 cells, or at least 20 randomly chosen ¢elds, was counted on each slide. Only those cells where the stain was clearly visible were counted. The fraction of live bacteria was calculated as the abundance of live cells divided by the total count obtained with the Live/Dead method. 2.3. Total and nucleoid-containing bacterial abundance and biomass We used DAPI for total bacterial counts. A portion (0.1 g) of sediment was ¢xed with 9.9 ml of 4% ¢ltersterilized (0.2 Wm) formaldehyde. This mixture was diluted 1000 times and a 5-ml subsample was taken and sonicated for 90 s as described above. DAPI was added to a ¢nal concentration of 10 mg l31 and incubated for 30 min. Filtration, mounting on slides and counting were performed as described above. The DAPI-stained cells were visualized in UV light using epi£uorescence microscopy. To estimate bacterial biomass, photos of slides with DAPI-stained bacteria were taken. Photos of samples from all eight layers from three cores were used. To be able to determine the size of bacteria, the photos and a micrometer scale were projected on the wall and 50 cells from each slide were measured. The bacterial biomass was then estimated using the abundance and average biovolume assuming a carbon content of 105 fg C Wm33 [8]. The resulting average cell carbon content was used in all biomass calculations. An additional DAPI-stained subsample was subjected to a washing step to remove unspeci¢c DAPI staining [6] using warm (60^70‡C, 1 ml) 2-propanol. Then the ¢lter was dried for 5 min before mounting on a slide as described above. The fraction of nucleoid-containing bacteria was calculated as the abundance of nucleoid-containing cells divided by the total counts obtained with the DAPI method. 2.4. Bacterial activity Bacterial production was estimated using [3 H]thymidine and [14 C]leucine incorporation, while heat production using direct calorimetry was used to determine total sediment metabolism. For the [3 H]thymidine incorporation, a mixture of labeled and unlabeled thymidine was used at a ¢nal concen-
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tration of 1000 nM. Previous studies of Lake Erken sediment have shown thymidine saturation at 1000 nM [3]. By mixing labelled (81.0^83.0 Ci mmol31 ) [3 H]thymidine (Amersham, TRK 686) and unlabelled thymidine (Sigma) the ¢nal speci¢c activity obtained was 43.45^44.02 Ci mmol31 . Subsamples of sediment (0.2 g wet weight) were incubated in 10-ml centrifugation tubes. There were three replicates and one control for each sediment layer and three cores were analyzed. The controls were killed by adding 5 ml of 80% ethanol. Thereafter, [3 H]thymidine was added to each tube, and the samples were incubated at in situ temperature (12‡C) for 40 min. The incubations were terminated by adding 5 ml of 80% ethanol. Samples were kept cool (4‡C) until further processing. The samples were processed according to Bell and Ahlgren [9] and the radioactivity was detected using a liquid scintillation counter (LKB Wallac 1219 Rackbeta). Bacterial production was estimated from thymidine incorporation rates using a conversion factor of 2.0U1018 cells mol31 thymidine [10]. Incorporation of [14 C]leucine was measured following the procedure for sediments established by van Duyl and Kop [11] with a few adaptations. 0.2 g of sediment was incubated for 30 min with [14 C]leucine (Amersham, CFB 67) (94 mCi mmol31 ) in 10-ml centrifuge tubes (¢nal concentration 1000 nM). The incubation was performed in triplicate with one additional control killed with 5 ml of 80% ethanol. Incubation was terminated by adding 5 ml of 80% ethanol and the samples were stored at 4‡C overnight. Samples were then centrifuged (7500 rpm, 8500Ug) for 20 min, the supernatant was discarded and the pellet was washed with 5 ml of 80% ethanol. This procedure was repeated twice. The pellets were then washed with 5 ml of ice-cold trichloroacetic acid (5%), and after centrifugation (at 4‡C) the supernatant was discarded. This procedure was repeated twice. 2 ml of 2 M NaOH was added to each pellet and the samples were placed in a boiling water bath for 2 h followed by centrifugation for 20 min (7500 rpm, 8500Ug). 1 ml of the supernatant was then transferred to a scintillation vial and scintillation cocktail was added. The leucine concentration (1000 nM) was chosen based on a saturation experiment performed on sediment samples from the sampling site. Final concentrations in the saturation experiment ranged from 500 to 9000 nM. We measured heat production of the total sediment metabolism using a heat conduction multichannel microcalorimeter (TAM 2277, Thermometric) according to Bostro«m and To«rnblom [12], with a modi¢cation in order to maintain anaerobic conditions. Instead of using a smaller amount of sediment and an overlying water phase, 3-ml ampoules were completely ¢lled with sediment using a syringe. All measurements were performed at 12‡C and heat production values were recorded after an equilibration time of 10 h.
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2.5. Statistical analysis One-way analysis of variance (ANOVA) was used to distinguish changes in the bacterial community with depth. Homogeneity of variances (Bartlett’s M2 ) and normal distribution of the dependent variable (Shapiro^Wilks test) were assured. When necessary, the data were log- or arcsin-transformed to achieve normal distribution. Spearman rank correlation was used when relating bacterial activity and abundance to depth.
3. Results The water content in the sediment surface layer (0^6 mm) was 95% and decreased to 86% in the deepest layer measured (222^228 mm) (Fig. 1A). The carbon content decreased from 10.3% at the surface to 7.7% of sediment dry weight in the deepest layer. The corresponding numbers for nitrogen content were 1.3% and 1.0%, respectively. The molar C:N ratio varied from 9.0 in the surface sediment to 8.7 at 48 mm depth and then it increased again to slightly over 9 in the deepest layer (Fig. 1B). The total number of bacteria followed the same general trend with both staining methods (DAPI and Live/Dead). Bacterial abundance was highest at the sediment surface and decreased with depth (P 6 0.001; b = 30.84 and b = 30.77 for DAPI total counts and Live/Dead total counts, respectively). The total bacterial abundance of DAPI-stained bacteria was 11.0U1010 cells (g dry weight)31 at the sediment surface, and 2.0U1010 cells (g dry weight)31 in the deepest layer (Fig. 2A). When adding
Fig. 1. (A) Water content (%) and (B) C:N ratio at di¡erent sediment depths (n = 7 for each depth). Error bars indicate standard deviation.
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Fig. 2. (A) Total bacterial counts (DAPI) and abundance of nucleoidcontaining cells (DAPI-destained) and (B) total bacterial counts (Live/ Dead) and abundance of live and dead cells at di¡erent sediment depths (n = 4 at each depth). Error bars indicate standard error.
the washing step with 2-propanol, the abundance decreased by more than 50%. The nucleoid-containing cell abundance at the surface was 4.3U1010 cells (g dry weight)31 and in the deepest layer 0.5U1010 cells (g dry weight)31 (Fig. 2A). The fraction of nucleoid-containing bacteria was not signi¢cantly di¡erent between depths (P = 0.083, ANOVA) and varied from 13 to 52% (Fig. 3) of the total counts obtained with DAPI staining. Total counts of bacteria using the Live/Dead method were lower than DAPI counts. The total abundance decreased from 4.0U1010 cells (g dry weight)31 in the most sur¢cial sediment to 1.8U1010 cells (g dry weight)31 in the deepest layer (Fig. 2B). The live cells were 2.5U1010 cells (g dry weight)31 at the surface and decreased to 0.7U1010 cells (g dry weight)31 in the deepest layer (Fig. 2B). The corresponding ¢gures for the dead bacteria were 1.5U1010
Fig. 3. Fraction of viable cells (%) at di¡erent sediment depths obtained with the Live/Dead method (¢lled symbols) and DAPI destaining method (open symbols) (n = 4 at each depth). Error bars indicate standard error.
cells (g dry weight)31 and 0.5U1010 cells (g dry weight)31 , respectively (Fig. 2B). The fraction of live or dead bacteria was not signi¢cantly di¡erent between depths (P = 0.31, ANOVA) but was rather similar at all sediment depths. The live fraction ranged from 57 to 63% of the total cell abundance obtained with the Live/Dead method (Fig. 3). DAPI staining resulted in considerably higher abundances compared to the Live/Dead method. This was visually apparent when the total DAPI counts were plotted against the Live/Dead total counts. At high abundances, the total DAPI counts were more than twice as large as the total Live/Dead counts (Fig. 4A). Interestingly, the nucleoidcontaining cells were strongly related to the live bacterial cells (r2 = 0.86) (Fig. 4B) although di¡erent total counts were used for the calculations. The slope of the regression was not signi¢cantly di¡erent from one (P 6 0.001, t-test). The bacterial activity generally decreased with depth (P 6 0.001; b = 30.80 and b = 30.68 for thymidine and leucine incorporation, respectively, P = 0.002; b = 30.53 for heat production) (Fig. 5). The [3 H]thymidine incorporation had a steeper decrease compared to [14 C]leucine incorporation, as seen from the decrease in the ratio between leucine and thymidine incorporation with increasing depth (Table 1). The [3 H]thymidine incorporation was 50.1 pmol (g dry weightUh)31 at the surface and declined to 3.8 pmol (g dry weightUh)31 at 222 mm depth. The corresponding values for [14 C]leucine incorporation were 3.0 and 0.27 nmol (g dry weightUh)31 , respectively. Heat production also decreased with depth but not in as pronounced a manner as the thymidine and leucine incorpo-
Fig. 4. (A) Total DAPI counts as a function of total Live/Dead counts and (B) nucleoid-containing cell abundance as a function of live cell abundance in a sediment pro¢le (n = 4 at each depth). Lines indicate 1:1 ratio.
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Fig. 5. Incorporation of (A) thymidine (n = 7 at each depth), (B) leucine (n = 4 at each depth) and (C) heat production (n = 5 at each depth) at di¡erent sediment depths. Error bars indicate standard error.
ration rates. At the surface heat production was 46.8 WW (g dry weight)31 and at 222 mm depth it declined to 26.7 WW (g dry weight)31 . The ratio between leucine incorporation and heat production £uctuated somewhat but it did not decline with depth until below the 102-mm layer (Table 1). The cell-speci¢c bacterial activity, as calculated by dividing the carbon production (derived from leucine incorporation) by the total carbon biomass (DAPI counts and cell volume estimates), varied from 0.04 to 0.22 day31 and was not signi¢cantly correlated with depth (P = 0.11; b = 30.29) (Fig. 6A). When the biomass of nucleoid-containing cells (Fig. 6B) or live cells (Fig. 6C) was used instead of the total bacterial biomass, cell-speci¢c activity Table 1 Ratio between mean leucine and thymidine incorporation (pmol (g dry weightUh)31 ) and mean leucine incorporation and heat production (WW (g dry weight)31 ) at di¡erent depths Depth (mm)
Leucine:thymidine
Leucine:heat
0 12 24 48 78 102 162 222
61 86 104 167 286 243 122 73
65 89 115 99 112 72 23 10
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Fig. 6. Cell-speci¢c production (BP:BB) at di¡erent sediment depths calculated by dividing bacterial production (leucine incorporation) by (A) the total bacterial biomass (DAPI), (B) the nucleoid-containing cell biomass, and (C) the biomass of live cells (Live/Dead) (n = 4 at each depth). Error bars indicate standard error.
was considerably higher. When the biomass of live cells was used the speci¢c activity ranged between 0.12 and 0.83 day31 , decreasing with depth (P = 0.006; b = 30.47) (Fig. 6C). Calculations with nucleoid-containing cells resulted in cell-speci¢c activities from 0.31 to 0.74 day31 and there was no signi¢cant decrease with depth (P = 0.75; b = 0.06) (Fig. 6B).
4. Discussion Bacterial abundance generally decreases with sediment depth [1,13,14]. Interestingly, we found no signi¢cant difference in the fraction of live or nucleoid-containing bacteria among depths (Fig. 3). Hence, there was no accumulation of dead bacterial cells with depth, a fact also reported by Miskin et al. [4]. Viable bacteria are present also in deep sediments of the oceans. Parkes et al. [5] found a constant percentage of bacterial cells in a dividing stage down to 518 m in the deep sediments of the Paci¢c Ocean. The fact that there was no di¡erence in the fraction of live or nucleoid-containing bacteria with sediment depth indicates that grazing or viruses are not important regulators of bacterial mortality in these sediments. There are often high densities of protozoa in aquatic sediments (e.g. [15]). However, most grazing studies show a minor impact of £agellate grazing on the bacterial community
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(e.g. [16]). In addition, Hamels et al. [16] found that the importance of £agellate grazing on bacterial mortality decreased with grain size of the sediment. Viruses have been found to be numerous in freshwater sediments but the virus-to-bacteria ratio has generally been close to one or lower, i.e. signi¢cantly lower than in pelagic environments [17,18]. Accordingly, the two mortality factors generally considered to be responsible for most of the mortality of bacteria in the water column may not be e¡ective in the sediments. This may contribute to the persistence of intact, active cells throughout the studied sediment pro¢le. Apparently, the Live/Dead BacLight bacterial viability kit has not been used on sediment bacteria before. Luna et al. [19], who applied a similar method using SYBR Green I instead of SYTO 9, obtained live bacterial fractions between 26 and 30% in a coastal marine sediment pro¢le. Metabolically active fractions of bacteria in surface sediments between 4 and 67% are reported using the tetrazolium salts 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) [3,20,21] or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride [22,23] and nalidixic acid [22]. In the study on the viability of prokaryote communities in a post-glacial profundal freshwater sediment pro¢le, close to 100% of the bacteria in the sediment were active throughout the sediment core down to about 6 m, using the CTC method [4]. Since the CTC incubation was performed together with nutrient additions, this probably re£ects the viable fraction of bacteria, rather than the fraction that is metabolically active in the sediment. The fraction of live bacteria obtained in this study (57^63%) was higher than most proportions of metabolically active bacteria reported in sediments, but since dormant bacteria with intact membranes should be included in the live fraction, this is not surprising. Total DAPI counts were up to more than twice the total abundance with the Live/Dead staining (Fig. 4A). This is consistent with the reports of unspeci¢c staining of bacteria lacking nucleoids and non-bacterial particles with DAPI [2,24], and DAPI staining of non-bacterial particles such as mucus, £agellate mucocysts and polyphosphate granules, some of which may be erroneously recorded as bacteria [25]. Destaining of the DAPI samples according to Zweifel and Hagstro«m [6] resulted in large standard errors in our sediment samples (Fig. 3). The abundance of nucleoid-containing cells was often higher than the abundance of live cells, using the Live/Dead method, suggesting that some cells with broken cell membranes (scored dead with the Live/Dead staining) contained nucleoids. Similarly, Choi et al. [26] report a higher percentage of nucleoid-containing cells than live cells (Live/Dead method) in marine bacterioplankton. When adding peptone, the fraction of nucleoid-containing bacteria increased, although the total bacterial count was constant, which led to the conclusion that cells without visible nucleoids are not necessarily empty ghost cells [26]. Hence, nucleoid-containing bacteria, as detected after DAPI de-
staining procedure, seem to include some cells that are not viable, while other cells that are viable are not detected by this procedure. We report fractions of nucleoid-containing cells between 13 and 52%. Luna et al. [19] found fractions between 1.5 and 6.2% in marine sediments. These considerably lower fractions in marine sediments may be attributed to lower nutrient concentrations in the marine environment compared to our freshwater sediment. The nucleoid-containing fraction increased upon nutrient enrichment [19] as also reported by Choi et al. [26]. Estimates of cell-speci¢c growth rate and productivity are usually based on the total bacterial biomass and the total bacterial production. Since this and several other studies report that only a fraction of the total bacteria are viable or active and capable of growth, the cell-speci¢c production obtained using the total biomass is most likely underestimated. We calculated cell-speci¢c activity using three di¡erent biomasses, i.e. the total bacterial biomass (DAPI), the biomass of nucleoid-containing cells, and the biomass of live cells. The two latter calculations produce speci¢c activity estimates up to eight times higher (Fig. 6). Hence, the cell-speci¢c production of the viable fraction of the bacteria is considerably higher than implied from estimates based on total bacterial counts. Like the bacterial abundance, total bacterial activity decreased with depth. Several studies from river sediments show a similar pattern [1,14,27]. Bacterial activity declined with depth independent of the method used, but the steepness of the decline di¡ered. Thymidine incorporation declined most rapidly, which may re£ect the shortcomings of this method for deeper anaerobic sediments. Many anaerobic bacteria lack the ability to incorporate externally added thymidine into their DNA [28,29], which leads to an underestimation of the bacterial activity in anaerobic environments. In contrast to thymidine, incorporation of leucine re£ects bacterial production equally in aerobic and anaerobic waters [30]. The increase in leucine:thymidine ratio with depth may be due to an increasing relative contribution of anaerobic bacteria that are unable to incorporate thymidine. McDonough et al. [31] also report higher leucine :thymidine ratios in anoxic compared to oxic waters. Direct calorimetry (heat production) provides non-speci¢c estimates of the total metabolism of the sediment community. It measures the enthalpy changes in biological and chemical processes independently of their function and origin. Thus, the technique is especially useful with sediments that contain mixed communities of anaerobes, fermenters, and aerobes [32], but the method is seldom used due to the unspeci¢city in distinguishing between metabolically and chemically produced heat [33]. The leucine :heat ratio was fairly constant in the upper decimeter of the sediment, but considerably lower further down (Table 1). This implies that the deeper bacterial communities use more of their energy for respiration (and thus dissipate heat) than for protein production as compared to the bac-
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teria in the overlying sediment. The C:N ratio was somewhat higher in deeper sediment, which indicates a more refractory carbon source [34]. As a consequence, the bacterial community at these depths should have lower cellspeci¢c production. This is consistent with our estimates of productivity based on the total bacterial biomass and the live fraction of the bacteria, but not on the nucleoidcontaining fraction (Fig. 6). In conclusion, bacterial abundance and total activity decreased with increasing sediment depth, but the viable fraction of the total bacteria was similar throughout the sediment pro¢le (Fig. 3). Thus, organic particles, recognizable as bacteria, seem to be lost during the diagenesis of organic carbon in the sediment rather than being accumulated. We report up to eight times higher cell-speci¢c production for the viable bacterial community compared to traditional calculations using the total bacterial biomass. Hence, estimates of productivity and speci¢c growth rates of sediment bacteria may be highly biased if the non-viable fraction of the bacterial community is not considered.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements This work was supported by funds from Ma«lardalen University and the Swedish Research Council.
[19]
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production estimates for coastal waters of British Columbia, Antarctica, and California. Appl. Environ. Microbiol. 39, 1085^1095. vanDuyl, F.C. and Kop, A.J. (1994) Bacterial production in North Sea sediments : Clues to seasonal and spatial variation. Mar. Biol. 120, 323^337. Bostro«m, B. and To«rnblom, E. (1990) Bacterial production, heat production and ATP-turnover in shallow marine sediments. Thermochim. Acta 172, 147^156. Ga«chter, R., Meyer, J.S. and Mares, A. (1988) Contribution of bacteria to release and ¢xation of phosphorus in lake sediments. Limnol. Oceanogr. 33, 1542^1558. Hendricks, S.P. (1996) Bacterial biomass, activity, and production within the hyporheic zone of a north-temperate stream. Arch. Hydrobiol. 136, 467^487. Hondeveld, B.J.M., Nieuwland, G., vanDuyl, F.C. and Bak, R.P.M. (1994) Temporal and spatial variations in heterotrophic nano£agellate abundance in North Sea sediments. Mar. Ecol. Prog. Ser. 109, 235^243. Hamels, I., Muylaert, K., Casteleyn, G. and Vyverman, W. (2001) Uncoupling of bacterial production and £agellate grazing in aquatic sediments: a case study from an intertidal £at. Aquat. Microb. Ecol. 25, 31^42. Maranger, R. and Bird, D.F. (1996) High concentrations of viruses in the sediments of Lac Gilbert, Quebec. Microb. Ecol. 31, 141^ 151. Ricciardi-Rigault, M., Bird, D.F. and Prairie, Y.T. (2000) Changes in sediment viral and bacterial abundances with hypolimnetic oxygen depletion in a shallow eutrophic Lac Brome (Quebec, Canada). Can. J. Fish. Aquat. Sci. 56, 1284^1290. Luna, G.M., Manini, E. and Danovaro, R. (2002) Large fraction of dead and inactive bacteria in coastal marine sediments: Comparison of protocols for determination and ecological signi¢cance. Appl. Environ. Microbiol. 68, 3509^3513. vanDuyl, F.C., deWinder, B., Kop, A.J. and Wollenzien, U. (1999) Tidal coupling between carbohydrate concentrations and bacterial activities in diatom-inhabited intertidal mud£ats. Mar. Ecol. Prog. Ser. 191, 19^32. Fischer, H. and Pusch, M. (1999) Use of the [14 C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton. Appl. Environ. Microbiol. 65, 4411^4418. Quinn, J.P. (1984) The modi¢cation and evaluation of some cytochemical techniques for the enumeration of metabolically active heterotrophic bacteria in the aquatic environment. J. Appl. Bacteriol. 57, 51^57. Flindt, M.R. and Nielsen, J.B. (1992) Heterotrophic bacterial activity in Roskilde Fjord sediment during an autumn sedimentation peak. Hydrobiologia 235/236, 283^293. Weinbauer, M.G., Beckmann, C. and Ho«£e, M.G. (1998) Utility of green £uorescent nucleic acid dyes and aluminum oxide membrane ¢lters for rapid epi£uorescence enumeration of soil and sediment bacteria. Appl. Environ. Microbiol. 64, 5000^5003. Coleman, A.W. (1980) Enhanced detection of bacteria in natural environments by £uorochrome staining of DNA. Limnol. Oceanogr. 25, 948^951. Choi, J.W., Sherr, E.B. and Sherr, B.F. (1996) Relation between presence-absence of a visible nucleoid and metabolic activity in bacterioplankton cells. Limnol. Oceanogr. 41, 1161^1168. Marxsen, J. (1996) Measurement of bacterial production in streambed sediments via leucine incorporation. FEMS Microb. Ecol. 21, 313^325. Gilmour, C.C., Leavitt, M.E. and Shiaris, M.P. (1990) Evidence against incorporation of exogenous thymidine by sulfate-reducing bacteria. Limnol. Oceanogr. 35, 1401^1409. Wellsbury, P., Herbert, R.A. and Parkes, R.J. (1993) Incorporation of [methyl-3 H]thymidine by obligate and facultative anaerobic bacteria when grown under de¢ned culture conditions. FEMS Microb. Ecol. 12, 87^95.
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[30] Bastviken, D. and Tranvik, L. (2001) The leucine incorporation method estimates bacterial growth eqally well in both oxic and anoxic lake waters. Appl. Environ. Microbiol. 67, 2916^2921. [31] McDonough, R.J., Sanders, R.W., Porter, K.G. and Kirchman, D.L. (1986) Depth distribution of bacterial production in a strati¢ed lake with an anoxic hypolimnion. Appl. Environ. Microbiol. 52, 992^ 1000.
[32] Pamatmat, M.M. (1982) Heat production by sediment: Ecological signi¢cance. Science 215, 395^397. [33] Pamatmat, M.M., Graf, G., Bengtsson, W. and Novak, C.S. (1981) Heat production, ATP concentration and electron transport activity of marine sediments. Mar. Ecol. Prog. Ser. 4, 135^143. [34] Wetzel, R.G. (1983) in: Limnology, 2nd edn., pp. 243^247. Saunders, Philadelphia, PA.
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www.fems-microbiology.org
Depth distribution of active bacteria and bacterial activity in lake sediment Ann-Louise Haglund b
a;b;
, Peter Lantz b , Erik To«rnblom b , Lars Tranvik
b
a Department of Public Technology, Ma«lardalen University, Box 883, SE-721 23 Va«sterafis, Sweden Department of Limnology, Evolutionary Biology Centre, Uppsala University, Norbyva«gen 20, SE-752 36 Uppsala, Sweden
Received 16 April 2003; received in revised form 10 June 2003; accepted 26 June 2003 First published online 12 August 2003
Abstract The bacterial activity in sediments is often low considering the generally high bacterial abundance. Still, a large fraction of bacteria have been found active even in deep sediments. These findings suggest that sediment bacteria have comparatively low cell-specific production. We studied bacterial activity and the active fraction of bacteria in a lake sediment profile. Bacterial production and metabolism were measured by thymidine and leucine incorporation and by microcalorimetry. In addition to counts of total bacteria, we estimated the nucleoid-containing fraction of the bacteria by adding a destaining step to the DAPI staining method, and the live fraction using the Live/ Dead Baclight bacterial viability kit. The bacterial activity and abundance decreased with sediment depth, while the proportion of active bacteria remained similar at all depths. Between 57 and 63% of the bacteria were scored viable, and 13^52% were scored as nucleoidcontaining cells. Consequently, there was no accumulation of dead bacterial cells in deeper sediments. Cell-specific production of sediment bacteria may be severely underestimated if the active fraction of the sediment bacterial community is not considered during enumeration. 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Bacterium ; Sediment ; Production; Metabolically active bacterium ; Cell-speci¢c production
1. Introduction Sediment bacteria constitute an important component of the benthic food web as well as of nutrient cycling and decomposition of organic matter. Bacterial activity and biomass are generally highest near the sediment surface and decrease with depth (e.g. [1]). Considering the generally high abundance of bacteria in sediments, the bacterial activity therein is often low. This indicates that the cell-speci¢c activity of sediment bacteria is low, or that only a small fraction of the bacterial community is metabolically active [2]. Several studies have reported relatively large proportions of active bacteria in sediments (e.g. [3]), which favors the hypothesis of low cell-speci¢c productivity. Even in deep sediments, there are viable bacteria (e.g. [4,5]), however, the metabolic state of sediment bacteria in depth gradients is not well known.
* Corresponding author. Tel. : +46 (18) 471 27 18; Fax : +46 (18) 53 11 34. E-mail address : [email protected] (A.-L. Haglund).
Fluorochromes such as acridine orange and 4P,6P-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) may overestimate the number of bacteria due to staining of dead cells and non-bacterial particles [2]. In addition, all cells are not actively participating in production and growth, which may lead to underestimates of cell-speci¢c growth rates using the traditional enumeration methods. There are several methods to distinguish live cells from dead cells but they have been used mostly on bacterioplankton. In this study, we employed two di¡erent methods to determine the number of viable bacteria, neither of which has been used on freshwater sediment bacteria before. We used Zweifel and Hagstro«m’s [6] method to detect cells containing nucleoids and the Live/Dead BacLight bacterial viability kit (Molecular Probes, Eugene, OR, USA) to separates live cells from dead cells. Zweifel and Hagstro«m [6] added a destaining step to the traditional DAPI staining procedure to avoid unspeci¢c staining of cells lacking nucleoids. The empty cells were referred to as ‘ghosts’ incapable of growth while the nucleoid-containing cells were considered viable and capable of metabolic activity.
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0168-6496(03)00190-9
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The Live/Dead BacLight bacterial viability kit is a twocolor £uorescence assay. Cells with intact membranes stain £uorescent green and are considered to be live while cells with damaged membranes stain £uorescent red. Combining several methods for bacterial activity and abundance, we examined the changes in the distribution of viable bacteria as well as the total and cell-speci¢c metabolic activity in depth pro¢le of a sediment.
2. Materials and methods 2.1. Sampling Seven sediment cores were taken in June 1999 at 12 m depth at an accumulation bottom in Lake Erken. Lake Erken is a relatively large mesotrophic lake situated 60 km north of Stockholm, Sweden (59‡51PN, 18‡35PE). The sediment cores were transported to the laboratory in darkness and stored at 4‡C until sliced. The cores were sliced at 6-mm intervals. Eight layers were saved for further analysis, taken at depths below the sediment surface of 0^6, 12^18, 24^30, 48^54, 78^84, 102^108, 162^168, and 222^228 mm. The sampling site is in an area with a sediment accumulation rate of 6 mm year31 (G. Weyhenmeyer, personal communication). Consequently, a core represents sediment accumulated over approximately the last 60 years, taking the compaction of the sediment into account. The sediments were muddy and had an organic content of approximately 18% [7]. After slicing, the sediment was analyzed within 24 h. Samples for DAPI staining were taken immediately after slicing and ¢xed with 4% formaldehyde. The rest of the staining and analyses was performed on live sediment. Water content was determined and content of carbon and nitrogen were measured using elemental microanalysis (LECO, CHNS 932) on all the layers in seven cores. 2.2. Abundance of live and dead bacteria We counted live and dead bacteria in the sediment using the Live/Dead BacLight bacterial viability Kit, which contains a mixture of SYTO0 9 and propidium iodide. The SYTO0 9 stain is a nucleic acid stain with green £uorescence, which labels all cells with intact cell membranes. Propidium iodide penetrates those cells that have damaged cell membranes, where it causes a red £uorescence and a reduction in the SYTO0 9 £uorescence when both dyes are present. Thus, when excited with blue light, the live cells, with intact cell membranes, will appear green. Dead cells, with damaged cell membranes, will appear red. Portions of 0.5 g of live sediment were transferred to glass vials and mixed with 4.5 ml of ¢lter-sterilized (0.2 Wm) water. The mixture was diluted 1000 times, and a 2.5ml subsample was mixed with 2.5 ml of water and soni-
cated for 90 s at 30 W using an ultrasonic cell disrupter (Microson1). Filter-sterilized (0.2 Wm) water was used in all dilutions. A 1:1 mixture of SYTO0 9 and propidium iodide was added (7.5 Wl), and the sample was incubated for 15 min. Thereafter, it was ¢ltered onto a 0.2-Wm black polycarbonate ¢lter (Micron Separations). The ¢lter was dried and placed on a slide in mounting oil (Molecular Probes) together with the 0.2-Wm cellulose acetate ¢lter (Lida), used as a drain disc during ¢ltration. Once mounted, ¢lters were immediately counted using epi£uorescence microscopy. A minimum of 400 cells, or at least 20 randomly chosen ¢elds, was counted on each slide. Only those cells where the stain was clearly visible were counted. The fraction of live bacteria was calculated as the abundance of live cells divided by the total count obtained with the Live/Dead method. 2.3. Total and nucleoid-containing bacterial abundance and biomass We used DAPI for total bacterial counts. A portion (0.1 g) of sediment was ¢xed with 9.9 ml of 4% ¢ltersterilized (0.2 Wm) formaldehyde. This mixture was diluted 1000 times and a 5-ml subsample was taken and sonicated for 90 s as described above. DAPI was added to a ¢nal concentration of 10 mg l31 and incubated for 30 min. Filtration, mounting on slides and counting were performed as described above. The DAPI-stained cells were visualized in UV light using epi£uorescence microscopy. To estimate bacterial biomass, photos of slides with DAPI-stained bacteria were taken. Photos of samples from all eight layers from three cores were used. To be able to determine the size of bacteria, the photos and a micrometer scale were projected on the wall and 50 cells from each slide were measured. The bacterial biomass was then estimated using the abundance and average biovolume assuming a carbon content of 105 fg C Wm33 [8]. The resulting average cell carbon content was used in all biomass calculations. An additional DAPI-stained subsample was subjected to a washing step to remove unspeci¢c DAPI staining [6] using warm (60^70‡C, 1 ml) 2-propanol. Then the ¢lter was dried for 5 min before mounting on a slide as described above. The fraction of nucleoid-containing bacteria was calculated as the abundance of nucleoid-containing cells divided by the total counts obtained with the DAPI method. 2.4. Bacterial activity Bacterial production was estimated using [3 H]thymidine and [14 C]leucine incorporation, while heat production using direct calorimetry was used to determine total sediment metabolism. For the [3 H]thymidine incorporation, a mixture of labeled and unlabeled thymidine was used at a ¢nal concen-
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tration of 1000 nM. Previous studies of Lake Erken sediment have shown thymidine saturation at 1000 nM [3]. By mixing labelled (81.0^83.0 Ci mmol31 ) [3 H]thymidine (Amersham, TRK 686) and unlabelled thymidine (Sigma) the ¢nal speci¢c activity obtained was 43.45^44.02 Ci mmol31 . Subsamples of sediment (0.2 g wet weight) were incubated in 10-ml centrifugation tubes. There were three replicates and one control for each sediment layer and three cores were analyzed. The controls were killed by adding 5 ml of 80% ethanol. Thereafter, [3 H]thymidine was added to each tube, and the samples were incubated at in situ temperature (12‡C) for 40 min. The incubations were terminated by adding 5 ml of 80% ethanol. Samples were kept cool (4‡C) until further processing. The samples were processed according to Bell and Ahlgren [9] and the radioactivity was detected using a liquid scintillation counter (LKB Wallac 1219 Rackbeta). Bacterial production was estimated from thymidine incorporation rates using a conversion factor of 2.0U1018 cells mol31 thymidine [10]. Incorporation of [14 C]leucine was measured following the procedure for sediments established by van Duyl and Kop [11] with a few adaptations. 0.2 g of sediment was incubated for 30 min with [14 C]leucine (Amersham, CFB 67) (94 mCi mmol31 ) in 10-ml centrifuge tubes (¢nal concentration 1000 nM). The incubation was performed in triplicate with one additional control killed with 5 ml of 80% ethanol. Incubation was terminated by adding 5 ml of 80% ethanol and the samples were stored at 4‡C overnight. Samples were then centrifuged (7500 rpm, 8500Ug) for 20 min, the supernatant was discarded and the pellet was washed with 5 ml of 80% ethanol. This procedure was repeated twice. The pellets were then washed with 5 ml of ice-cold trichloroacetic acid (5%), and after centrifugation (at 4‡C) the supernatant was discarded. This procedure was repeated twice. 2 ml of 2 M NaOH was added to each pellet and the samples were placed in a boiling water bath for 2 h followed by centrifugation for 20 min (7500 rpm, 8500Ug). 1 ml of the supernatant was then transferred to a scintillation vial and scintillation cocktail was added. The leucine concentration (1000 nM) was chosen based on a saturation experiment performed on sediment samples from the sampling site. Final concentrations in the saturation experiment ranged from 500 to 9000 nM. We measured heat production of the total sediment metabolism using a heat conduction multichannel microcalorimeter (TAM 2277, Thermometric) according to Bostro«m and To«rnblom [12], with a modi¢cation in order to maintain anaerobic conditions. Instead of using a smaller amount of sediment and an overlying water phase, 3-ml ampoules were completely ¢lled with sediment using a syringe. All measurements were performed at 12‡C and heat production values were recorded after an equilibration time of 10 h.
33
2.5. Statistical analysis One-way analysis of variance (ANOVA) was used to distinguish changes in the bacterial community with depth. Homogeneity of variances (Bartlett’s M2 ) and normal distribution of the dependent variable (Shapiro^Wilks test) were assured. When necessary, the data were log- or arcsin-transformed to achieve normal distribution. Spearman rank correlation was used when relating bacterial activity and abundance to depth.
3. Results The water content in the sediment surface layer (0^6 mm) was 95% and decreased to 86% in the deepest layer measured (222^228 mm) (Fig. 1A). The carbon content decreased from 10.3% at the surface to 7.7% of sediment dry weight in the deepest layer. The corresponding numbers for nitrogen content were 1.3% and 1.0%, respectively. The molar C:N ratio varied from 9.0 in the surface sediment to 8.7 at 48 mm depth and then it increased again to slightly over 9 in the deepest layer (Fig. 1B). The total number of bacteria followed the same general trend with both staining methods (DAPI and Live/Dead). Bacterial abundance was highest at the sediment surface and decreased with depth (P 6 0.001; b = 30.84 and b = 30.77 for DAPI total counts and Live/Dead total counts, respectively). The total bacterial abundance of DAPI-stained bacteria was 11.0U1010 cells (g dry weight)31 at the sediment surface, and 2.0U1010 cells (g dry weight)31 in the deepest layer (Fig. 2A). When adding
Fig. 1. (A) Water content (%) and (B) C:N ratio at di¡erent sediment depths (n = 7 for each depth). Error bars indicate standard deviation.
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Fig. 2. (A) Total bacterial counts (DAPI) and abundance of nucleoidcontaining cells (DAPI-destained) and (B) total bacterial counts (Live/ Dead) and abundance of live and dead cells at di¡erent sediment depths (n = 4 at each depth). Error bars indicate standard error.
the washing step with 2-propanol, the abundance decreased by more than 50%. The nucleoid-containing cell abundance at the surface was 4.3U1010 cells (g dry weight)31 and in the deepest layer 0.5U1010 cells (g dry weight)31 (Fig. 2A). The fraction of nucleoid-containing bacteria was not signi¢cantly di¡erent between depths (P = 0.083, ANOVA) and varied from 13 to 52% (Fig. 3) of the total counts obtained with DAPI staining. Total counts of bacteria using the Live/Dead method were lower than DAPI counts. The total abundance decreased from 4.0U1010 cells (g dry weight)31 in the most sur¢cial sediment to 1.8U1010 cells (g dry weight)31 in the deepest layer (Fig. 2B). The live cells were 2.5U1010 cells (g dry weight)31 at the surface and decreased to 0.7U1010 cells (g dry weight)31 in the deepest layer (Fig. 2B). The corresponding ¢gures for the dead bacteria were 1.5U1010
Fig. 3. Fraction of viable cells (%) at di¡erent sediment depths obtained with the Live/Dead method (¢lled symbols) and DAPI destaining method (open symbols) (n = 4 at each depth). Error bars indicate standard error.
cells (g dry weight)31 and 0.5U1010 cells (g dry weight)31 , respectively (Fig. 2B). The fraction of live or dead bacteria was not signi¢cantly di¡erent between depths (P = 0.31, ANOVA) but was rather similar at all sediment depths. The live fraction ranged from 57 to 63% of the total cell abundance obtained with the Live/Dead method (Fig. 3). DAPI staining resulted in considerably higher abundances compared to the Live/Dead method. This was visually apparent when the total DAPI counts were plotted against the Live/Dead total counts. At high abundances, the total DAPI counts were more than twice as large as the total Live/Dead counts (Fig. 4A). Interestingly, the nucleoidcontaining cells were strongly related to the live bacterial cells (r2 = 0.86) (Fig. 4B) although di¡erent total counts were used for the calculations. The slope of the regression was not signi¢cantly di¡erent from one (P 6 0.001, t-test). The bacterial activity generally decreased with depth (P 6 0.001; b = 30.80 and b = 30.68 for thymidine and leucine incorporation, respectively, P = 0.002; b = 30.53 for heat production) (Fig. 5). The [3 H]thymidine incorporation had a steeper decrease compared to [14 C]leucine incorporation, as seen from the decrease in the ratio between leucine and thymidine incorporation with increasing depth (Table 1). The [3 H]thymidine incorporation was 50.1 pmol (g dry weightUh)31 at the surface and declined to 3.8 pmol (g dry weightUh)31 at 222 mm depth. The corresponding values for [14 C]leucine incorporation were 3.0 and 0.27 nmol (g dry weightUh)31 , respectively. Heat production also decreased with depth but not in as pronounced a manner as the thymidine and leucine incorpo-
Fig. 4. (A) Total DAPI counts as a function of total Live/Dead counts and (B) nucleoid-containing cell abundance as a function of live cell abundance in a sediment pro¢le (n = 4 at each depth). Lines indicate 1:1 ratio.
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Fig. 5. Incorporation of (A) thymidine (n = 7 at each depth), (B) leucine (n = 4 at each depth) and (C) heat production (n = 5 at each depth) at di¡erent sediment depths. Error bars indicate standard error.
ration rates. At the surface heat production was 46.8 WW (g dry weight)31 and at 222 mm depth it declined to 26.7 WW (g dry weight)31 . The ratio between leucine incorporation and heat production £uctuated somewhat but it did not decline with depth until below the 102-mm layer (Table 1). The cell-speci¢c bacterial activity, as calculated by dividing the carbon production (derived from leucine incorporation) by the total carbon biomass (DAPI counts and cell volume estimates), varied from 0.04 to 0.22 day31 and was not signi¢cantly correlated with depth (P = 0.11; b = 30.29) (Fig. 6A). When the biomass of nucleoid-containing cells (Fig. 6B) or live cells (Fig. 6C) was used instead of the total bacterial biomass, cell-speci¢c activity Table 1 Ratio between mean leucine and thymidine incorporation (pmol (g dry weightUh)31 ) and mean leucine incorporation and heat production (WW (g dry weight)31 ) at di¡erent depths Depth (mm)
Leucine:thymidine
Leucine:heat
0 12 24 48 78 102 162 222
61 86 104 167 286 243 122 73
65 89 115 99 112 72 23 10
35
Fig. 6. Cell-speci¢c production (BP:BB) at di¡erent sediment depths calculated by dividing bacterial production (leucine incorporation) by (A) the total bacterial biomass (DAPI), (B) the nucleoid-containing cell biomass, and (C) the biomass of live cells (Live/Dead) (n = 4 at each depth). Error bars indicate standard error.
was considerably higher. When the biomass of live cells was used the speci¢c activity ranged between 0.12 and 0.83 day31 , decreasing with depth (P = 0.006; b = 30.47) (Fig. 6C). Calculations with nucleoid-containing cells resulted in cell-speci¢c activities from 0.31 to 0.74 day31 and there was no signi¢cant decrease with depth (P = 0.75; b = 0.06) (Fig. 6B).
4. Discussion Bacterial abundance generally decreases with sediment depth [1,13,14]. Interestingly, we found no signi¢cant difference in the fraction of live or nucleoid-containing bacteria among depths (Fig. 3). Hence, there was no accumulation of dead bacterial cells with depth, a fact also reported by Miskin et al. [4]. Viable bacteria are present also in deep sediments of the oceans. Parkes et al. [5] found a constant percentage of bacterial cells in a dividing stage down to 518 m in the deep sediments of the Paci¢c Ocean. The fact that there was no di¡erence in the fraction of live or nucleoid-containing bacteria with sediment depth indicates that grazing or viruses are not important regulators of bacterial mortality in these sediments. There are often high densities of protozoa in aquatic sediments (e.g. [15]). However, most grazing studies show a minor impact of £agellate grazing on the bacterial community
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(e.g. [16]). In addition, Hamels et al. [16] found that the importance of £agellate grazing on bacterial mortality decreased with grain size of the sediment. Viruses have been found to be numerous in freshwater sediments but the virus-to-bacteria ratio has generally been close to one or lower, i.e. signi¢cantly lower than in pelagic environments [17,18]. Accordingly, the two mortality factors generally considered to be responsible for most of the mortality of bacteria in the water column may not be e¡ective in the sediments. This may contribute to the persistence of intact, active cells throughout the studied sediment pro¢le. Apparently, the Live/Dead BacLight bacterial viability kit has not been used on sediment bacteria before. Luna et al. [19], who applied a similar method using SYBR Green I instead of SYTO 9, obtained live bacterial fractions between 26 and 30% in a coastal marine sediment pro¢le. Metabolically active fractions of bacteria in surface sediments between 4 and 67% are reported using the tetrazolium salts 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) [3,20,21] or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride [22,23] and nalidixic acid [22]. In the study on the viability of prokaryote communities in a post-glacial profundal freshwater sediment pro¢le, close to 100% of the bacteria in the sediment were active throughout the sediment core down to about 6 m, using the CTC method [4]. Since the CTC incubation was performed together with nutrient additions, this probably re£ects the viable fraction of bacteria, rather than the fraction that is metabolically active in the sediment. The fraction of live bacteria obtained in this study (57^63%) was higher than most proportions of metabolically active bacteria reported in sediments, but since dormant bacteria with intact membranes should be included in the live fraction, this is not surprising. Total DAPI counts were up to more than twice the total abundance with the Live/Dead staining (Fig. 4A). This is consistent with the reports of unspeci¢c staining of bacteria lacking nucleoids and non-bacterial particles with DAPI [2,24], and DAPI staining of non-bacterial particles such as mucus, £agellate mucocysts and polyphosphate granules, some of which may be erroneously recorded as bacteria [25]. Destaining of the DAPI samples according to Zweifel and Hagstro«m [6] resulted in large standard errors in our sediment samples (Fig. 3). The abundance of nucleoid-containing cells was often higher than the abundance of live cells, using the Live/Dead method, suggesting that some cells with broken cell membranes (scored dead with the Live/Dead staining) contained nucleoids. Similarly, Choi et al. [26] report a higher percentage of nucleoid-containing cells than live cells (Live/Dead method) in marine bacterioplankton. When adding peptone, the fraction of nucleoid-containing bacteria increased, although the total bacterial count was constant, which led to the conclusion that cells without visible nucleoids are not necessarily empty ghost cells [26]. Hence, nucleoid-containing bacteria, as detected after DAPI de-
staining procedure, seem to include some cells that are not viable, while other cells that are viable are not detected by this procedure. We report fractions of nucleoid-containing cells between 13 and 52%. Luna et al. [19] found fractions between 1.5 and 6.2% in marine sediments. These considerably lower fractions in marine sediments may be attributed to lower nutrient concentrations in the marine environment compared to our freshwater sediment. The nucleoid-containing fraction increased upon nutrient enrichment [19] as also reported by Choi et al. [26]. Estimates of cell-speci¢c growth rate and productivity are usually based on the total bacterial biomass and the total bacterial production. Since this and several other studies report that only a fraction of the total bacteria are viable or active and capable of growth, the cell-speci¢c production obtained using the total biomass is most likely underestimated. We calculated cell-speci¢c activity using three di¡erent biomasses, i.e. the total bacterial biomass (DAPI), the biomass of nucleoid-containing cells, and the biomass of live cells. The two latter calculations produce speci¢c activity estimates up to eight times higher (Fig. 6). Hence, the cell-speci¢c production of the viable fraction of the bacteria is considerably higher than implied from estimates based on total bacterial counts. Like the bacterial abundance, total bacterial activity decreased with depth. Several studies from river sediments show a similar pattern [1,14,27]. Bacterial activity declined with depth independent of the method used, but the steepness of the decline di¡ered. Thymidine incorporation declined most rapidly, which may re£ect the shortcomings of this method for deeper anaerobic sediments. Many anaerobic bacteria lack the ability to incorporate externally added thymidine into their DNA [28,29], which leads to an underestimation of the bacterial activity in anaerobic environments. In contrast to thymidine, incorporation of leucine re£ects bacterial production equally in aerobic and anaerobic waters [30]. The increase in leucine:thymidine ratio with depth may be due to an increasing relative contribution of anaerobic bacteria that are unable to incorporate thymidine. McDonough et al. [31] also report higher leucine :thymidine ratios in anoxic compared to oxic waters. Direct calorimetry (heat production) provides non-speci¢c estimates of the total metabolism of the sediment community. It measures the enthalpy changes in biological and chemical processes independently of their function and origin. Thus, the technique is especially useful with sediments that contain mixed communities of anaerobes, fermenters, and aerobes [32], but the method is seldom used due to the unspeci¢city in distinguishing between metabolically and chemically produced heat [33]. The leucine :heat ratio was fairly constant in the upper decimeter of the sediment, but considerably lower further down (Table 1). This implies that the deeper bacterial communities use more of their energy for respiration (and thus dissipate heat) than for protein production as compared to the bac-
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teria in the overlying sediment. The C:N ratio was somewhat higher in deeper sediment, which indicates a more refractory carbon source [34]. As a consequence, the bacterial community at these depths should have lower cellspeci¢c production. This is consistent with our estimates of productivity based on the total bacterial biomass and the live fraction of the bacteria, but not on the nucleoidcontaining fraction (Fig. 6). In conclusion, bacterial abundance and total activity decreased with increasing sediment depth, but the viable fraction of the total bacteria was similar throughout the sediment pro¢le (Fig. 3). Thus, organic particles, recognizable as bacteria, seem to be lost during the diagenesis of organic carbon in the sediment rather than being accumulated. We report up to eight times higher cell-speci¢c production for the viable bacterial community compared to traditional calculations using the total bacterial biomass. Hence, estimates of productivity and speci¢c growth rates of sediment bacteria may be highly biased if the non-viable fraction of the bacterial community is not considered.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements This work was supported by funds from Ma«lardalen University and the Swedish Research Council.
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