Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm³). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm. Received: 21 January 1999; Accepted: 12 April 1999     

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.     

The relationship between species richness and productivity in metazoan parasite communities

Biodiversity is not distributed homogeneously in space, and it often covaries with productivity. The shape of the relationship between diversity and productivity, however, varies from a monotonic linear increase to a hump-shaped curve with maximum diversity values corresponding to intermediate productivity. The system studied and the spatial scale of study may affect this relationship. Parasite communities are useful models to test the productivity-diversity relationship because they consist of species belonging to a restricted set of higher taxa common to all host species. Using total parasite biovolume per host individual as a surrogate for community productivity, we tested the relationship between productivity and species richness among assemblages of metazoan parasites in 131 vertebrate host species. Across all host species, we found a linear relationship between total parasite biovolume and parasite species richness, with no trace of a hump-shaped curve. This result remained after corrections for the potential confounding effect of the number of host individuals examined per host species, host body mass, and phylogenetic relationships among host species. Although weaker, the linear relationship remained when the analyses were performed within the five vertebrate groups (fish, amphibians, reptiles, mammals and birds) instead of across all host species. These findings agree with the classic isolationist-interactive continuum of parasite communities that has become widely accepted in parasite ecology. They also suggest that parasite communities are not saturated with species, and that the addition of new species will result in increased total parasite biovolume per host. If the number of parasite species exploiting a host population is not regulated by processes arising from within the parasite community, external factors such as host characteristics may be the main determinants of parasite diversity.     

Heterogeneous Cell Density and Genetic Structure of Bacterial Pools Associated with Various Soil Microenvironments as Determined by Enumeration and DNA Fingerprinting Approach (RISA)

Abstract The cell density and the genetic structure of bacterial subcommunities (further named pools) present in the various microenvironments of a silt loam soil were investigated. The microenvironments were isolated first using a procedure of soil washes that separated bacteria located outside aggregates (outer part) from those located inside aggregates (inner part). A nondestructive physical fractionation was then applied to the inner part in order to separate bacteria located inside stable aggregates of different size (size fractions, i.e., two macroaggregate fractions, two microaggregate fractions, and the dispersible day fraction). Bacterial densities measured by acridine orange direct counts (AODC) and viable heterotrophic (VH) cell enumerations showed the heterogeneous quantitative distribution of cells in soil. Bacteria were preferentially located in the inner part with 87.6% and 95.4% of the whole AODC and VH bacteria, respectively, and in the microaggregate and dispersible clay fractions of this part with more than 70% and 80% of the whole AODC and VH bacteria, respectively. The rRNA intergenic spacer analysis (RISA) was used to study the genetic structure of the bacterial pools. Different fingerprints and consequently different genetic structures were observed between the unfractionated soil and the microenvironments, and also among the various microenvironments, giving evidence that some populations were specific to a given location in addition to the common populations of all the microenvironments. Cluster and multivariate analysis of RISA profiles showed the weak contribution of the pools located in the macroaggregate fractions to the whole soil community structure, as well as the clear distinction between the pool associated to the macroaggregate fractions and the pools associated to the microaggregate ones. Furthermore, these statistical analyses allowed us to ascertain the influence of the clay and organic matter content of microenvironments on the genetic structure relatedness between pools. Received: 15 December 1999; Accepted: 5 April 2000; Online Publication: 19 May 2000     

Accuracy of Biovolume Formulas for CMEIAS Computer-Assisted Microscopy and Body Size Analysis of Morphologically Diverse Microbial Populations and Communities

Cell biovolume is a commonly used metric of microbial abundance analyzed by computer-assisted microscopy, but the accuracies of most biovolume formulas have not been validated by ground truth data. We examined the accuracy of 17 biovolume formulas by comparing the computed volumes of 3D models representing 11 microbial morphotypes (cocci, spirals, curved rods, U-shaped rods, regular straight rods, unbranched filaments, ellipsoids, clubs, prosthecates, rudimentary branched rods, and branched filaments) to the volume displacement of the same objects as ground truth. As anticipated, formula accuracy was significantly influenced by the morphotype examined. A few formulas performed very accurately (> 95 %), especially those that adapted to the cell’s shape, whereas others were consistently inaccurate or only accurate for one or two morphotypes. As an example of application, indices of morphological diversity in a freshwater biofilm assemblage were shown to be significantly different when microbial abundance among morphotype classes was measured as biovolume body mass rather than cell counts. Spatial analysis of biovolume body mass can also provide insights on the in situ ecophysiological attributes among individuals in microbial populations and communities, including their spatially autocorrelated allometric scaling interrelationships between body size, metabolic activity, resource apportionment and use, food web dynamics, and various cell-cell interactions affecting their growth and colonization behavior within spatially structured biofilm landscapes. This improved computing technology of biovolume algorithms with proven accuracy identifies which formula(s) should be used to compute microbial biovolumes in 2D images of morphologically diverse communities acquired by conventional phase-contrast light microscopy at single-cell resolution.     

Genetic Diversity of Viable, Injured, and Dead Fecal Bacteria Assessed by Fluorescence-Activated Cell Sorting and 16S rRNA Gene Analysis

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.     

Benthic algal response to N and P enrichment along a pH gradient

Nutrient enrichment and its effect on benthic algal growth, community composition, and average cell size was assessed across two sites of differing pH within a single habitat. Nutrients were added using in situ substrata, which released either N, P, or no additional nutrients (controls) at each site for 21 days. Upon collection, chlorophyll and biovolume standing stocks of the attached algal microflora were measured. Chlorophyll concentration was different among all treatments, accumulating greatest on P, followed by N, and the least on C substrata (P < 0.001) and was highest at site-2 (P < 0.001), while total algal biovolume was highest on P compared to both N and C substrata (P < 0.05) and did not vary between sites. Increased growth on P substrata was due to the enhanced biovolume of filamentous green algae, although the affected taxa varied between sites. Biovolume to cell density ratios (as a measure of average cell size) were highest on P substrata over both N-enriched and control substrata (P < 0.05) and this pattern was similar between sites. Progression towards a community composed of larger cells following P enrichment observed along this pH gradient, seems to be related to the dominance of larger celled filamentous green algae. Thus, nutrients exhibited greater control on benthic algal growth than did changes in hydrogen ion concentration.Contribution number 581, Great Lakes Environmental Research LaboratoryContribution number 581, Great Lakes Environmental Research Laboratory     

Phytoplankton community of floodplain lakes of the Araguaia River, Brazil, in the rainy and dry seasons

This work analyses the attributes of the phytoplankton community(species richness, biovolume, diversity, equitability, abundance,dominance and size structure) associated with some physical,chemical and climatic variables of floodplain lakes of the AraguaiaRiver, central Brazil, in the rainy and dry seasons of 2000and 2001. The lakes in 2000 and 2001 presented different limnologicalcharacteristics between the periods of high and low water. Thephytoplankton comprised 292 taxa, mainly Chlorophyceae and nanoplankton.In the dry seasons of both years, the functional group Y (Cryptophyceae)was the most prominent association. In high-water periods, differentfunctional groups dominated the lakes. The canonical correspondenceanalysis (CCA) demonstrated a separation between the rainy anddry seasons of both years. The high and low water presenteddifferent dynamics in 2 years due to the influence of the floodpulse.     

Diatoms and biomonitoring: should cell size be accounted for?

Despite the fact that biovolume calculation is a common procedure in most phytoplankton and periphyton studies, diatom community analyses are usually based on relative abundance data. In a biomonitoring context, a community metric that accounts for cell size could be of interest due to the potential differences that might exist in nutrient uptake between large and small-sized species. This paper addresses the question of whether diatom community analysis should be based on relative abundance, biovolume or cell surface. The results show that although community structure expressed as relative proportion of taxa varied according to the metric used, the ordinations conducted with each metric were similar. The explained percentage of species variance was slightly higher with the relative abundance metric compared to the metrics based on relative biovolume or cell surface area. Partial CCAs showed that each water chemistry variable generally explained a higher portion of species variance when the relative abundance was used. The analyses conducted with two size groups (small and large taxa) expressed as relative abundance and relative biovolume showed similar results. Moreover, our data showed that there is no significant relationship between diatom size and total phosphorus. According to these results, it seems that relative abundance would be the most appropriate metric to use for biomonitoring purposes. The biovolume and cell surface area calculations added substantially to the total analysis time due to the numerous measurements required, but did not improve the variance explained in community structure, and site ordinations were not significantly different. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.     

Infracommunity structure of parasites of Hemigymnus melapterus (Pisces: Labridae) from Lizard Island, Australia: the importance of habitat and parasite body size

This study describes the community of all metazoan parasites from 14 individuals of thicklip wrasse, Hemigymnus melapterus, from Lizard Island, Australia. All fish were parasitized, and 4,649 parasite individuals were found. Twenty-six parasite species were identified although only 6 species were abundant and prevalent: gnathiid isopods, the copepod Hatschekia hemigymni, the digenean Callohelmis pichelinae, and 3 morphotypes of tetraphyllidean cestode larvae. We analyzed whether the body size and microhabitat of the parasites and size of the host affected understanding of the structure of the parasite community. We related the abundance, biovolume, and density of parasites with the host body size and analyzed the abundances and volumetric densities of some parasite species within microhabitats. Although the 2 most abundant species comprised 75% of all parasite individuals, 4 species, each in similar proportion, comprised 85% of the total biovolume. Although larger host individuals had higher richness, abundance, and biovolume of parasites than smaller individuals, overall parasite volumetric density actually decreased with the host body size. Moreover, parasites exhibited abundances and densities significantly different among microhabitats; some parasite species depended on the area available, whereas others selected a specific microhabitat. Parasite and habitat size exhibited interesting relationships that should be considered more frequently. Considerations of these parameters improve understanding of parasite community structure and how the parasites use their habitats.     

施肥和刈割对亚高山草甸物种多样性与生产力及其关系的影响

施肥和刈割分别对植物群落物种多样性和生产力有重要的影响。采用不同施肥水平和刈割频度, 研究了施肥和刈割对亚高山草甸植物群落物种多样性与生产力及其关系的影响。结果显示, 5年的施肥和刈割处理对群落地上生物量均有显著影响, 但对物种多样性影响不显著; 物种多样性与生产力之间的关系因施肥和刈割次数的不同而不同, 有负相关、二次函数关系和不相关等几种类型, 多数为不相关。此结果表明施肥和刈割并不总是一致性地影响群落物种多样性与生产力。因此, 不同施肥和刈割处理下的亚高山草甸植物群落生产力与物种多样性之间并没有确定的关系。     

Cell size, cell cycle and transition probability in mouse fibroblasts

This paper describes the relationship between cell size and cell division in two situations. In the first, quiescent cells were sorted on the basis of cell size using a fluorescence-activated cell sorter and returned to culture. The results of this type of experiment are compatible with the idea that once cells have completed a size-dependent lag, the rate of entry of cells into S phase is controlled by a rate-limiting random event (or transition).The second kind of experiment follows the kinetics of complete cell cycles in rapidly proliferating cells whose mothers had been sorted on the basis of cell size. The cells born of small mother cells have longer cycle times than cells derived from large mothers. The difference in the cycle time of these two classes was due to differences in the B phase of the cell cycle [containing S, G2, M and part of G1 (G1B)], transition probability being the same in both size classes. Our results show that S, G2 and M are unaffected by size, thus confining the effect of size to G1B. It seems probable that the variability of B phase in cloned cell populations is partly due to variations of cell size at division, and correlations between the cycle times of sister cells result because sibling cells are more similar in size than unrelated cells. The major factor controlling cell division in mouse fibroblasts is shown, however, to be the transition probability; size has a more minor role.     

The relationship of diversity and biomass in phytoplankton communities weakens when accounting for species proportions

The relationship between productivity (or biomass) and species diversity in ecological communities remains a hotly debated topic. While much is already known about vascular plants, little is known in other types of organisms. We used a broad and standardized database of phytoplankton samples from the Czech Republic, containing 413 samples of various types of stagnant waters to evaluate this relationship. Biomass was characterized by the total biovolume/ml, the total number of individuals/ml and cells/ml all giving similar results. All these indicators spanned over five orders of magnitude while the number of species ranged between 1 and 57. Diversity was characterized by indices of Hill’s unified notation series progressively accounting for species proportion effects. The number of species showed an asymmetric unimodal relationship with biomass. The relationship weakened when considering diversity indices including species proportions. At very low productivity values (characterized by low biomass), diversity was probably restricted by the ability of algae and cyanobacteria to survive a lack of nutrients, in high productivities, by the competition for light. Medium productivities, where maximum diversity was found, exhibited large variability of diversity values (including very low ones), suggesting that low diversity of phytoplankton samples can be caused by multitude of factors.     

Spatial variation of arbuscular mycorrhizal fungi in two vegetation types in Gurbantonggut Desert

Geostatistical techniques were used to assess the spatial patterns of spores densities and biovolume of arbuscular mycorrhizal fungi (AMF) in soils from two contrasting vegetation communities: an Ephedra distachya-ephemeral plant vegetation community and an Eremurus anisopteris vegetation community. Also evaluated the relationship between the spatial distribution of spore densities and biovolume of AMF and soil properties. Spatial dependence of spore densities and biovolume of AMF were exhibited further by kriged maps. The results showed spore density and biovolume indicated strong spatial autocorrelation and a patchy distribution within both sites. However, the patch size of genera and biovolume of AMF differed between the two communities. The correlation between distribution of spore and biovolume of AMF and distribution of soil parameters was expressed by Spearman rank-correlations coefficients. These results suggest that spore or biovolume distribution of AMF was affected significantly by some soil properties.     

Effects of fertilization and clipping on species diversity,productivity and their relationship in subalpine meadow

施肥和刈割分别对植物群落物种多样性和生产力有重要的影响。采用不同施肥水平和刈割频度, 研究了施肥和刈割对亚高山草甸植物群落物种多样性与生产力及其关系的影响。结果显示, 5年的施肥和刈割处理对群落地上生物量均有显著影响, 但对物种多样性影响不显著; 物种多样性与生产力之间的关系因施肥和刈割次数的不同而不同, 有负相关、二次函数关系和不相关等几种类型, 多数为不相关。此结果表明施肥和刈割并不总是一致性地影响群落物种多样性与生产力。因此, 不同施肥和刈割处理下的亚高山草甸植物群落生产力与物种多样性之间并没有确定的关系。     

Stressor fluxes alter the relationship between beta‐diversity and regional productivity

Dispersal of organisms can influence the relationship between beta‐diversity and regional productivity in heterogeneous environments. However, many ecosystems are also linked by fluxes of stressors, with an unknown influence on this relationship. In this study, we assess the relationship between beta‐diversity (measured as Bray–Curtis dissimilarity) and regional productivity (measured as biovolume) under various levels of a stressor flux in meta‐ecosystems that were composed of two marine micro‐algae communities. We created heterogeneity by exposing one of the two communities to a herbicide and manipulated regional diversity by applying a dispersal gradient, which decreased beta‐diversity. We applied four stressor flux levels, which homogenized the herbicide concentration between the communities over time. The stressor flux changed the relationship between beta‐diversity and regional productivity by changing the effect of dispersal on regional productivity. In absence of the stressor flux, the relationship between beta‐diversity and regional productivity was mostly not significant, but positive at the end of the experiment. This positive relationship was generated by a negative effect of dispersal on regional productivity, probably because dispersal disrupted local dynamics by removing organisms from the most‐productive unstressed community. In presence of the stressor flux, the relationship between beta‐diversity and regional productivity was often negative as dispersal now increased regional productivity. Dispersal increased regional productivity by increasing the productivity of the stressed community. This positive effect was stronger in the presence than in the absence of the stressor flux because the stressor flux reduced the concentration of the herbicide in the stressed community, where it facilitated recovery. Our study shows that stressor fluxes can strongly interact with the effects of dispersal on productivity and thus influence diversity–productivity relationships.     

Tree Diversity,Environmental Heterogeneity,and Productivity in a Mexican Tropical Dry Forest1

Species diversity–environmental heterogeneity (D–EH) and species diversity–productivity (D–P) relationships have seldom been analyzed simultaneously even though such analyses could help to understand the processes underlying contrasts in species diversity among sites. Here we analyzed both relationships at a local scale for a highly diverse tropical dry forest of Mexico. We posed the following questions: (1) are environmental heterogeneity and productivity related?; (2) what are the shapes of D–EH and D–P relationships?; (3) what are individual, and interactive, contributions of these two variables to the observed variance in species diversity?; and (4) are patterns affected by sample size, or by partitioning into average local diversity and spatial species turnover? All trees (diameter at breast height ≥5 cm) within twenty‐six 0.2‐ha transects were censused; four environmental variables associated with water availability were combined into an environmental heterogeneity index; aboveground standing biomass was used as a productivity estimator. Simple and multiple linear and nonlinear regression models were run. Environmental heterogeneity and productivity were not correlated. We found consistently positive log‐linear D–EH and D–P relationships. Productivity explained a larger fraction of among‐transect variance in species diversity than did environmental heterogeneity. No effects of sample size were found. Different components of diversity varied in sensitivity to environmental heterogeneity and productivity. Our results suggest that species' differentiation along water availability gradients and species exclusion at the lowest productivity (driest) sites occur simultaneously, independently, and in a scale‐dependent fashion on the tree community of this forest.     

Physiological Status and Community Composition of Microbial Mats of the Ebro Delta, Spain, by Signature Lipid Biomarkers

Abstract Physiological status of microbial mats of the Ebro Delta (Tarragona, Spain) based on the extraction of lipids considered ``signature lipid biomarkers' (SLB) from the cell membranes and walls of microorganisms has been analyzed. Data from a day–night cycle show significant differences in viable cells countings (PLFA cells counts) ranging from 1.5 × 10¹⁰ to 5.0 × 10¹⁰ cells g⁻¹ of sediment. Minimum values were observed at 18:00 and 6:00, when physicochemical conditions change drastically. The diversity of the microbial community was assessed by GC/MS analysis of phospholipid fatty acids (PLFA). The ratio of PLFA, representative of Gram-negative bacteria, comprises 47.8% of the total PLFA of the microbial mat community. The remaining PLFA was representative of Gram-positive (10.0%), anaerobic (5.7%), and eukaryotic microorganisms (5.7%), and other common lipids. Two different approaches were used as a comparative study to assess the physiological status of the microbial mats. Two parameters (cyclopropane fatty acids/ω7c monoenoic fatty acids, and measurement of the trans/cis monoenoic PLFA ratio) showed a minimum at midnight, suggesting the highest microbial activity. Higher values were observed at 18:00 and 6:00, coinciding with lower PLFA cell counts. Received: 14 May 1999; Accepted: 6 September 1999; Online Publication: 24 March 2000     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997