Structural and functional microbial diversity along a eutrophication gradient of interconnected lakes undergoing anthropopressure.

We present the results of an analysis of the 16S rRNA-based taxonomical structure of bacteria together with an analysis of carbon source utilization ability using EcoPlate (Biolog, USA) metabolic fingerprinting assessment against the backdrop of physicochemical parameters in fifteen interconnected lakes. The lakes exhibit a wide spectrum of trophic gradients and undergo different intensities of anthropopressure. Sequences of V3-V4 16S rRNA genes binned by taxonomic assignment to family indicated that bacterial communities in the highly eutrophicated lakes were distinctly different from the bacterial communities in the meso-eutrophic lakes (ANOSIM r = 0.99, p = 0.0002) and were characterized by higher richness and more diverse taxonomical structure. Representatives of the Actinobacteria, Proteobacteria, Cyanobacteria, Planctomycetes, Verrucomicrobia, Bacteroides phyla predominated. In most cases their relative abundance was significantly correlated with lake trophic state. We found no similar clear relationship of community-level physiological profiling with lake trophic state. However, we found some significant links between the taxonomic and metabolic structure of the microbes in the studied lakes (Mantel's correlation r = 0.22, p = 0.006). The carbon source utilization ability of the studied microorganisms was affected not only by the taxonomic groups present in the lakes but also by various characteristics like a high PO43- concentration inhibiting the utilization of phosphorylated carbon.

Quantitative description of respiration processes in meso-eutrophic and eutrophic freshwater environments.

We propose a modification of measurement methodology allowing the overall respiration rate (VResp) close to the in situ conditions; size of the labile, respirable organic matter pool (OMResp); and its turnover time (Tt) to be calculated. In addition to the respiration of dissolved substrates by free-living bacteria, the respiration of attached bacteria and other planktonic organisms is also taken into account. In case study we evaluated the modified, quantitative description of respiration processes in surface waters of lakes of different trophic status: mezzo-eutrophic and eutrophic. In both types of studied environments, VResp oscillated between 1.0 µmol C l-1 h-1 and 3.0 µmol C l-1 h-1, and the size of the OMResp pool varied from 39.3 µM C to 828.7 µM C. Despite of higher OMResp concentrations in eutrophic lakes, we found a lower susceptibility of OM to respiration processes in eutrophic than in meso-eutrophic lakes but similar VResp in both types of lakes. We conclude that the proposed method allows a fast quantitative description of labile organic matter utilization by aerobic aquatic microorganisms.

Coomassie Blue G250 for Visualization of Active Bacteria from Lake Environment and Culture.

Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary production or maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances for bacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.

Persistence of bacterial proteolytic enzymes in lake ecosystems.

This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems.

The dynamics of protein decomposition in lakes of different trophic status--reflections on the assessment of the real proteolytic activity in situ.

Abstract The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ proteolytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins (C(LAB)), and their half-life time (T(1/2)). Moreover, it gives more realistic information on resultant activity in situ (V(T(1/2))) of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are generally well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.

Urea and ureolytic activity in lakes of different trophic status.

Urea and uraease (U-ase) activity were determined in water samples taken from the surface layers of 17 lakes of different trophic status. Urea concentrations were inversely correlated with the trophic status of the studied lakes and varied from below the detection limit to 25 micromol l(-1). Maximal potential ureolytic activity (V(max)) ranged from 0.2 to 7.0 micromol l(-1) h(-1). The highest urea concentrations and the lowest U-ase activities were recorded in the spring, whereas the lowest urea concentrations and the highest rates of urea hydrolysis were observed late in summer, during heavy phytoplankton blooms. Since in the majority of the Great Mazurian Lakes microplankton growth was limited by nitrogen supply, urea was an important N source for both auto- and heterotrophic planktonic microorganisms throughout the growth period. U-ase activity was mainly related to the seston. Only up to 25% of total activity could be attributed to free enzymes dissolved in lake water. In epilimnetic water samples the bulk of the ureolytic activity originated from seston-attached bacteria. However, a positive, statistically significant correlation between ureolytic activity and chlorophyll a (Chl(a)) concentrations suggests that phytoplankton may also be responsible for at least a some of the observed ureolytic activity in the highly eutrophic Great Mazurian Lakes.