Diel Rhythm Does Not Shape the Vertical Distribution of Bacterial and Archaeal 16S rRNA Transcript Diversity in Intertidal Sediments: a Mesocosm Study.

In intertidal sediments, circadian oscillations (i.e., tidal and diel rhythms) and/or depth may affect prokaryotic activity. However, it is difficult to distinguish the effect of each single force on active community changes in these natural and complex intertidal ecosystems. Therefore, we developed a tidal mesocosm to control the tidal rhythm and test whether diel fluctuation or sediment depth influence active prokaryotes in the top 10 cm of sediment. Day- and nighttime emersions were compared as they are expected to display contrasting conditions through microphytobenthic activity in five different sediment layers. A multiple factor analysis revealed that bacterial and archaeal 16S ribosomal RNA (rRNA) transcript diversity assessed by pyrosequencing was similar between day and night emersions. Potentially active benthic Bacteria were highly diverse and influenced by chlorophyll a and phosphate concentrations. While in oxic and suboxic sediments, Thaumarchaeota Marine Group I (MGI) was the most active archaeal phylum, suggesting the importance of the nitrogen cycle in muddy sediments, in anoxic sediments, the mysterious archaeal C3 group dominated the community. This work highlighted that active prokaryotes organize themselves vertically within sediments independently of diel fluctuations suggesting adaptation to physicochemical-specific conditions associated with sediment depth.

Comparative sensitivity to the fungicide tebuconazole of biofilm and plankton microbial communities in freshwater ecosystems.

Stream and lake ecosystems in agricultural watersheds are exposed to fungicide inputs that can threaten the structure and functioning of aquatic microbial communities. This research analyzes the impact of the triazole fungicide tebuconazole (TBZ) on natural biofilm and plankton microbial communities from sites presenting different degrees of agricultural contamination. Biofilm and plankton communities from less-polluted (LP) and polluted (P) sites were exposed to nominal concentrations of 0 (control), 2 and 20 µg TBZ L(-1) in 3-week microcosm experiments. Descriptors of microbial community structure (bacterial density and chlorophyll-a concentration) and function (bacterial respiration and production and photosynthesis) were analyzed to chart the effects of TBZ and the kinetics of TBZ attenuation in water during the experiments. The results showed TBZ-induced effects on biofilm function (inhibition of substrate-induced respiration and photosynthetic activity), especially in LP-site communities, whereas plankton communities experienced a transitory stimulation of bacterial densities in communities from both LP and P sites. TBZ attenuation was stronger in biofilm (60-75%) than plankton (15-18%) experiments, probably due to greater adsorption on biofilms. The differences between biofilm and plankton responses to TBZ were likely explained by differences in community structure (presence of extracellular polymeric substances (EPS) matrix) and microbial composition. Biofilm communities also exhibited different sensitivity levels according to their in-field pre-exposure to fungicide, with P-site communities demonstrating adaptation capacities to TBZ. This study indicates that TBZ toxicity to non-targeted aquatic microbial communities essentially composed by microalgae and bacteria was moderate, and that its effects varied between stream and lake microbial communities.

Phage bacteriolysis, protistan bacterivory potential, and bacterial production in a freshwater reservoir: coupling with temperature.

Phage abundance and infection of bacterioplankton were studied from March to November 2003 in the Sep Reservoir (Massif Central, France), together with temperature, chlorophyll, bacteria (abundance and production), and heterotrophic nanoflagellates (abundance and potential bacterivory). Virus abundance (VA) ranged from 0.6 to 13 x 10(10) viruses l(-1), exceeding bacterial abundance (BA) approximately sixfold on average. In terms of carbon, viruses corresponded to up to 25% of bacterial biomass. A multiple regression model indicated that BA was the best predictor for VA (R(2) = 0.75). The frequency of infected bacteria (estimated from the percentage of visibly infected cells) varied from 1% to 32% and was best explained by a combination of temperature (R(2) = 0.20) and bacterial production (R(2) = 0.25). Viruses and flagellates contributed about equally to bacterial mortality. Both factors destroyed 55% of bacterial production, with a shift from phage bacteriolysis in early spring to protistan bacterivory in late summer. The vertical differences in most of the biological variables were not significant, contrasting with the seasonal differences (i.e., spring vs. summer-autumn). All biological variables under study were indeed significantly coupled to temperature. We regarded this to be the consequence of the enhanced discharge of the reservoir in 2003 (compared to previous years). This substantially weakened the stability and the thermal inertia of the water column, thereby establishing temperature as a stronger forcing factor in setting the conditions for optimal metabolic activity of microbial communities.

Microbial population dynamics in the sediments of a eutrophic lake (Aydat, France) and characterization of some heterotrophic bacterial isolates.

The bacterial populations of anoxic sediments in a eutrophic lake (Aydat, Puy-de-Dôme-France) were studied by phospholipid fatty acid analysis (PLFA) and also by culturing heterotrophic bacteria under strictly anaerobic conditions. The mean PLFA concentrations of prokaryotes and microeukaryotes were 5.7 +/- 2.9 mgC g(-1) DS and 9.6 +/- 6.7 mgC g(-1) DS, respectively. The analysis of bacterial PLFA markers was used to determine the dynamics of the Gram-positive and Gram-negative species of anaerobic bacteria, Clostridiae, and sulfate-reducing bacteria. Throughout the sampling period the concentrations of i15:0 (from 20 nmol g(-1) DS to 130 nmol g(-1) DS), markers of Gram-positive bacteria, were higher than those for Gram-negative bacteria. The dynamics of Clostridiae (Cy15:0) paralleled those of sulfate-reducing bacteria that were marked by i17:1omega7. Partial 16S rDNA sequencing and the physiological study of the various fermenting strains, whose abundance in the superficial sediment layer was 1.1 +/- 0.4 x 10(6) cells mL(-1), showed that all the isolates belonged to the Clostridiae and related taxa ( Lactosphaera pasteurii, Clostridium vincentii, C. butyricum, C. algidixylanolyticum, C. puniceum, C. lituseburense, and C. gasigenes). All the isolates were capable of metabolizing a wide range of organic substrates.

Regulation of b- and a-Glycolytic Activities in the Sediments of a Eutrophic Lake.

Temporal changes in a- and b-glucosidase activities, dissolved organic matter content, and bacterial biomass were studied in the superficial sediment layer of a eutrophic lake during the period of anoxia. The mean a- and b-glucosidase activities were 30.7 +/- 11.0 and 15.1 +/- 6.2 nmol h-1 g-1 of dry sediment, respectively. The specifc b-glucosidase activity seemed to be stimulated by carbohydrates (r = 0.80, P <0.05), whereas the specifc a-glucosidase activity was negatively correlated with the dissolved protein concentration (r = -0.72, P <0.10). To test the effect of organic matter on hydrolytic activities under controlled conditions, changes in specific activities were studied in relation to the concentrations of different types of organic matter: phytoplankton, polymers (proteins, cellobiose, and starch) and monomers (glucose and amino acids). The specifc a- and b-glucosidase activities were strongly induced by their natural substrates (starch and cellobiose, respectively) (P <0.05) and were not inhibited by glucose. Proteins inhibited these activities (P <0.05), whereas supplementation with amino acids had no effect on specifc glycolytic activities.

In vitro study of molecular weight, hydrophobicity and amino acid composition of peptides during breakdown of a casein hydrolysate by two strains of Prevotella ruminicola.

The molecular weight, amino acid composition and hydrophobicity of the peptide residue produced by hydrolysis of protein by two strains of Prevotella ruminicola (23 and S17/3) were determined. These last two characteristics could play a role in the control of proteolysis. Both strains produced dipeptidyl peptidases (DAP-1) but only P. ruminicola 23 synthesised alanine aminopeptidase. The area of 3-5 kDa peptides decreased, while the peptides directly assimilable by bacteria (0.5-1 kDa and < 0.5 kDa) increased with strain S17/3, but decreased with P. ruminicola 23. The amino acid compositions showed that the proportions of these compounds changed little with time and there was proline enrichment. Similarly, reverse phase HPLC showed no evidence of enrichment of the culture medium by hydrophobic peptides during the growth phase of P. ruminicola. These experiments show that the changes in the various peptide classes resulting from the hydrolysis and uptake of peptides by P. ruminicola differed with time and depended on the strain used. The nature of the enzyme activity and the use of other nitrogen sources may explain the difference between the two strains.

Breakdown of peptides from a casein hydrolysate by rumen bacteria. Simultaneous study of enzyme activities and physicochemical parameters.

The breakdown of a pancreatic hydrolysate of casein (tryptone) by an inoculum of ruminal mixed bacteria was studied in vitro. Peptides were degradated at 33% after 5 h. The dipeptidyl aminopeptidase type 1 (DAP-1), exoaminopeptidase and leucine aminopeptidase (LAP) like activities were measured using, respectively, Gly-Arg-MNA, Ala-pNa and Leu-pNA as substrates. While the total proteolytic activity remained stable throughout peptide breakdown, the DAP-1 already present at the beginning of fermentation increased until two times its early activity. This peptidase activity is a major index for the presence of Prevotella ruminicola. A peak of exoaminopeptidase and LAP activities were observed at 3 h incubation. This latter activity, which was associated with the production of lactate after 3 h, suggested that Streptococcus bovis was present in the environment. With the use of high-performance size exclusion chromatography (HPSEC), the size of the peptides during their breakdown could be measured, showing that high molecular weight peptides (4 to 2 kDa) were apparently more rapidly broken down to the profit of small-sized peptides (< 0.5 kDa), which increased. These degradation abilities were closely linked to DAP-1 activity. Separation of tryptone peptides by reverse-phase high-performance liquid chromatography (HPLC) showed that more hydrophilic peptides disappeared than hydrophobic peptides. Moreover, a few peaks eluting for one in the hydrophilic area and for the others in the hydrophobic area resisted the bacterial breakdown. In contrast, amino acid profiles indicated that hydrophobic amino acids were not taken up significantly faster than the hydrophobic amino acids by mixed ruminal bacterial.

Interactions between proteolytic and cellulolytic rumen bacteria during hydrolysis of plant cell wall protein.

During the degradation of the plant cell wall protein of dried alfalfa, interactions may occur between hydrolytic activities of cellulolytic (Ruminococcus albus or Fibrobacter succinogenes) and proteolytic (Prevotella ruminicola or Butyrivibrio fibrisolvens) bacteria. In vitro the hydrolysis of these protein compounds begins after the depolymerization of the cell wall polysaccharides has started. Maximal degradation of cell wall protein of dried alfalfa (37.2%) was obtained with cocultures of Prevotella ruminicola and Ruminococcus albus.