The composition and bacterial utilization of DOC released by phytoplankton

The primary production, quantity and molecular weight distribution of DOC released by phytoplankton, and the subsequent utilization and transformation of these compounds by bacteria in an estuary was studied. High primary production rate and DOC release was observed. The amount and composition of DOC released by phytoplankton aried with time and changing algal population. Low molecular weight products with MW less than 500, fraction with MW 10000 - 30000, and high molecular weightcompounds greater than 300000 dominated in the algal DOC. Bacteria utilized a significant portion of released DOC. The role of bacteria in the transformation of released DOC by algae is discussed

Microbial enzymatic assays in environmental water samples: impact of Inner Filter Effect and substrate concentrations

As microbial enzymatic activities initiate the mineralization of organic matter through the microbial loop, it is important to correctly measure those activities and be able to perform inter-study comparisons. Enzymatic activity assays are typically carried out using fluorogenic substrate analogs, such as 4-Methylumbelliferone and 7-Amino-4-methylcoumarin linked to sugar monomers, phosphate group or amino acids. However, methodological divergences can be found in aquatic science literature, potentially leading to misestimated activities. To highlight some of those methodological key points, we first addressed the potential occurrence of an Inner Filter Effect (IFE), a fluorometric artifact that affects the relationship between fluorophore concentration and fluorescence intensity, due to absorption of exciting or emitted light. It has never been considered in the context of environmental waterstudies before, despite significantly affecting measured activities. IFE occurred with 2 out of 3 tested spectrofluorometers when assaying proteases, although no IFE was detected for phosphatase assays. We also evaluated how substrate concentration ranges might affect kinetic parameters estimation, revealing that a many existing studies might use insufficient maximum substrate concentration. Finally, for single substrate concentration assays, we argued for the use of saturating substrate concentration, as naturally occurring substrates might compete with the fluorogenic analog at trace level. The amendment of a molecule mimicking natural substrates generated a significant inhibition of natural seawater phosphatases and proteases assayed with trace concentrations of fluorogenic substrate, while almost no inhibition occurred at higher concentrations. Those key points need to be addressed in order to assess enzymatic rates and allow inter-study comparison.

Input of Protein to Lake Water Microcosms Affects Expression of Proteolytic Enzymes and the Dynamics of Pseudomonas spp.

The objective of this study was to determine how an input of protein to lake water affects expression of a proteolytic potential and influences the abundance and composition of a specific group of bacteria. Pseudomonas spp. were chosen as a target group that can be recovered on selective growth media and contain both proteolytic and nonproteolytic strains. Amendment with 2 mg of casein per liter increased total proteinase activity (hydrolysis of [(3)H]casein) by 74%, leucine-aminopeptidase activity (hydrolysis of leucine-methyl-coumarinylamide) by 133%, bacterial abundance by 44%, and phytoplankton biomass (chlorophyll a) by 39%. The casein amendment also increased the abundance of culturable Pseudomonas spp. by fivefold relative to control microcosms but did not select for proteolytic isolates. Soluble proteins immunochemically related to the Pseudomonas fluorescens alkaline proteinase, AprX, were detected in amended microcosms but not in the controls. The expression of this class of proteinase was confirmed exclusively for proteolytic Pseudomonas isolates from the microcosms. The population structure of Pseudomonas isolates was determined from genomic fingerprints generated by universally primed PCR, and the analysis indicated that casein amendment led to only minor shifts in population structure. The appearance of AprX-like proteinases in the lake water might thus reflect a general induction of enzyme expression rather than pronounced shifts in the Pseudomonas population structure. The limited effect of casein amendment on Pseudomonas population structure might be due to the availability of casein hydrolysates to bacteria independent of their proteinase expression. In the lake water, 44% of the total proteinase activity was recovered in 0.22-μm-pore-size filtrates and thus without a direct association with the bacteria providing the extracellular enzyme activity. Since all Pseudomonas isolates expressed leucine-aminopeptidase in pure culture, proteolytic as well as nonproteolytic pseudomonads were likely members of the bacterial consortium that metabolized protein in the lake water