Microbial Extracellular Enzymes in Marine Sediments: Methods Development and Potential Activities in the Baltic Sea Deep Biosphere

The deep biosphere is defined as the subsurface ecosystem in which little energy is available to microorganisms and microorganisms can live for thousands of years. Heterotrophic microbes survive in the deep biosphere even though organic matter is limited and highly recalcitrant in nature. Measuring microbial extracellular enzyme activity provides a potential means to evaluate the rate at which microorganisms are performing carbon remineralization in the energy limited sediment beneath the seafloor. Extracellular enzymes breakdown organic compounds so that the nutrients can move inside the cell and be used for energy. This study explored the role extracellular enzymes play in the microbial metabolisms of the deep biosphere by 1) developing reliable methodology for microbial extracellular enzyme assays in aquatic sediments; and 2) measuring the activities of multiple carbon-degrading extracellular enzymes in Baltic Sea sediment. Test assays with fluorophore standards were performed on a BioTek Cytation 3 96-well plate reader and a Promega Glomax multi JR single cuvette fluorimeter and results were compared to determine which yielded the most high resolution and dependable results. The Promega single-cuvette fluorimeter proved to be the more precise method because instrumental drift over time, intrawell variation among replicates, and settling of slurry homogenate over long incubations were observed using the BioTek Cytation 3. Manually measuring fluorescence one cuvette at a time with a Promega Glomax multi JR lead to little instrumental drift, less variation among replicates, and a consistent shading effect that does not alter final results. Saturation curves created with the Promega Glomax multi JR obeyed Michaelis-Menten kinetics. Next, enzymatic activity measured in the Baltic Sea deep biosphere supported the hypothesis that microbes in the Baltic Sea subsurface utilize extracellular enzymes to acquire organic carbon, nitrogen, and phosphorus. Data suggest the microbial community in the Baltic Sea deep biosphere is seeking nitrogen and phosphorus via amino acids and phosphate more than it is seeking carbon. Enzyme activity is strongly correlated with organic matter content and depth. Residual activity in autoclaved sediments suggest that some enzymes can survive intense heat implying stability and possible hundred-year lifetimes in the deep subsurface biosphere of the Baltic Sea Basin.