The Reinfelder lab and high school students from Malcolm X Shabazz High School (MXSHS) in Newark, NJ were awarded a Rutgers Raritan River Consortium Mini-grant to conduct field studies of ecoenzymes along the Raritan River Bay. Uptake experiments of the neurotoxic methylmercury (MeHg) into a freshwater phytoplankton using natural river water were conducted to evaluate the influence of enzyme hydrolysis on MeHg bioavailability. Participating researchers included Professor John Reinfelder (Dept. of Environmental Sciences, SEBS), Professor Jeffra Schaefer (Dept. of Environmental Sciences, SEBS), Professor Grace Saba (Dept. of Marine Science, SEBS), Dr. Philip Sontag (Dept. of Environmental Sciences, SEBS), undergraduate student intern Harold Ofori, and high school instructor Pat Murry and the MXSHS Biogeochemistry team.
Ecoenzymes are produced by microbes, located outside of cells (Sinsabaugh et al. 2009), and are catalysts for hydrolysis reactions which are responsible for organic matter degradation (Arnosti et al. 2009). Methylmercury, an organic and toxic form of mercury (Hg), undergoes bioaccumulation in phytoplankton cells which can be related to the chemical conditions of aquatic ecosystems (Watras and Bloom 1992). Dissolved organic matter (DOM) is known for controlling speciation of Hg in aqueous environments. The conversion of large molecular weight DOM into smaller, more usable sizes by ecoenzymes may change the availability of dissolved MeHg and other toxic metals bound to these compounds in the environment.
The scientific motivation for this research is to evaluate the influence of Raritan River discharge on ecoenzyme activity in both filtered and unfiltered Raritan River-Bay water and test the effect of a model enzyme (β-glucosidase) spike on the entrance of MeHg into phytoplankton cells using filtered Raritan River water in laboratory experiments. We propose that increased Raritan River discharge into Raritan Bay will bring an abundant source of organic matter and nutrients thus increasing microbial enzyme activity. Hydrolysis of Raritan River DOM by β-glucosidase may increase the bioavailability of MeHg and thus uptake of this compound into phytoplankton cells.
Fluorescence of model alpha (α-glucosidase) and beta (β-glucosidase) glucose sugar enzyme substrates and a single peptidase enzyme substrate leucine-aminomethylmcoumarin (leu-aminopeptidase) were measured during hydrolysis of these model compounds to determine the ecoenzymes’ activities in the Raritan River-Bay. Multi-year measurements of enzymes activities in the Raritan Bay were used to assess the impact of seasonal Raritan River discharge on the activities of these enzymes in 2017 and 2018 spring/summer and the biogeochemistry of organic matter between these two years. Ecoenzyme activities were used as a measure of the microbial demand for carbon and nutrients available along the Raritan River. The relatively high β-glucosidase activity measured in the dissolved phase at site RR3.5 was chosen for laboratory incubations in which Raritan River water with natural DOM was spiked with HeMg only (control) or subjected to β-glucosidase hydrolysis for >48 hours prior to MeHg addition. These experimental manipulations were designed to assess the effect of the hydrolysis of Raritan DOM on the availability of DOM-bound MeHg to phytoplankton.
The educational motivations for this study include the continuation of ongoing ecoenzyme research with high school students in underserved communities and the employment of an undergraduate intern to gain experience in field sampling, enzyme and molecular biology. Future aims for student work are to continue to optimize a molecular-screening protocol for mercury resistance and to sample along the Raritan River during different spring and summer seasons.
For more information, contact Dr. Philip Sontag at philip.sontag@rutgers.edu.
Graphics provide by Dr. Sontag.
Dr. Sontag presented preliminary findings on this research at the 10th Annual Sustainable Raritan River Conference and Awards Ceremony, June 8, 2018. Click on the link to view his presentation titled, Examining Influence of Raritan River-Bay Dissolved Organic Matter (DOM) and Extracellular Enzymes on Methylmercury (MeHg) in Phytoplankton.