Methylmercury Degradation Activity Assay

September 20th, 2014

Methylmercury testing with encapsulated bacteria: Trial 1 – Purpose of experiment: determine if the mer operon in pBBRBB performs similar to pDU1358.

Added 7 mL of LB + Km (50μg/mL) to acid-washed glass Balch tubes

0.5 mL of encapsulated material containing the following strains were added to individual tubes: Strains tested: E. coli K12 pBBRBB::mer, P. putida pBBRBB::mer Negative Controls: E. coli K12 pBBRBB::gfp, P. putida pBBRBB::gfp Positive Control: P. putida pDU1358

Cultures were incubated with shaking at 250 rpm and 37C and 30C for E. coli and P. putida, respectively.

Samples were taken after 36 hours of incubation and diluted one-million fold for concentration analysis. Methylmercury concentrations were measured with a Tekran model 2700 Automated Methyl Mercury Analyzer using EPA method 1630 without distillation. All quality assurance and quality control measures were taken as outlined in EPA method 1630. All MeHg standards (ongoing precision recoveries) were within the acceptable range averaging 96%.

October 1, 2014

Methylmercury testing with encapsulated and non-encapsulated cells: Trial 2 – purpose of experiment: determine rate of methylmercury degradation by encapsulated and non-encapsulated cells

Added 7 mL of LB + Km (50μg/mL) to acid-washed glass Balch tubes

Inoculation: - For tubes containing encapsulated cells: 0.5 mL of encapsulated cell material were added to each tube - For tubes containing non-encapsulated cells: overnight cultures were washed with minimal medium and brought to an OD = 1.0. Tubes were then inoculated to an initial OD ~0.08. Strains tested: E. coli K12 pBBRBB::mer, E. coli K12 pBBRBB::gfp, P. putida pBBRBB::mer, P. putida pBBRBB::gfp, - Negative controls: Abiotic encapsulation material (to determine amount of methylmercury absorbed by beads), Abiotic medium (to determine the natural degradation rate for methylmercury), and E. coli K12 pBBRBB::gfp, P. putida pBBRBB::gfp -

Methylmercury chloride(0.1M) was added to each tube at 1 mg/L. Non-encapsulated cells were also tested at 1 mg/mL and 4 mg/mL starting concentrations to determine degradation rate at differing starting concentrations.

Cultures were incubated at 250 rpm and 37C or 30C for P. putida and E. coli K12, respectively.

Samples were removed after 1, 2, 4, 8, and 24 hours. Methylmercury concentrations were measured with a Tekran model 2700 Automated Methyl Mercury Analyzer using EPA method 1630 without distillation. All quality assurance and quality control measures were taken as outlined in EPA method 1630. All MeHg standards (ongoing precision recoveries) were within the acceptable range averaging 96%.