Abstract:
Enzyme activity, dissolved oxygen, particulate organic carbon (POC), dissolved organic carbon (DOC), total organic carbon (TOC) and particle size were measured on the last day of two mesocosm experiments performed with open ocean and coastal water. Enzyme activity, polysaccharide, proteins, uronic acids, POC, TOC and DOC were measured in the top, middle and bottom layer of the tanks, dissolved oxygen was measured only on the top and bottom layer while particle size was measured only from the middle layer.
Suggested Citation:
Manoj Kamalanathan, Chen Xu. 2017. Extracellular Enzyme Activity Profile in Chemically Enhanced Water Accommodated Fraction of Surrogate Oil from BP Oil Spill. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7348HTQ
Publications:
Kamalanathan, M., Xu, C., Schwehr, K., Bretherton, L., Beaver, M., Doyle, S. M., … Quigg, A. (2018). Extracellular Enzyme Activity Profile in a Chemically Enhanced Water Accommodated Fraction of Surrogate Oil: Toward Understanding Microbial Activities After the Deepwater Horizon Oil Spill. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.00798
Kamalanathan, M., Chiu, M.-H., Bacosa, H., Schwehr, K., Tsai, S.-M., Doyle, S., … Quigg, A. (2019). Role of Polysaccharides in Diatom Thalassiosira pseudonana and its Associated Bacteria in Hydrocarbon Presence. Plant Physiology, 180(4), 1898–1911. doi:10.1104/pp.19.00301
Purpose:
Measurement of enzyme activity allowed for determination of active conversion of the exopolysaccaride to particulate organic matter in control and Chemically Enhanced Water Accommodated Fraction of Oil (CEWAF) treatments.
Data Parameters and Units:
Relative fluorescence for enzyme activity, % saturation for Dissolved oxygen, mg/L for POC, TOC and DOC and polysaccharide proteins and uronic acids, and micrometer (um) for particle size. CA, CB and CC are three replicates of control MA, MB, and MC are three replicates of CEWAF-Chemically Enhanced Water Accommodated Fraction of Oil M3 = Mesocosm3 = GOMOO, a mesocosm using Gulf Of Mexico Open Ocean water, description in GRIIDC dataset doi:10.7266/N78P5XZD M4 = Mesocosm4 = GOMCOAST, a mesocosm using Gulf Of Mexico COASTal water; description in GRIIDC dataset doi:10.7266/N74X568X
Methods:
Enzyme assays. Enzyme activities for α and β glucosidase, alkaline phosphatase, leucine amino-peptidase and lipase were measured using substrates at a final substrate concentration of 0.2 mM that were dissolved in milli-Q. 0.4 mL of the substrates were added to 1.6 mL of seawater (control) and CEWAF samples in triplicate. The samples were then incubated at room temperature in dark for 3 hours. After incubation, the reactions were stopped by the addition of 1 mL aliquot of borate buffer solution (0.4 M) adjusted to pH 8.0 for AMC-tagged substrates or 10.0 MUF-tagged substrates. The fluorescence intensity was then measured at excitation/emission wavelengths (nm) of 380/440 (AMC) or 365/448 (MUF) for the hydrolytic products AMC or MUF using a spectrofluorometer (Shimadzu RF-5300). The measurements were then blanked with the values obtained using heated seawater samples (80°C for 15 min) in duplicate at the beginning of the incubation. Carbohydrate, protein and uronic acid analysis. EPS composition was determined in terms of carbohydrate, protein and uronic acid content. EPS from the samples were extracted by ultrafiltration using 1% EDTA. The anthrone method was used to determine the carbohydrate concentration in the EPS with glucose as the standard. Pierce BCA protein assay kit (Cat. # 23225) based on a modified bicinchoninic acid (BCA) method was used to measure the protein content of EPS with bovine serum albumin (BSA) as the standard. Estimation of uronic acids was done by addition of sodium borate (75 mM) in concentrated sulfuric acid and m-hydroxydiphenyl according to with glucuronic acid as the standard for this assay. Total Organic Carbon (TOC), Dissolved organic carbon (DOC), Particulate organic carbon (POC) analysis. Carbon contents were determined with a Perkin-Elmer CHNS 2400 analyzer. Acetanilide (70.09% C, 6.71% H, 10.36 %N) was used as analytical standard. The calibration was checked with Standard Reference Material 1941b and the error was well within 5% of the reported value (Xu et al., 2011). For dissolved organic carbon measurement, samples were measured with a Shimadzu TOC-L analyzer (Xu et al., 2011). Dissolved oxygen (DO) concentration. A calibrated 556 MPS YSI meter (Yellow Springs, Ohio) fitted with a DO/Temperature sensor (5563-10) was used to measure the DO in the surface and bottom of each mesocosm tank on the last day of each mesocosm experiment. Particle sizes. Aggregate analysis were performed using Z1 dual-threshold Coulter counter (Beckman Coulter). 15mL samples were taken every 24h and analyzed immediate. Particles of three different ranges (10-20µm, 20-50µm and >50µm) were counted with a 100µm aperture. Particles above the upper limit of the size range were also counted. A sample of filtered seawater was used as blank, less than 10 particles were counted, this ensured no contamination of the instrument. Samples were diluted with filtered seawater if the particle coincidence at the aperture exceeded 5%.
Error Analysis:
One way annova with tukey test was used
Provenance and Historical References:
Arnosti, C. (2011). Microbial extracellular enzymes and the marine carbon cycle. Annual review of marine science 3, 401-425. Hoppe, H.-G. (1993). Use of fluorogenic model substrates for extracellular enzyme activity (EEA) measurement of bacteria. Handbook of methods in aquatic microbial ecology, 423-431. Xu, C.; Zhang, S. J.; Chuang, C. Y.; Miller, E. J.; Schwehr, K. A.; Santschi, P. H., Chemical composition and relative hydrophobicity of microbial exopolymeric substances (EPS) isolated by anion exchange chromatography and their actinide-binding affinities. Marine Chemistry 2011, 126, (1-4), 27-36.
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