Abstract:

The data includes the protein and carbohydrate concentration of soluble extracellular polymeric substance (EPS) from Sagitulla Stellata (ss) of treatments in ASW (artificial seawater) during sunlight irradiation and in darkness for aggregates (> 0.2 µm) and the filtrate. Additionally, the data reflect the flow cytometry data for the ss for these light and dark ASW aggregates.

Suggested Citation:

Luni Sun,Luni Sun, Chen Xu, Saijin Zhang, Peng Lin, Kathleen A. Schwehr, Antonietta Quigg, Meng-Hsuen Chiu, Wei-Chun Chin, Peter H. Santschi. 2017. EPS mechanisms which aid in aggregation: A test of light to induce bacterial EPS aggregation, particle size of the EPS solution; protein and carbohydrate composition of aggregates and colloidal filtrate. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7BC3WKT

Purpose:

To examine the possibility of light-induced aggregation of bacterial EPS, we conducted irradiation experiments on a well-characterized protein-containing EPS from ss, a lignin-transforming marine gram-negative aerobic bacterium. The possible mechanisms are revealed by the ratios of protein to CHO.

Data Parameters and Units:

EPS= extracellular polymeric substance; ss= Sagitulla stellata; ASW= artificial seawater Dark: no UV exposure Light: under irradiation CHO = total carbohydrate concentration in mg/L Protein concentration in mg/L For the flow cytometry, time is in seconds; FSC=forward scattering, where A= area, H= height, W= width in arbitrary units; similarly, SSC=side scattering, where A= area, H= height, W= width in arbitrary units; FITC = Fluorescein isothiocyanate fluorescent dye; Alexa Fluor 647 is a fluorescent dye; # events signifies the number of particles were collected in the indicated time.

Methods:

CHO concentrations were determined by the anthrone method (Morris, 1948). 0.1 mL of sample was added in test tube, 0.2 mL of anthrone solution (1 g/L anthrone in concentrated sulfuric acid) was added to the sample or standard, the tube was heated in the heating block at 100 °C for 10 min. Then the samples were measured by UV-vis spectrometer at 620nm. Protein concentrations were determined by the Lowry Protein Assay Kit (Pierce, 23240, USA). The samples were measured by UV-vis spectrometer at 562nm. Irradiation. The ss EPS was dissolved in ASW with final concentration of 10 mg/L. The samples were filtered through 0.2 μm polycarbonate filter. The samples were irradiated in 250 mL borosilicate or quartz flasks outdoor in a water bath. The samples were irradiated under natural sunlight in the noon or in dark as control. Sample analysis. After irradiation, the samples were filtered through 0.2 μm filter, and the filter was washed with milli-Q water twice. Excessive salt was removed by ultrafiltration via Amicon Ultra-4 Centrifugal Filter Unit with a 3 kDa cut-off membrane (Millipore), and the samples were concentrated to 2 mL. Carbohydrate (CHO) and protein concentrations of this colloidal filtrate were measured. The filters from duplicate samples were combined for EPS composition analysis by re-suspending them in 4 mL 1% EDTA solution, followed by the incubation at 4 °C for 3 h on an orbital shaker at 150 rpm. After the extraction, the particles were removed using a Flipmate 100 System (0.45 µm PES, polyethersulfone, Environmental Express). Excessive EDTA was removed by ultrafiltration and the samples were concentrated to 500 µL. CHO and protein concentrations in this EDTA extractable aggregates (> 3 kDa) were measured. Flow cytometer particle size was captured using a laser set for natural fluorescence of green 515-545 nm (Ding et al., 2008). The collected data set was analyzed with FlowJo data analysis software package (TreeStar, USA).

Instruments:

UV-Vis: Biotech-Epoch UV-vis spectrometer Particle-size was determined by LSR2 flow cytometry (BD Biosciences; MoFlo, DakoCytomation; and inFlux, Cytopeia, Inc.).

Error Analysis:

Two measurements for filtrate, one measurement for aggregates. No standard deviation.

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