Extraction, Purification, and Estimation of Exopolysaccharide Production Under Salt and Arsenic Stresses

For estimation of EPS production under As stress, 100 μl of 24-h old culture of Halomonas sp. Exo1 was inoculated (0.1% v/v) into 250 ml Erlenmeyer flasks containing 100 ml of As-free and As-supplemented (2, 4, 6, 8 mM) liquid 8% TSMGM, mixed thoroughly and placed on a rotary shaker (125 rpm) at 30 ± 2°C for 5 days. For determination of the effect of salt stress on EPS yield of Halomonas sp. Exo1, 100 ml Erlenmeyer flasks each containing 50 ml of LB broth with varying concentrations of NaCl (0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 22.5 %) were inoculated with the bacterial strain. For extraction of EPS in all the treatments, 5-day old cultures were centrifuged at 7,000 rpm for 20 min. The supernatants were vacuum-filtered through cellulose nitrate filter. The EPS was precipitated from the cell-free supernatant by the addition of ice-cold ethanol (100%, Merck) in 1:3 ratios. The cell-free extract-ethanol mixture was shaken vigorously and incubated overnight at 4°C. For removal of salt and impurities, EPS was dialyzed for 24–48 h using 12–14 kDa MW cut-off dialysis bags against deionized H₂O at 4°C. A fraction of the partially purified EPS solution was dried at 60°C and weighed to determine crude EPS yield (Corzo et al., 1994). For further purification of EPS, methods described by Bales et al. (2013) were followed. In brief, the EPS solution was mixed with ice-cold 20% (w/v) trichloroacetic acid (TCA) solution to precipitate proteins and nucleic acids. After 30 min of incubation, the solution was centrifuged at 15,000 rpm for 1 h at 4°C. To the supernatant, 1.5 volume of 95% ethanol was added and the mixture was kept at −20°C for 24 h to facilitate precipitation of EPS from lipids. To collect the precipitated EPS, the solution was centrifuged at 15,000 rpm for 1 h at 4°C and the EPS pellet was re-suspended in Milli-Q H₂O and dialyzed against the same for 24 h at 4°C using a 12–14 kDa MW cut-off dialysis membrane to remove low molecular weight impurities. The remaining retentate containing purified EPS was lyophilized overnight and stored in sterile containers at −20°C until further analyses. The lyophilized powder of purified EPS was weighed again to determine pure EPS yield. In order to extract cell-bound EPS, the bacterial cell-pellet was re-suspended in 300 μl EDTA solution (10 mM EDTA + 1.5 mM NaCl) and heated at 50°C in water bath for 3 min. The bacterial cell suspension was again centrifuged and cell-free supernatant was decanted and EPS was purified as above. Finally, to understand the exact time-point of getting highest EPS yield, total EPS yield was calculated from cultures grown for 0, 3, 6, 9, and 12 days.