Extraction and Purification of Exopolysaccharides from Geobacillus

The Geobacillus sp. strain WSUCF1 media was prepared in a 1000 mL flask containing 6 g of shredded corn stover, 20 g yeast extract, 3 g NaCl, and 1000 mL of distilled (DI) water [9 (link)]. The corn stover, yeast extract, and NaCl solution was first made homogeneous using a magnetic stir bar with the pH set to 7.0, then allowed to autoclave at 121 °C for 20 min. Once the media had cooled, the flask was inoculated with the WSUCF1 bacteria and placed in a shaker set at 60 °C for 24 h. After 24 h, the liquid culture was passed through a Büchner funnel with the bottom of the funnel lined with a 0.22 µm pore size Whatman paper filter to separate the corn stover from the culture.
The bacterial cells were separated from the biofilm matrix-containing supernatant, which includes the EPS, via centrifugation at 8000× g for 20 min in 50 mL tubes or sometimes 500 mL centrifuge bottles. To separate the media from the EPS further, an optional step involves allowing the supernatant to evaporate via rotary evaporator until approximately 250–300 mL of water was removed. To precipitate the crude EPS, a 1:1 ratio of absolute ethanol was added to the remaining supernatant and allowed to sit in a freezer overnight at −20 °C. Lastly, the crude EPS was obtained by centrifuging the ethanol/supernatant solution at 8000× g for 40 min [9 (link)]. Following collection, the crude EPS was stored at −20 °C without a cryoprotectant.