Alginate purification and quantification and free-uronic acid assay.

Two milliliters of bacterial overnight culture grown in LB medium supplemented with the appropriate antibiotic was sedimented, and cells were washed twice with saline solution. Cells were suspended in 1 ml of saline solution, and 200 μl of cell suspension (optical density at 600 nm [OD₆₀₀], 3.0) was plated onto a thick layer of PIA medium (40 ml in each plate) containing 300 μg/ml of gentamicin and then incubated at 37°C for 72 h, an incubation period that had previously been described and found to be optimal for cell growth and alginate quantification, avoiding nutrient deprivation and desiccation stress (11). Grown cells were scraped off from agar plates and suspended in saline solution until a homogenous suspension was formed. Then, suspensions were pelleted and supernatants containing alginate were precipitated with equal volumes of ice-cold isopropanol. The alginate precipitants were freeze-dried and then redissolved in 50 mM Tris-HCl (pH 7.4)–10 mM MgCl₂ to a final concentration of 0.5% (wt/vol), followed by incubation with 15 μg/ml DNase I and 15 μg/ml RNase I at 37°C for 6 h. Then, pronase E was added to a final concentration of 20 μg/ml, and the mixture was incubated at 37°C for a further 18 h. Alginate solutions were dialyzed (molecular mass cutoff, 12 to 14 kDa; ZelluTrans/Roth mini dialyzer; Carl Roth GmbH & Co.) against 5 liters of ultrapure H₂O for 48 h. Finally, alginates were precipitated with equal volumes of ice-cold isopropanol and freeze-dried for uronic acid assay and biochemical analysis, which was previously described in detail (9, 11, 48).

Assessment of free uronic acids was performed using 2 ml of overnight liquid culture. Cells were pelleted, and supernatants were filtered through a Vivaspin-500 (GE Healthcare) filter device with a molecular mass cutoff of 10 kDa. The uronic acids in the flowthrough, which consist of free uronic acids and short alginate degradation products, and total sample were measured according to a previously described procedure (49).