Gravimetric Dry Weight Estimation

Biomass production in milligrams dry weight (mg dw)/L of culture was estimated for flask cultures by collecting 10 ml of stationary phase cultures (19 dpi; WT, 29 dpi; LOG OX1) for three biological replicates each by a method described in an earlier study (Chioccioli et al., 2014). The culture was centrifuged (1,500 × g, 5 min) in sterile 15-ml tubes (Tarsons). The pellet was then gently washed with 2 ml of sterilized deionized water to remove the culture medium. The sample was pelleted again (1,500 × g, 5 min). The supernatant was carefully removed, and 1.5 ml of sterilized deionized water was used in several aliquots to re-suspend the pellet into pre-weighed 1.5-ml Eppendorf tubes. Cells were pelleted at 1,500 × g for 5 min, and the supernatant was discarded. The pellets were dried in a hot air oven (ThermoScientific, United States) at 60°C. Tubes were weighed on a precision balance (Shimadzu, Japan) to estimate the weight of the dried biomass. This experiment was repeated three times.