The methods for cellular disruption of S. obliquus biomass and
sugar extraction were performed using 2.5 g of wet biomass
(approximately 0.5 g of dried weight).
The biomass was processed with physical methods (sonication,
bead-beating, autoclaving (temperature with pressure) and
homogenization), and with physicochemical processes consisting
in alkaline and acid hydrolysis (NaOH, HCl and H2SO4) in autoclave.
Sonication experiments were done with the biomass suspended
in 5 mL water and submitted to a sonication apparatus Labsonic L.
B. Braun (with probe 1000 L B. Braun at 200 W), for 30 s, followed
by an ice bath for 10 min. This procedure was done five times for
each sample.
The bead-beating treatment was carried out in a biomass suspension
in 10 mL of water to which 900 mg of glass beads were
added (Sigma, 425–600 lm). The biomass was stirred in a vortex
for 1 min followed by 1 min in an ice bath. This method was repeated
five times.
The homogenization assays were done suspending the biomass
in 5 mL of water and then processing in a Heidolph DIAX homogeneizer
at 24,000 rpm for 2.5 min, followed by 10 min in an ice bath.
This mode was repeated two times.
In the physico-chemical cell disruption methods, the biomass
was suspended in 5 mL of NaOH, HCl or H2SO4 in closed test tubes.
The suspensions were autoclaved at 120 C and 1.2 bar for 30 min.
A similar procedure using 5 mL of water allowed evaluating the
combined effect of temperature and pressure.
All the described treatments were performed in duplicate.
In order to evaluate the effect of biomass moisture on cell disruption,
the previously mentioned methods were also performed
with dried biomass (oven at 80 C until constant weight).