Materials and methods
Isolation of xylose assimilation yeast strains
Yeast strains were isolated from different natural
sources, including flowers, fruits, wood, and soil,
obtained from the Kyoto area in Japan (approximately
100 samples in total). The samples were collected in
sterilized polypropylene bottles of 15 mL capacity (Becton
Dickinson, Franklin Lakes, NJ, USA) and suspended
in 10 mL of SX liquid medium (3% xylose and 0.67%
YNB without amino acid; Difco, Detroit, MI, USA) containing
chloramphenicol at a concentration of 100 μg/
mL. The samples were cultivated for 4 days at 30°C in
static culture with the lids slightly opened. Aliquots (200
μL) of the culture supernatants were spread onto SX
agar medium and incubated aerobically at 30°C for 3
days. Yeasts were purified using single-colony isolation.
The yeast strains were routinely maintained on YPD
agar plates (2% glucose, 2% peptone [Difco], 1% yeast extract
[Difco] and 1.5% agar) and grown at 30°C. YPX
medium (4% xylose, 2% peptone [Difco] and 1% yeast
extract [Difco]) was used for the screening of xylosefermenting
yeast.
Selection of xylose-fermenting yeast with
thermotolerance
Prior to the fermentation experiments, strain ATY839
was inoculated into 3 mL of YPD medium in test tubes
and incubated overnight at 30°C with reciprocal shaking
at 150 opm (preculture). The preculture was suspended
to 25 mL of synthetic glucose (SG) medium (2% glucose
and 0.67% YNB without amino acid) or synthetic xylose
(SX) medium (2% xylose and 0.67% YNB without amino
acid) in a 50 mL Erlenmeyer flask to a cell optical density
of 0.1 at 600 nm (OD600) and then cultured at 35°C,
37°C, 38°C, or 39°C for 48 h at 120 rpm. Sugars and
ethanol concentrations of the culture supernatants were
determined by following the procedures detailed below.
Cell growth was determined at OD600 using a spectrophotometer.
All experiments were performed in
triplicate.
Materials and methods
Isolation of xylose assimilation yeast strains
Yeast strains were isolated from different natural
sources, including flowers, fruits, wood, and soil,
obtained from the Kyoto area in Japan (approximately
100 samples in total). The samples were collected in
sterilized polypropylene bottles of 15 mL capacity (Becton
Dickinson, Franklin Lakes, NJ, USA) and suspended
in 10 mL of SX liquid medium (3% xylose and 0.67%
YNB without amino acid; Difco, Detroit, MI, USA) containing
chloramphenicol at a concentration of 100 μg/
mL. The samples were cultivated for 4 days at 30°C in
static culture with the lids slightly opened. Aliquots (200
μL) of the culture supernatants were spread onto SX
agar medium and incubated aerobically at 30°C for 3
days. Yeasts were purified using single-colony isolation.
The yeast strains were routinely maintained on YPD
agar plates (2% glucose, 2% peptone [Difco], 1% yeast extract
[Difco] and 1.5% agar) and grown at 30°C. YPX
medium (4% xylose, 2% peptone [Difco] and 1% yeast
extract [Difco]) was used for the screening of xylosefermenting
yeast.
Selection of xylose-fermenting yeast with
thermotolerance
Prior to the fermentation experiments, strain ATY839
was inoculated into 3 mL of YPD medium in test tubes
and incubated overnight at 30°C with reciprocal shaking
at 150 opm (preculture). The preculture was suspended
to 25 mL of synthetic glucose (SG) medium (2% glucose
and 0.67% YNB without amino acid) or synthetic xylose
(SX) medium (2% xylose and 0.67% YNB without amino
acid) in a 50 mL Erlenmeyer flask to a cell optical density
of 0.1 at 600 nm (OD600) and then cultured at 35°C,
37°C, 38°C, or 39°C for 48 h at 120 rpm. Sugars and
ethanol concentrations of the culture supernatants were
determined by following the procedures detailed below.
Cell growth was determined at OD600 using a spectrophotometer.
All experiments were performed in
triplicate.
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