Microorganism
Saccharomyces cerevisiae strain CCTCC M206111 was separated
from wine lees in our laboratory. The yeast strain was maintained
on malt wort agar medium at 4 C.
2.2. Inoculum culture
The yeast strain was added to 250 mL flasks containing 100 mL
of sterilized medium. The medium composition was as follows:
100 g/L glucose, 1.3 g/L (NH4)2SO4, 0.1 g/L MgSO4 and 8.5 g/L yeast
extract. The culture was incubated at 30 C for approximately 14 h.
The liquid cultures had an initial cell dry weight of 3.0e3.5 g/L.
2.3. Enzymes
The commercial enzymes for liquefaction (Liquozyme Supra,
alpha-amylase) and for saccharification (Suhong GA II, glucosidase)
were purchased from Novozymes. The amount of enzyme that can
hydrolyze 5.26 g soluble starch per hour at pH 5.6 and 37 C is
defined as 1 KNU (Kilo Novo alpha-amylase Unit). The amount of
enzyme that cleaves 1 mmol of maltose per min at pH 4.3 and 25 C
is defined as 1 AGU (Amyloglucosidase Novo Unit) (Novo, 1981).
Liquozyme Supra is a heat-stable a-amylase (EC 3.2.1.1) from
Bacillus licheniformis with a declared activity of 90 KNU/g. Suhong
GA II is a glucoamylase (EC 3.2.1.1) from Aspergillus niger with
a declared activity of 500 AGU/g.
2.4. Raw materials and fermentation culture
Fresh sweet potatoes were collected from Nanchong Agricultural
Research Institute, Sichuan, China. The sweet potato crops
were planted on May 29th, 2008 and harvested after growing for
100, 130 or 160 d.
The fresh sweet potatoes were cleaned and crushed into small
pieces (<3 mm). Then, water was impregnated in the various
hydromoduli of different varieties of sweet potato, which corresponded
to the initial total fermentable sugar concentration of
180 g/kg in all fermenters. After low-temperature cooking at 85 C
for 10 min, the mixture was liquefied using Liquozyme Supra
(10 min, 85 C, pH 5.2, 120 KNU/kg starch). All fermentations were
carried out using 250 mL Erlenmeyer flasks containing 100 g fresh
sweet potato hydrolysis. The initial pH of all media was adjusted to
5 with 1 M NaOH and 1 M HCl. The flasks with media were sterilized
by autoclaving at 115 C for 20 min prior to inoculation. Glucoamylase
was added at a dosage of 750 AGU/kg starch at the time
of inoculation. The ethanol fermentations were carried out at 30 C
under anaerobic conditions with agitation at 150 rpm.
2.5. Analytical methods
The total fermentable sugar content of the fresh sweet potatoes
was determined by acid hydrolysis (Wang et al., 2007) in which the
samples were treated with HCl at 100 C for 2 h and the amount of
reducing sugar was measured by the DNS method (3,5-dinitro
salicylic acid) using glucose as the standard (Maldonado and
Strasser de Saad, 1998). The crude fiber in the fresh sweet potato
is the acid and alkali-insoluble residue analyzed according to the
Chinese Standard GB/T 5009.10-2003. The pectic substances in
fresh sweet potato were extracted using the ethanol and water
method and determined via galacturonic acid (Walter et al., 1997).
Fermentation samples were taken from the fermenter and
centrifuged at 8000 rpm to remove any solids from the media. The
ethanol concentration was determined by gas chromatography, in
which 0.8 mL of a 10% sample supernatant filtered through
a 0.45 mm membrane filter (Millipore, USA) was mixed with 0.2 mL
of 10% n-propanol. All determinations were performed using
standard curves. A gas chromatograph (model FULI 9790; FULI
Corp., China) fitted with a flame ionization detector was operated
under the following conditions: gas column stainless steel column
(3.2 mm 2 m) packed with Chromosorb (FULI Corp., China);
temperature of injector and detector, 200 C; nitrogen carrier gas
flow rate, 30 mL/min; and temperature of column oven, 160 C (Liu
et al., 2007). The fermentation residue was measured by the
gravimetric method at 105 C until a constant weight was achieved.
The viscosity was measured using a rotational viscometer (DV-IIþ
PRO, Brookfield, USA) equipped with a recirculating water-bath (TC
200, Brookfield, USA) for control of the sample-container temperature.
The viscosity changes were determined at 30 C with
a paddle speed of 100 rpm. Every experiment was conducted in
duplicate, and the data presented represent mean values.
2.6. Statistical methods and calculations
The limitation of using root and tuber mashes for ethanol
production is attributed to their highly viscous nature. High
viscosity caused resistance to solideliquid separation and lower
fermentation efficiency (Srikanta et al., 1992), which will increase
the cost of ethanol production. Therefore, choosing a sweet potato
with low viscosity as the feedstock may be beneficial for reducing
the cost of ethanol production. According to several studies, the
viscosity of a pl
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