Characterization of SSF of pretreated switchgrass by K. marxianus
IMB strains and S. cerevisiae D5A
The glucose concentration in all SSFs, shown in Fig. 1, increased
during the first 4 h indicating that hydrolysis of glucan to glucose
was occurring faster than ethanol production shown in Fig. 1. After
4 h, ethanol production occurred faster than hydrolysis, resulting
in a decrease in glucose concentration. At 48 h, IMB 2 had reduced
the glucose concentration to <0.01 g/L. IMB 1 and IMB 3 also reduced
glucose concentrations below 0.3 g/L at 48 h. IMB 1 and
IMB 2 maintained these glucose concentrations through 72 h,
while IMB 3 maintained low glucose concentration through 96 h,
indicating longer fermentation than other IMB strains. In all IMB
SSFs, hydrolysis continued throughout the entire experiment. After
96 h, the glucose concentration in all IMB SSFs increased until the
end of the experiment. The lowest glucose concentration after
168 h was 2.5 g/L in IMB 3 SSFs. The highest glucose concentration
was 8.8 g/L in IMB 4 SSFs.
Glucose concentrations in SSFs carried out with S. cerevisiae D5A
were similar to glucose concentrations in IMB 1, IMB 2, and IMB 3
through 72 h. After 72 h, however, the glucose concentration in S.
cerevisiae D5A SSFs continued to decrease and reached <0.01 g/L
at 144 h. The concentration remained at <0.01 g/L until the end
of the experiment, indicating fermentation by S. cerevisiae D5A
had not ceased.
Cellobiose concentrations in SSFs, shown in Fig. 1, were similar
to the glucose concentrations before 72 h. The cellobiose concentration
increased rapidly from 0 to 4 h and then decreased as ethanol
production increased. Unlike glucose in IMB SSFs, cellobiose
concentrations continued to decrease after ethanol fermentation
had slowed because of b-glucosidase activity in the Fibrilase enzyme
mixture. All SSFs had final cellobiose concentrations of
<0.5 g/L.
Acetic acid (Fig. 1), glycerol (not shown), and succinic acid (not
shown) were produced by all strains at concentrations below 2 g/L.
IMB 1 accumulated the most acetic acid, 1.7 g/L, at 168 h. The most
glycerol produced was 1.3 g/L at 96 h by IMB 3. The most succinic
acid produced was 0.6 g/L by IMB 5 at 72 h. The maximum percentage
of glucose consumed to produce metabolic products was 85.7%,IMB 1; 78.0%, IMB 2; 90.1%, IMB 3; 74.9%, IMB 4; 67.6%, IMB 5; and
98.3%, D5A. The percentage of glucose recovered as products or
remaining in solution as glucose or cellobiose was 95.2%, IMB 1;
88.9%, IMB 2; 95.1%, IMB 3; 94.3%, IMB 4; 94.7%, IMB 5; and
99.1%, D5A.
The pH of each SSF was recorded at 168 h and the mean pH for
each strain was calculated. The final mean pH values ± one standard
deviation in increasing order were IMB 1, 4.56 ± 0.02; IMB
2, 4.60 ± 0.05; IMB 3, 4.64 ± 0.07; IMB 5, 4.67 ± 0.03; IMB 4,
4.71 ± 0.03 (mean of only two pH values); S. cerevisiae D5A,
4.79 ± 0.06. One SSF with IMB 4 resulted in a failed fermentation.
The final concentration of acetic acid produced by each strain in order from highest to lowest was in the same order as increasing pH.
This suggests that acetic acid production is partially responsible for
pH reduction during SSF.
All five of the IMB strains produced more than 15 g/L of ethanol
at 72 h (Fig. 1). The highest ethanol concentration achieved by an
IMB strain was 19.5 g/L by IMB 3 at 144 h. All strains, except IMB
4, had a similar pattern for ethanol production during the initial
72 h. At 24 h, the concentration of ethanol produced by IMB 4,
5.9 g/L, was lower than the other four strains, which were between
8 and 10 g/L. However at 48 and 72 h, all five strains produced similar
concentrations of ethanol. Fermentation by IMB 1, IMB 2, IMB
4, and IMB 5 slowed after 72 h. This was reflected by glucose concentrations,
which begin to increase after 72 h (Fig. 1), indicating
glucan hydrolysis was occurring faster than fermentation. IMB 3
fermentation remained faster than hydrolysis until 96 h, as is evidenced
by the glucose concentration remaining near 0 g/L. Ethanol
production by S. cerevisiae proceeded similarly to the IMB strains
through 72 h, after which, ethanol production continued until the
end of the experiment, reaching a maximum concentration of
21.9 g/L at 168 h.
At 72 h, glucan to ethanol yields by all IMB yeasts had achieved
between 60% and 70% MTY (Table 1). IMB 3 achieved an ethanol
yield of 77% MTY at 96 h, but 96 h ethanol yields by IMB 1, IMB
2, IMB 4, and IMB 5 remained below 70% MTY. At 168 h, ethanol
yields for the IMB strains ranged from 67% MTY for IMB 4 to 80%
MTY for IMB 3. S. cerevisiae D5A achieved ethanol yields of 83%
MTY at 96 h and 92% MTY at 168 h. At 72 h and afterward, ethanol
yield for S. cerevisiae D5A was greater than ethanol yields for IMB 1,
IMB 2, IMB 4, and IMB 5 (p < 0.05) (Table 1). At 72 h, ethanol yield
for S. cerevisiae D5A was also greater than that for IMB 3. At 96 and
120 h, ethanol yield for S. cerevisiae D5A was not different than ethanol
yield for IMB 3, but at 144 and 168 h, S. cerevisiae D5A’s ethanol
yield was greater than IMB 3’s. IMB 3 was chosen for use in
subsequent experiments because it continued fermentation longer
than the other IMB strains and compared best to the S. cerevisiae
D5A control in terms of ethanol yield.
Previous research has found similar ethanol production results
at elevated temperatures to those of the K. marxianus strains used
in this study. Lark et al. (1997) performed SSF on recycled paper
sludge using a strain of K. marxianus at 38 C and reported an ethanol
yield of 72% MTY from cellulose after 72 h. Ballesteros et al.
(2004) achieved ethanol yields ranging from 50% to 72% MTY from
glucan using K. marxianus CECT 10875 in SSFs of lignocellulosic
material at 42 C. Using K. marxianus IMB 3 at 45 C in SSFs of pretreated
straw, Barron et al. (1997) achieved ethanol yields between
75% and 86% MTY using cellulose. In Suryawati et al. (2008), SSFs of
switchgrass with similar conditions using IMB 4 resulted in 16.6 g/
L of ethanol at 72 h and a 79% MTY at 168 h. Ethanol production
using IMB 4 in this study was slightly lower achieving 15.3 g/L of
ethanol at 72 h and a 67% MTY at 168 h.
The reasons that the IMB strains did not maintain fermentation
activity throughout the duration of these experiments are not
clear. The cause is unlikely ethanol inhibition because the IMB
strains have shown the ability to maintain cell growth and ethanol
production in solutions containing more than 75 g/L ethanol (Barron
et al., 1995), more than three times the concentration produced
in these experiments. Deficiency of certain nutrients may explain
the shortened fermentation time, however, in a previous study
using the same media used here, increasing the nutrient concentration
threefold reduced ethanol production during SSF by K.
marxianus IMB 4 (Suryawati et al., 2008). The effect of individual
media components on ethanol production by K. marxianus IMB
strains has not been reported. Increasing the number of cells added
at the beginning of the SSF may also improve ethanol yield and
increase fermentation time due to the increased number of viable
cells.
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