0.5 g L1. In the SHF variant of experiment yeast did consume half
of the available sugars in the fermentation feed after 24 h of the
process and almost all available sugars after subsequent days.
HPLC analysis of carbohydrates in media during the fermentation
also shown that almost all available glucose was utilized by yeast
by the second day of the process (Table 1). The content of maltotriose
throughout the fermentation was below 1 g L1 regardless
from the experimental variant. The dextrins were not completely
hydrolyzed and eventually utilized by yeast by the end of fermentation,
however its concentration decreased between the
24th and 48th hour and did not significantly change by the end
of the process. The changes in dissolved solids in the fermentation
media were similar to changes in the concentration of reducing
sugars in particular variants of experiment (Fig. 1b). The nondissolved
solids content after the first day of fermentation was
similar in all studied variants of experiment (ca. 30 g kg1) and
it did not significantly change by the end of the process
(Fig. 1c). The changes in ethanol formation during fermentation
tests were shown in Fig. 2a. It was observed that within the first
24 h of fermentation of enzymatically pretreated waste bread
with GSHE and separate hydrolysis and fermentation variant,
yeast produced the highest amount of ethanol (ca. 43.5 g L1).
Lower concentration of ethanol (ca. 38.8 g L1) was found after
the same time in unpretreated and sonificated waste bread samples,
while the lowest amount of alcohol (ca. 32.6 g L1) was
determined in media with subsequently microwave irradiated
waste bread. The production of ethanol by yeast in studied fermentation
tests lasted by the second day of the process and did
not significantly change by the third day. The lowest concentration
of ethyl alcohol was found in unpretreated samples
(51.58 g L1), different methods of raw material pretreatment
and the SHF process resulted in higher amount of ethanol by
the end of fermentation (ca. 55–58 g L1). The concentration of
fermentation by-products, glycerol and lactic acid were shown
in Fig. 2b and c respectively. Within the first 24 h of fermentation
yeast did produce ca. 90% of total glycerol in all studied variants
of experiment, however less glycerol was formed in media with
unpretreated and microwaved waste bread than in the case of
other studied methods. The final glycerol concentration was similar
in all studied fermentation media (ca. 6.0–6.5 g L1). The lactic
acid content in fermentation media was similar in all studied
variants of experiment throughout the process (ca. 1 g L1) without
major changes.
The improvement in fermentation dynamics, as described by
CO2 emission from fermentation media, for SHF and enzymatically
prehydrolyzed waste bread was probably caused by hydrolysis of
proteins present in raw material to short-chained peptides and
amino acids, which are essential for proper yeast metabolism [7]
and increased amount of fermentable sugars in comparison to
other variants. Previous study on the utilization of waste bread
for ethanol production proved that application of Ceremix 6X MG
enzyme blend, prior to a-amylase aided liquefaction, improved
the dynamics and ethanol yield of separate hydrolysis and fermentation
process [12]. Balcerek and Pielech-Przybylska [22] also
proved that addition of proteases increase the ethanol formation
rate in the native starch hydrolysis and fermentation process, especially
in its early stages. The increase in reducing sugars concentration
in GSHE aided fermentations in the early stages of
fermentation was typical for the simultaneous saccharification
and fermentation process. This was observed earlier [23,32] and
was caused by higher rate of enzymatic hydrolysis of sugars than
utilization of them by yeast. It was observed that not all dextrins
in the fermentation media were hydrolyzed and fermented by
the end of fermentation. This could be caused by enzyme
inactivation by high concentration of ethanol or decrease in the
0.5 g L1. In the SHF variant of experiment yeast did consume half
of the available sugars in the fermentation feed after 24 h of the
process and almost all available sugars after subsequent days.
HPLC analysis of carbohydrates in media during the fermentation
also shown that almost all available glucose was utilized by yeast
by the second day of the process (Table 1). The content of maltotriose
throughout the fermentation was below 1 g L1 regardless
from the experimental variant. The dextrins were not completely
hydrolyzed and eventually utilized by yeast by the end of fermentation,
however its concentration decreased between the
24th and 48th hour and did not significantly change by the end
of the process. The changes in dissolved solids in the fermentation
media were similar to changes in the concentration of reducing
sugars in particular variants of experiment (Fig. 1b). The nondissolved
solids content after the first day of fermentation was
similar in all studied variants of experiment (ca. 30 g kg1) and
it did not significantly change by the end of the process
(Fig. 1c). The changes in ethanol formation during fermentation
tests were shown in Fig. 2a. It was observed that within the first
24 h of fermentation of enzymatically pretreated waste bread
with GSHE and separate hydrolysis and fermentation variant,
yeast produced the highest amount of ethanol (ca. 43.5 g L1).
Lower concentration of ethanol (ca. 38.8 g L1) was found after
the same time in unpretreated and sonificated waste bread samples,
while the lowest amount of alcohol (ca. 32.6 g L1) was
determined in media with subsequently microwave irradiated
waste bread. The production of ethanol by yeast in studied fermentation
tests lasted by the second day of the process and did
not significantly change by the third day. The lowest concentration
of ethyl alcohol was found in unpretreated samples
(51.58 g L1), different methods of raw material pretreatment
and the SHF process resulted in higher amount of ethanol by
the end of fermentation (ca. 55–58 g L1). The concentration of
fermentation by-products, glycerol and lactic acid were shown
in Fig. 2b and c respectively. Within the first 24 h of fermentation
yeast did produce ca. 90% of total glycerol in all studied variants
of experiment, however less glycerol was formed in media with
unpretreated and microwaved waste bread than in the case of
other studied methods. The final glycerol concentration was similar
in all studied fermentation media (ca. 6.0–6.5 g L1). The lactic
acid content in fermentation media was similar in all studied
variants of experiment throughout the process (ca. 1 g L1) without
major changes.
The improvement in fermentation dynamics, as described by
CO2 emission from fermentation media, for SHF and enzymatically
prehydrolyzed waste bread was probably caused by hydrolysis of
proteins present in raw material to short-chained peptides and
amino acids, which are essential for proper yeast metabolism [7]
and increased amount of fermentable sugars in comparison to
other variants. Previous study on the utilization of waste bread
for ethanol production proved that application of Ceremix 6X MG
enzyme blend, prior to a-amylase aided liquefaction, improved
the dynamics and ethanol yield of separate hydrolysis and fermentation
process [12]. Balcerek and Pielech-Przybylska [22] also
proved that addition of proteases increase the ethanol formation
rate in the native starch hydrolysis and fermentation process, especially
in its early stages. The increase in reducing sugars concentration
in GSHE aided fermentations in the early stages of
fermentation was typical for the simultaneous saccharification
and fermentation process. This was observed earlier [23,32] and
was caused by higher rate of enzymatic hydrolysis of sugars than
utilization of them by yeast. It was observed that not all dextrins
in the fermentation media were hydrolyzed and fermented by
the end of fermentation. This could be caused by enzyme
inactivation by high concentration of ethanol or decrease in the
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