Accepted Manuscript A novel steam explosion sterilization improving solid-state fermentation performance Zhi-Min Zhao, Lan Wang, Hong-Zhang Chen PII: DOI: Reference:
S0960-8524(15)00781-6 http://dx.doi.org/10.1016/j.biortech.2015.05.099 BITE 15070
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
17 April 2015 29 May 2015 30 May 2015
Please cite this article as: Zhao, Z-M., Wang, L., Chen, H-Z., A novel steam explosion sterilization improving solidstate fermentation performance, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech. 2015.05.099
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1
A novel steam explosion sterilization improving solid-state
2
fermentation performance
3
Zhi-Min Zhao a, b, Lan Wang a,*, Hong-Zhang Chen a
4
a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
5 6
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
7 8 9 10 11 12 13 14 15 16 17
Abbreviations: SE, steam explosion; SIT, sterilization indicator tape; SSF,
18
solid-state fermentation; SmF, submerged fermentation; SM, solid medium; SES, steam
19
explosion sterilization; CTS, conventional thermal sterilization; CFU, colony-forming
20
units; FTIR, Fourier transform infrared spectroscopy; DM, dry medium.
21
*Corresponding author. Tel.: 86-01082544978.
22
E-mail address: [email protected] (Wang L.)
1
1
2
Abstract Traditional sterilization of solid medium (SM) requires lengthy time, degrades
3
nutrients, and even sterilizes inadequately compared with that of liquid medium due to
4
its low thermal conductivity. A novel sterilization strategy, high-temperature and
5
short-time steam explosion (SE), was exploited for SM sterilization in this study.
6
Results showed that SE conditions for complete sterilization were 172 °C for 2 min and
7
128 °C for 5 min. Glucose and xylose contents in medium after SE sterilization
8
increased by 157% and 93% respectively compared with those after conventional
9
sterilization (121 °C, 20 min) while fermentation inhibitors were not detected. FTIR
10
spectra revealed that the mild SE conditions helped to release monosaccharides from the
11
polysaccharides. Bacillus subtilis fermentation productivity on medium after SE
12
sterilization was 3.83 times of that after conventional sterilization. Therefore, SE
13
shortened sterilization time and improved SM nutrition, which facilitated fermentability
14
of SM and should promote economy of solid-state fermentation.
15
Keywords: sterilization; steam explosion; nutrition; solid medium; image processing
16
17
18
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2
1
1. Introduction
2
Sterilization is an operation essential to all industrial fermentation processes
3
requiring pure culture maintenance (Deindoerfer, 1957). It is defined as the complete
4
elimination or destruction of all forms of microbial life by either physical or chemical
5
processes (Liu et al., 2013b). In submerged fermentation (SmF), moist heat sterilization
6
is widely used and it is effective to lead to the microbes death due to the high thermal
7
conductivity of liquid medium. Solid-state fermentation (SSF) is considered as a
8
promising process for production of various products such as fuel, feed, industrial
9
chemicals and pharmaceutical products (Pandey, 2003). Lignocellulosic biomass
10
converted by SSF for biofuels or biomaterials production has attracted substantial
11
interests from both the government and private sectors due to the abundance and
12
renewability of raw materials as well as the urgent demand for energy (Li & Chen, 2014;
13
Liu et al., 2013a). Unlike liquid medium, the complete sterilization of SSF medium has
14
issue itself due to its low thermal conductivity and multi-compositions, especially in the
15
industrial process.
16
Many sterilization methods including dry heat sterilization, moist heat sterilization,
17
ultrasonic sterilization, microwave sterilization, radiations, and chemical disinfectants
18
sterilization are currently being used according to the property of medium. Mechanisms
19
and characteristics of these common sterilization methods are shown in Table 1.
20
Because heating the medium, usually by steam, is the most reliable and the easiest to
21
control on a large scale, it is the method of choice throughout the fermentation industry. 3
1
Spherical digester has been commonly used for sterilization of solid medium (SM) in an
2
industrial scale. The SM is heated to 121 °C and kept for at least 20 min by the injected
3
saturated steam in spherical digester, and then naturally cooled. The spherical digester
4
rotates to accelerate heat transfer during the whole process, in which the
5
microorganisms are killed by the moist heat. However, the main drawbacks of this
6
technique include long cycle time, incomplete discharge, and energy waste, which
7
results in high capital cost and low process efficiency (Zhang et al., 2007). Previous
8
study also pointed out that heat processing, especially for a long time, has an adverse
9
effect on nutrients since thermal degradation of nutrients can and does occur (Lund,
10
1988). Therefore, the lengthy time, degraded nutrients, and energy waste are the main
11
problems of traditional thermal sterilization of SM.
12
According to the Arrhenius equation, rate constant of chemical degradation is less
13
influenced by temperature than that of microbial inactivation due to the fact that the
14
activation energy of chemical degradation is lower than that of microbial inactivation
15
(Mann et al., 2001; Mei et al., 1999). Therefore, the sterilization process using a high
16
temperature and short time strategy could make it possible to minimize the breakdown
17
of nutrients while kill the microorganisms. Mann et al. (2001) studied the thermal
18
sterilization of heat-sensitive products. It was suggested that the levels of degradation
19
can be substantially reduced by employing high-temperature short-time sterilization.
20
The work of Kjellstrand et al. also suggested that high-temperature short-time strategy
21
should be used for heat sterilization to minimize the development of the breakdown
4
1
products (Kjellstrand et al., 1995). Steam explosion (SE) is one of the most efficient
2
treatment methods for lignocellulosic biomass and has been developed into commercial
3
scale (Chen & Liu, 2007). Solid materials in SE reactor are heated by high-pressure
4
saturated steam for a relatively short time (normally 2-8 min). Until reaching the desired
5
residence time the reaction system was depressurized instantaneously. This is a typical
6
high-temperature short-time process. In SE process, there are high-temperature steam
7
heating step and explosion step (Liu et al., 2013c). Besides the high-temperature heating,
8
the structure change of materials by physical collision in explosion step may also help
9
to destroy the microbial cells.
10
In this study, we aim to exploit a novel and potential sterilization strategy,
11
high-temperature and short-time steam explosion (SE) process, for solid medium.
12
Effects of SE temperature and time on sterilization performance were studied and the
13
SE conditions for complete sterilization were determined. Kinetics of SE sterilization
14
was established and the action of explosion step in SE was investigated. Nutrients
15
content in medium was considered as an important metric to evaluate the sterilization
16
performance. Additionally, fermentation performance was compared between SE and
17
conventional thermal sterilization (121 °C, 20 min) to examine the feasibility of SE
18
sterilization. Finally, sterilization efficiency and energy utilization were analyzed from
19
the point of industrial scale.
5
1
2. Materials and methods
2
2.1. Experimental materials
3
The SM prepared for sterilization consisted of 200 g wheat bran, 20 g soybean
4
meal, 5 g glucose, 10 g CaCO3 and 200 mL of 2.5% KH2PO4, 1.25% (NH4)2SO4, 0.05%
5
MnSO4 inorganic salt solution. The initial moisture content of the SM was adjusted to
6
60% (w/w). Chemicals were purchased from Beijing Chemical works.
7
2.2. Steam explosion sterilization (SES)
8 9
Steam explosion sterilization (SES) was performed in a 2.0 L self-designed batch vessel which mainly consisted of a reactor chamber, a reception chamber and a steam
10
generator (Figure 1). During sterilization, the SM was top-loaded into the reactor
11
chamber and treated at constant saturated steam pressure until reaching the set residence
12
time. The reaction system was then terminated with a suddenly explosion by the ball
13
valve into the sterilized reception chamber. The materials were then prepared for
14
analysis. The SE severity factor can be calculated according to the following equation
15
(Liu et al., 2013d; Overend et al., 1987):
16
= ∗ exp (
.
)
(1)
17
Where R0 is the severity factor; t is the residence time, min; and T is the holding
18
temperature, °C. Table 2 lists the experimental conditions of SE and the corresponding
19
severity factor.
20
Conventional thermal sterilization (CTS) was performed as control. The SM with 6
1
same components was loaded into a 2.0 L Erlenmeyer flask and put into a vertical
2
autoclave (YXQ-LS-50A, Shanghai Boxun Industry & Commerce Co., Ltd). The
3
temperature and holding time were set to 121 °C for 20 min and 128 °C for 5 min,
4
respectively. All these experiments were conducted with two replicates.
5
2.3. Determination of viable cell number
6
5 g of SM after sterilization treatment was mixed with sterilized water (1:20, w/v)
7
in a 250 mL Erlenmeyer flask and shaken at 150 rpm for 30 min at 37 °C. Then, 100 µL
8
of the mixture was inoculated in duplicate in PDA and LB plate’s surface. After 36 h of
9
incubation at 37 °C, the number of colonies was determined and expressed as
10
colony-forming units (CFU).
11
2.4. Characterization of color-changing of sterilization indicator tape (SIT)
12
Two sterilization indicator tapes (purchased from 3M Company) with about 6 cm
13
length which were embedded in the SM were put into the SE reactor. Color of the heat
14
sensitive area would change if the SIT withstood moist heat by steam. It is known that
15
the gray value of the white is 255 while that is 0 of black in RGB images. The darker
16
the color is, the smaller the gray value is. Digital image processing was used to analyze
17
the color-changing of SIT. The sterilization indicator tapes before and after sterilization
18
treatment were photographed using a FUJIFILM FinePix Z20fd camera with same focal
19
length and under constant illumination. Images were acquired at a resolution 3648×
20
2736 pixels. The digital image processing was carried out by using the commercial 7
1
software ImageJ 1.44 (National Institutes of Health, USA) and MatLab 7.1 (MathWorks
2
Inc., USA). The schematic representation of image processing and analysis procedure
3
was illustrated in Figure 2A. The first step was threshold segmentation of the RGB image
4
of SIT after sterilization treatment according to the brightness by using ImageJ 1.44.
5
Through the segmentation, brightness of the heat sensitive area was retained while that
6
of background was changed to be 255. Once the threshold segmentation was performed,
7
the heat sensitive area and the background were distinguished significantly. Then the
8
image was converted into grayscale image. The average gray value of heat sensitive
9
area was calculated by automated protocol which was developed in MatLab 7.1 (Couri
10
et al., 2006; Duan et al., 2012).
11
2.5. Analysis of nutrients and inhibitors after sterilization
12
4 mL of the mixture in Section 2.3 was centrifuged at 8000 rpm for 10 min at room
13
temperature. The contents of glucose, xylose and inhibiting compounds such as acetic
14
acid, formic acid, furfural, and hydroxymethylfurfural (HMF) in the supernatants were
15
analyzed by high-performance liquid chromatography (HPLC; Agilent 1200, USA)
16
equipped with a refractive index detector. 10 µL samples were loaded on an Aminex
17
HPX-87H column (6.2 ×.250 mm, 5 µm), eluted with 0.5 mM H2SO4 solution at a
18
flow rate of 0.6 mL/min and operated at 55 °C. The content of each compound was
19
expressed as g/g dry medium (DM).
8
1
2
2.6. Fourier transform infrared spectroscopy (FTIR)
In order to identify and characterize the SM before and after sterilization, FTIR
3
spectra were studied between 4000 and 400 cm-1 using a FTIR-8400S spectrometer
4
(Shimadzu, Japan). Discs were prepared by mixing 2 mg of dried SM samples with 200
5
mg of KBr in an agate mortar and pressing 150 mg of the resultant mixture at 8 ton for 8
6
min. Discs were analyzed using 32 scans at a resolution of 4 cm-1. The characteristic
7
bands were assigned according to the literatures. In addition, a semi-quantitative
8
analysis of the FTIR spectra was carried out. In this analysis, the band at 1515 cm-1
9
(aromatic skeletal vibration) was considered as the reference band. Thus, the Ax/A1515
10
ratio represented the relative intensity of the specific band to the reference band at 1515
11
cm-1 (Casas et al., 2012).
12
2.7. Fermentation experiment
13
Bacillus subtilis PFK1302 used in this study was kindly provided by Hunan
14
PERFLY-Bio Co., Ltd., China. It was stored in LB medium slant at 4 °C. Bacillus
15
subtilis PFK1302 was precultivated in 100 mL liquid medium including 4% (w/w)
16
glucose, 1% (w/w) peptone, 1% (w/w) yeast extract, 1% (w/w) CaCO3 and 0.05% (w/w)
17
MgSO4 at 37 °C and 150 rpm for 24 h before inoculation.
18
25~40 g of SM after sterilization was cooled down and inoculated by adding 0.4
19
mL seed solution per gram of dry medium. Solid-state fermentation were carried out in
20
100 mL Erlenmeyer flasks and incubated at 37 °C for 72 h. At intervals of 24 h, 3~5 g
9
1
of SM were sampled to determine the bacteria amount and nutrients content similarly as
2
described in Sections 2.3 and 2.5.
3
3. Results and discussion
4
3.1. Evaluation of sterilization effect by SIT image processing
5
In order to evaluate the sterilization effect conveniently and choose a proper
6
parameter to predict the sterilization performance, the color-changing of SIT under
7
different SE conditions were investigated. SIT is exposed in steam moist heat in the SE
8
sterilization process. The greater the heat intensity, the more obvious is the
9
color-changing of SIT, which results in the smaller gray value of the heat sensitive area
10
in SIT. Therefore, the average gray value of the heat sensitive area in SIT could be
11
relevant to the sterilization performance. Figure 2B shows that the gray value decreased
12
obviously after either SES or CTS. In SES, the gray value decreased monotonically with
13
the increase of SE temperature at the same residence time. Results showed that the
14
complete sterilization of SES was obtained above 172 °C for 2 min and above 128 °C
15
for 5 min and 8 min, while the corresponding average gray value of the sensitive area in
16
SIT was 41.30, 59.06, and 58.71, respectively. It was noticed that the average gray
17
value under 172 °C for 2 min was obviously lower than that under 128 °C for 5 min and
18
8 min. This phenomenon may be attributed to that the color-changing of SIT is more
19
sensitive to the temperature than time. Nevertheless, when the SE residence time was 2
20
min, the average gray value of the sensitive area in SIT was not above 41.30 for
10
1
meaning complete sterilization. Correspondingly, the threshold value was 59.06 and
2
58.71 for complete sterilization when the SE residence time was 5 min and 8 min
3
respectively. Therefore, an effective parameter, average gray value of the sensitive area
4
in SIT, was successful to indicate SE sterilization effect conveniently and rapidly.
5
3.2. Determination of SE conditions for complete sterilization
6
3.2.1. Effects of SE temperature and explosion on sterilization efficiency
7
Effects of SE temperature and explosion on sterilization efficiency were
8
investigated. As shown in Table 2, the sterilization efficiency improved with the
9
increase of SE temperature at the same residence time. The reason for this result was
10
that coagulation and denaturation of proteins in microbes became serious at higher
11
temperature. Therefore, microbes were more likely to be killed, resulting in the better
12
sterilization efficiency at the higher temperature. It is interesting to note from number 7
13
and 15 in Table 2 that the SM was completely sterilized by SES with 128 °C for 5 min
14
while not by CTS with the same temperature and time. This phenomenon clearly
15
indicated that the explosion step in SE helped to destroy the microbial cells and thus
16
improved the sterilization efficiency. Overall, Table 2 shows that the complete
17
sterilization conditions by SES were above 172 °C for 2 min, and above 128 °C for 5
18
min and 8 min. Compared with the commonly used CTS condition (121 °C, 20 min),
19
the time of SES was shortened dramatically, showing the advantage of improving
20
sterilization efficiency by SES.
11
1
2
3.2.2. Effect of SE residence time on sterilization efficiency Residence time of SE is another key factor determining the sterilization effect. It
3
was found in Table 2 that to achieve the complete sterilization, the temperature needs to
4
reach 172 °C with the residence time of 2 min while that was 128 °C with the residence
5
time of 5 min. The results demonstrated that the required temperature for complete
6
sterilization decreased apparently with longer residence time. The Arrhenius equation
7
can be used to estimate the inactivation of microorganisms for heat sterilization process
8
(Mann et al., 2001).
9
k = A * exp (−
∆
)
(2)
10
Where k is the microorganisms inactivation rate constant, s-1; A is the Arrhenius
11
frequency factor, s-1; ∆ is the activation energy, J/mol; R is the universal gas constant,
12
J/(mol·K); and T is the absolute temperature, K. According to the Arrhenius equation,
13
higher sterilization temperature T resulted in larger microorganisms inactivation rate
14
constant k, requiring shorter residence time. In turn, longer residence time required
15
lower sterilization temperature, which was consistent with the results described above.
16
On the other hand, severity factor which combines temperature and residence time
17
is a concept to express the severity of a given treatment by SE. It was reported that
18
severity factor was a potential parameter for SE scale-up (Heitz et al., 1991; Iroba et al.,
19
2014; Overend et al., 1987). As seen from number 2 and 8 in Table 2, the temperature
20
and time combination of 134 °C * 5 min resulted in better sterilization effect than that of
21
148 °C * 2 min with almost the same SE severity factor R0. The possible reason was that
12
1
although the temperature was higher in group 2, the heat could not transfer evenly in the
2
medium within 2 min due to the low thermal conductivity of SM, which led to the
3
incomplete sterilization. While for group 8, despite the lower temperature, heat could
4
transfer uniformly during the longer residence time. Therefore, there were no blind
5
spots within the medium. The complete sterilization was therefore achieved. It also can be seen in number 7 and 11 from Table 2 that the sterilization effect of
6 7
group 11 with the temperature and time combination of 124 °C * 8 min was worse than
8
that of group 7 with 128 °C * 5 min, although the residence time was longer and the SE
9
severity factor was larger of group 11 than that of group 7. Number 11 and 12 showed
10
that temperature needed to reach 128 °C for complete sterilization with the residence
11
time of 8 min. These results implied that when the time was longer enough, it was no
12
longer the limiting factor for sterilization efficiency. Overall, combining the effects of
13
SE temperature and residence time on sterilization efficiency, it can be concluded that
14
using SE technique for SM sterilization was effective. SE conditions for complete
15
sterilization were temperature and time combination of 172 °C * 2 min and 128 °C * 5
16
min.
17
3.2.3. SE sterilization kinetics
18
The logarithm of microorganism survivors in SM after SE treatment at 128 °C was
19
plotted in Figure 3. It was shown that the inactivation curve exhibited a non-linear
20
behavior. Microorganism inactivation by SE progressed rapidly during pressure-hold
21
for 1 min. 5.1-log reduction was observed after SE treatment at 128 °C for 1 min, while
13
1
greater holding times had comparatively limited effect. There was no CFU observed
2
when the holding time reached to 5 min. Similar trends in inactivation have been
3
observed in other work involving high pressure and temperature treatments of
4
Clostridium sporogenes spores in ground beef and Bacillus stearothermophilus spores
5
in egg patties (Rajan et al., 2006; Zhu et al., 2008). The nonlinear survivor curve may
6
indicate that SE has multiple targets of action on microbial cells. The observed tailing
7
phenomenon could be attributed to cell damage by the instant pressure release. Overall,
8
it was noticed that the survival rate of SE treatments at 128 °C for more than 1 min were
9
all lower than 0.001, which was generally acceptable in sterilization evaluation. Rajan et
10
al. (2006) reported that only 1.5-log reduction was observed under the conventional
11
thermal treatment of Bacillus stearothermophilus spores in egg patties at 121 °C for 15
12
min. Therefore, it was indicated that using SE treatment for SM sterilization could result
13
in a much quicker processing and better sterilization effect.
14
3.3. Nutrients contents before and after sterilization
15
Heat processing also has a detrimental effect on nutrients since thermal
16
degradation of nutrients can and does occur (Lund, 1988). The effects of SES and CTS
17
on nutrients contents in medium were investigated. As shown in Figure 4, both glucose
18
and xylose contents decreased apparently after CTS than those of raw medium,
19
suggesting that CTS was adverse for nutrients. It was noted that the glucose and xylose
20
contents after CTS at 128 °C for 5 min were higher than those after CTS at 121 °C for
14
1
20 min, which confirmed that the high-temperature short-time heat process decreased
2
the nutrients breakdown. While for SES, the effects on nutrients contents were
3
complex. With the same residence time of SE, glucose and xylose contents increased
4
initially and then decreased with the temperature increase. This phenomenon implied
5
that SE may affect nutrients contents on two aspects: degrading polysaccharides into
6
glucose and xylose and degrading glucose and xylose into small molecules. Firstly, the
7
polysaccharides were degraded more and more intensely as SE severity increased thus
8
to release more glucose and xylose. However, when the SE severity reached a certain
9
value, the degradation of glucose and xylose began to increase drastically, leading to
10
the reduction of glucose and xylose contents in medium. Nevertheless, results clearly
11
showed that SES improved glucose and xylose contents in medium, especially
12
compared with CTS. It is interesting to note that the glucose and xylose contents after
13
SES at 128 °C for 5 min increased by 80.0% and 58.8% respectively than those after
14
CTS with the same temperature and time. This result revealed that the instant pressure
15
relief in SE helped to release glucose and xylose from the SM efficiently. From the
16
perspective of nutrients and efficiency, combination of temperature and residence time
17
of 128 °C * 5 min was chosen as the preferable SE condition for sterilization, under
18
which the glucose and xylose contents were 2.57 and 1.93 times of those after the
19
effective CTS condition (121 °C, 20 min). The result clearly indicated that SES
20
improved the SM nutrition.
21
In order to reveal the effect of SES on glucose release explicitly, the medium
15
1
containing only wheat bran and soybean meal with moisture of 60% (w/w) were
2
employed for SE treatment. Figure 5 shows that SES obviously converted glucan into
3
glucose in the medium. At the residence time of 2 min, the glucose content increased
4
firstly and then began to decrease with the temperature increase, of which the trends
5
coincided with the results described above. The possible reason was that the released
6
glucose was further degraded under the higher SE severity. Additionally, it was noticed
7
that the CTS (121 °C, 20 min) could also lead to glucan conversion to glucose, but this
8
effect was negligible compared with SES. Overall, SES increased nutrients contents in
9
medium, which should be beneficial to the subsequent fermentation.
10
Previous works have also suggested that inhibiting compounds, such as weak acids,
11
furan derivatives may generate during the steam treatment of the lignocelluloses
12
(Palmqvist & Hahn-Hagerdal, 2000; Wang & Chen, 2011). These compounds are
13
inhibitory to microorganisms and limit the fermentation performance. Negro et al.
14
observed that inhibitors generated in the liquid fractions of steam exploded olive tree
15
pruning, of which the concentrations of acetic acid, formic acid, furfural and HMF
16
ranged from 2.7-3.5 g/L, 0.1-0.4 g/L, 0.7-1.4 g/L and 0.1-0.3 g/L respectively under the
17
SE conditions of 175 °C and 195 °C for 10 min. The liquid fractions were then
18
subjected to fermentation with the xylose fermenting microorganism S. stipitis for
19
ethanol production. However, neither ethanol nor growth was observed, indicating that
20
the presence of toxic compounds inhibited both growth and fermentation of
21
microorganisms (Negro et al., 2014). While in the present work, the inhibitors were not
16
1
detected, which was beneficial to the subsequent fermentation process. The possible
2
reason was that the SE severity for sterilization in the present work was much lower
3
than that for pretreatment. Therefore, the generation reactions of inhibitors were not
4
stimulated.
5
3.4. FTIR analysis of solid medium before and after sterilization
6
FTIR, which is a quite convenient and frequently used analysis technique for
7
structural characterization, was applied in this study to investigate the structural changes
8
of SM before and after sterilization. As shown in Table 3, the spectra presented peaks at
9
3342 cm-1 (OH), 1658 cm-1 (CO, CN), 1541 cm-1 (CN, NH), 1421 cm-1 (CH2) and 1103
10
cm-1 (OH), which indicated the presence of cellulose (3342 cm-1 and 1421 cm-1),
11
polysaccharides (3342 cm-1 and 1103 cm-1) and proteins (1658 cm-1and 1541 cm-1)
12
(Wang et al., 2012). A semi-quantitative analysis of the FTIR spectra was established
13
and the band at 1515 cm-1 (aromatic skeletal stretching) was used as a reference band to
14
estimate the relative intensities of other bands (Nada et al., 1998; Wang & Chen, 2014).
15
Table 3 shows that some bands underwent detectable relative intensity changes as a
16
result of different sterilization methods. The relative intensity A3342/A1515 of band
17
attributed to the hydroxyl groups in polysaccharides decreased from 1.4652 of raw SM
18
to 1.2673 of SM after SES, which indicated that SES degraded polysaccharides and may
19
help to release glucose. Whereas the A3342/A1515 value of SM after CTS was 1.4616,
20
which was little lower than that of raw SM. The information showed that CTS can also
17
1
help to degrade polysaccharides, but the effect was little to neglect, which was
2
consistent with the results described in Figure 5. Moreover, the relative intensities of the
3
1421 cm−1 band originating from CH2 bending vibrations of cellulose and 1103 cm−1
4
band attributed to the C-OH skeletal vibration decreased more obviously after SES,
5
which confirmed the polysaccharides degradation by SES again. Both SES and CTS
6
reduced the intensities of the 1658 cm-1 and 1541 cm-1 bands attributed to the C=O
7
stretching, C-N stretching and N-H bending vibrations respectively of amide groups in
8
proteins or protein-like compounds. The information indicated that SES and CTS could
9
break down the long chain protein into the short, of which the effect by SES was more
10
obvious. This can be confirmed by the relatively lower absorbance of C-N stretching
11
vibration band at 1155 cm−1 in SM after SES compared with that after CTS. This effect
12
may make that microbes utilized amino acids in SM more effectively after SES. In
13
addition, SES obviously reduced the intensity of the 1242 cm-1 band attributed to the
14
C-O stretching band (guaiacyl units), which indicated that the relative content of
15
guaiacyl lignin units after SES had significant changes compared with that after CTS.
16
SES also reduced the intensity of peak at 1034 cm−1 attributed to the C-O-C stretching
17
typical of glucan and xylan significantly. The modification may correspond to the
18
degradation of glucan and xylan, which facilitated the release of glucose and xylose.
19
Thus, these results indicated that SES helped to disrupt the structure of polysaccharides
20
more efficiently than CTS, which contributed to the nutrients improvement in SM. This
21
information also corresponded to the data in Figure 4 that the glucose and xylose
18
1
contents increased in SM after SES than those after CTS.
2
3.5. Comparison of fermentation performance on SES and CTS medium
3
Fermentation performance is the key metric to evaluate the SES and CTS. As
4
shown in Figure 6, Bacillus subtilis fermented on SES medium grew fast after the
5
fermentation time of 24 h, and the maximum number reached 2.66 * 1011/g DM at 48 h
6
and decreased thereafter. While for CTS medium, the bacteria number increased slowly
7
during the fermentation process and reached 6.94 * 10 10/g DM at 48 h. It was reported
8
that the optimum fermentation period for Bacillus subtilis growth was 48 h by Fu et al.
9
(2010) and Joshi et al. (2008). The number of Bacillus subtilis fermented on CTS
10
medium reached the maximum of 6.67 * 1010/g DM at 48 h (Fu et al., 2010), which was
11
in accordance with the result in the present study. Therefore, the bacteria number on
12
SES medium was 2.83 times larger than that of CTS medium, which implied that SES
13
improved the fermentation performance apparently. Figure 6 also shows that the glucose
14
content of SES medium was 2.28 times of that of CTS medium at the fermentation time
15
of 24 h. Then it was decreased due to the consumption by Bacillus subtilis growth, and
16
the value was 1.92 times of that of CTS medium at 48 h. Finally, the glucose content of
17
SES medium was 1.08 times of that of CTS medium when the fermentation time
18
reached 72 h. In view of these above results, it was found that the glucose content of
19
SES medium was higher than that of CTS medium during the fermentation process.
20
That is, the medium after SES was more nutritional for Bacillus subtilis fermentation,
19
1
which should be an important reason for the enhanced Bacillus subtilis growth on SES
2
medium than that on CTS medium. Therefore, the results demonstrated that SES
3
facilitated the fermentability of SM and improved SSF performance efficiently.
4
3.6. Comparison of production efficiency between SES and CTS on large scale
5
Sterilization time and energy consumption affect the production efficiency and
6
economic cost, especially for industrialization. The sterilization time and energy
7
utilization of CTS for SM on large scale, spherical digester, and SES were analyzed and
8
compared. Table 4 shows the process flow of the conventional spherical digester
9
sterilization and SES. The significant difference of these two processes occurred on the
10
following two aspects. First, there was no cooling stage for SES. In spherical digester
11
sterilization, pressure inside the spherical digester was about 0.11 MPa (gauge pressure)
12
after the temperature maintenance stage. Therefore, the spherical digester should be
13
cooled for 30 min before discharge for security. It is noted that the cooling operation not
14
only consumes time, but also wastes the energy for heating. As for SES, the high
15
pressure was utilized to transport the materials aseptically and help to destroy the
16
microbe cells. After the instant pressure relief, the temperature of the moist air inside
17
the SE reactor was still high, which saved energy for heating in the next batch operation.
18
Besides, the time for temperature maintenance was shortened by 75% of SES than that
19
of spherical digester due to the enhanced sterilization performance. It was indicated
20
from Table 4 that the time needed for a batch operation of spherical digester sterilization
20
1
was 63 min while the time was 19 min of SES. That means 69.8% of time was saved by
2
SES, suggesting that the utilization efficiency of equipment obviously increased in SES
3
process. Therefore, SES could not only save energy efficiently, but also reduce
4
operation time dramatically. This is conductive to improving industrial production
5
efficiency and reducing the economic cost.
6
4. Conclusions
7
This study clearly showed the feasibility of SE technology for SM sterilization.
8
Owing to the higher temperature and physical collision effect, SES shortened
9
sterilization time by 69.8%. Meanwhile, it was worth noting that the glucose and xylose
10
contents in SM after SES were 2.57 and 1.93 times of those after CTS (121 °C, 20 min),
11
which suggested that SES improved the fermentability of SM. Bacillus subtilis
12
fermentation productivity on SES medium increased by 2.83 times than that on CTS
13
medium. Therefore, SE technology could be a new path in further SM sterilization,
14
which should promote the economy of solid-state fermentation.
15
Acknowledgements
16
This work was financially supported by the National Basic Research Program of
17
China (973 Project, No. 2011CB707401), the National High Technology Research and
18
Development Program (863 Program, 2012AA021302) and Open Funding Project of
19
the State Key Laboratory of Biochemical Engineering (No. 2013KF-01).
21
1
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steam-exploded corn stover for butanol production by removal of fermentation
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33
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34
beef at elevated temperatures. Int. J. food Microbiol. 126, 86-92.
35 36 24
1
Figure captions
2
Figure 1 Schematic diagram of the experimental apparatus for steam explosion
3
sterilization.
4
Figure 2 (A) Schematic representation of the image processing procedure. SIT,
5
sterilization indicator tape. (B) Average gray value of the sensitive area in SIT before
6
and after sterilization. CTS, conventional thermal sterilization; SES, steam explosion
7
sterilization.
8
Figure 3 Survival curve of microorganisms in solid medium after steam explosion
9
treatment at 128 °C. The blue horizontal line represents the survival rate of 0.001, below
10
which is acceptable for sterilization.
11
Figure 4 Effects of steam explosion sterilization (SES) on nutrients contents in medium
12
compared with conventional thermal sterilization (CTS).
13
Figure 5 Effects of steam explosion sterilization on glucose generation in wheat bran
14
and soybean meal. CTS, conventional thermal sterilization; SES, steam explosion
15
sterilization.
16
Figure 6 Fermentation kinetics on solid medium after steam explosion sterilization
17
(SES, 128 °C, 5 min) and conventional thermal sterilization (CTS, 121 °C, 20 min).
18
25
1
Table 1 Mechanisms and characteristics of different sterilization methods Sterilization methods Dry heat sterilization
Mechanisms
Coagulation of proteins thus destroy microorganisms by hot air Moist heat Denaturation of proteins in sterilizuxiaation microorganisms by steam
Ultrasonic sterilization
Microwave sterilization
Radiation sterilization
Killing microorganisms by radiation pressure, ultrasonic pressure, heat effect, cavitation effect and chemical effect Denaturation of protein by thermal effect and changing cell membrane permeability by nonthermal effect Penetrate microorganism cells by radiations
Advantages and disadvantages References Simple operation; no corrosion; lengthy time (about 1 h); nutrients degradation Large latent heat; strong penetrating power; low operation cost; lengthy time for solid materials; nutrients degradation Convenient operation; no pollution; high cost
(Halpern et al., 2014) (Mei et al., 1999)
(Wang, 2003)
Short time; convenient (Jeng et al., operation; non uniform action; 1987) harmful to human body
Simple operation; limited (Rai et al., application area; harmful to 2013) human body Chemical Oxidation of microorganisms Applicable to materials that (Halpern et disinfectants or damaging cells by cannot be heated; pollution to al., 2014) sterilization chemical disinfectants materials Steam explosion Deactivation of Short time; improving nutrition This study sterilization microorganisms by the high of solid medium; energy saving; temperature steam; destroy applicable to solid materials cells by the instant pressure relief 2
26
1
Table 2 Effects of temperature and residence time of steam explosion on sterilization
2
performance Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14b 15c 16d
3
a
Residence time (min) 2 2 2 2 2 5 5 5 5 5 8 8 8 20 5 −
Temperature (°C)
LgR0
135 148 158 165 172 124 128 134 137 141 124 128 134 121 128 −
1.33 1.71 2.01 2.21 2.42 1.41 1.52 1.70 1.79 1.91 1.61 1.73 1.90 − − −
+++ means lawn; ++ means CFU ≧30 but lawn was not formed; + means 30 > CFU > 0; ND means not determined (CFU = 0).
4
Colony-forming units (CFU)a ++ ++ ++ + ND + ND ND ND ND + ND ND ND + +++
5
b
Conventional thermal sterilization (CTS) at 121 °C for 20 min.
6
c
CTS at 128 °C for 5 min.
7
d
Medium without sterilization treatment.
27
1
Table 3 FTIR semi-quantitative analysis of raw solid medium (SM), SM after
2
conventional thermal sterilization (CTS, 121 °C, 20 min) and SM after steam explosion
3
sterilization (SES, 128 °C, 5 min) Bond (cm-1) 3342 1658
1541
1515
1421 1242
1155
1103 1034
4
a
Ax/A1515a Assignment
Raw SM
SM after CTS (121 °C, 20 min)
SM after SES (128 °C, 5 min)
1.4652
1.4616
1.2673
1.3930
1.2732
1.2095
0.9937
0.9828
0.9627
1.0000
1.0000
1.0000
1.4704
1.3843
1.3788
1.1528
1.1129
1.0379
1.6630
1.5930
1.4803
1.8662
1.7187
1.5758
2.1208
2.0341
1.8460
OH stretching and hydrogen bonds (Wang et al., 2012) CO stretching of amide groups in proteins or protein-like compounds, i.e., amide I (Wang et al., 2012) CN stretching and NH bending vibrations of amide groups in proteins or protein-like compounds, i.e., amide II (Wang et al., 2012) Aromatic skeletal vibration (Nada et al., 1998; Wang & Chen, 2014) CH2 bending vibrations of cellulose (Wang et al., 2012) CO stretching in the acetyl and phenolic groups (Liu et al., 2013d; Verma et al., 2012) CN stretching vibration of the protein fractions (Wang & Chen, 2009) OH skeletal vibration (Wang et al., 2012; Wang & Chen, 2009) COC stretching typical of glucan and xylan (Liu et al., 2013d)
Relative intensity of the specific band to the reference band at 1515 cm-1 .
5
28
1
Table 4 Processes and time consumption of conventional spherical digester sterilization
2
and novel steam explosion sterilization (SES) in a batch operation (data in this table
3
with the unit of min) Feedstock Steam heating Conventional spherical digester sterilization Steam explosion sterilization
Temperature Cooling Discharge maintenance
Total
5
5 (heating to 121 °C)
20
30
3
63
5
6 (heating to 128 °C)
5
0
3
19
4 5
29
1
2
Figure 1 Schematic diagram of the experimental apparatus for steam explosion
3
sterilization.
4
30
1
2 3
Figure 2 (A) Schematic representation of the image processing procedure. SIT,
4
sterilization indicator tape. (B) Average gray value of the sensitive area in SIT before
5
and after sterilization. CTS, conventional thermal sterilization; SES, steam explosion
6
sterilization. 31
1 2
Figure 3 Survival curve of microorganisms in solid medium after steam explosion
3
treatment at 128 °C. The blue horizontal line represents the survival rate of 0.001, below
4
which is acceptable for sterilization.
5 6 7
32
1 2
Figure 4 Effects of steam explosion sterilization (SES) on nutrients contents in medium
3
compared with conventional thermal sterilization (CTS).
4
5 6
33
1 2
Figure 5 Effects of steam explosion sterilization on glucose generation in wheat bran
3
and soybean meal. CTS, conventional thermal sterilization; SES, steam explosion
4
sterilization.
5 6
34
1 2
Figure 6 Fermentation kinetics on solid medium after steam explosion sterilization
3
(SES, 128 °C, 5 min) and conventional thermal sterilization (CTS, 121 °C, 20 min).
4
35
Highlights
1
2
A novel SE sterilization improves SSF performance.
3
SE conditions for complete sterilization are 172 °C * 2 min and 128 °C * 5 min.
4
Instant pressure relief in SE helps to destroy microbial cells and release glucose.
5
SE sterilization improves SM nutrition and increases SSF productivity by 2.83
6
times.
7
This work would benefit for improving economy of SM sterilization and further
8
SSF.
9
36
S0960-8524(15)00781-6 http://dx.doi.org/10.1016/j.biortech.2015.05.099 BITE 15070
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
17 April 2015 29 May 2015 30 May 2015
Please cite this article as: Zhao, Z-M., Wang, L., Chen, H-Z., A novel steam explosion sterilization improving solidstate fermentation performance, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech. 2015.05.099
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1
A novel steam explosion sterilization improving solid-state
2
fermentation performance
3
Zhi-Min Zhao a, b, Lan Wang a,*, Hong-Zhang Chen a
4
a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
5 6
b
University of Chinese Academy of Sciences, Beijing 100049, PR China
7 8 9 10 11 12 13 14 15 16 17
Abbreviations: SE, steam explosion; SIT, sterilization indicator tape; SSF,
18
solid-state fermentation; SmF, submerged fermentation; SM, solid medium; SES, steam
19
explosion sterilization; CTS, conventional thermal sterilization; CFU, colony-forming
20
units; FTIR, Fourier transform infrared spectroscopy; DM, dry medium.
21
*Corresponding author. Tel.: 86-01082544978.
22
E-mail address: [email protected] (Wang L.)
1
1
2
Abstract Traditional sterilization of solid medium (SM) requires lengthy time, degrades
3
nutrients, and even sterilizes inadequately compared with that of liquid medium due to
4
its low thermal conductivity. A novel sterilization strategy, high-temperature and
5
short-time steam explosion (SE), was exploited for SM sterilization in this study.
6
Results showed that SE conditions for complete sterilization were 172 °C for 2 min and
7
128 °C for 5 min. Glucose and xylose contents in medium after SE sterilization
8
increased by 157% and 93% respectively compared with those after conventional
9
sterilization (121 °C, 20 min) while fermentation inhibitors were not detected. FTIR
10
spectra revealed that the mild SE conditions helped to release monosaccharides from the
11
polysaccharides. Bacillus subtilis fermentation productivity on medium after SE
12
sterilization was 3.83 times of that after conventional sterilization. Therefore, SE
13
shortened sterilization time and improved SM nutrition, which facilitated fermentability
14
of SM and should promote economy of solid-state fermentation.
15
Keywords: sterilization; steam explosion; nutrition; solid medium; image processing
16
17
18
19
2
1
1. Introduction
2
Sterilization is an operation essential to all industrial fermentation processes
3
requiring pure culture maintenance (Deindoerfer, 1957). It is defined as the complete
4
elimination or destruction of all forms of microbial life by either physical or chemical
5
processes (Liu et al., 2013b). In submerged fermentation (SmF), moist heat sterilization
6
is widely used and it is effective to lead to the microbes death due to the high thermal
7
conductivity of liquid medium. Solid-state fermentation (SSF) is considered as a
8
promising process for production of various products such as fuel, feed, industrial
9
chemicals and pharmaceutical products (Pandey, 2003). Lignocellulosic biomass
10
converted by SSF for biofuels or biomaterials production has attracted substantial
11
interests from both the government and private sectors due to the abundance and
12
renewability of raw materials as well as the urgent demand for energy (Li & Chen, 2014;
13
Liu et al., 2013a). Unlike liquid medium, the complete sterilization of SSF medium has
14
issue itself due to its low thermal conductivity and multi-compositions, especially in the
15
industrial process.
16
Many sterilization methods including dry heat sterilization, moist heat sterilization,
17
ultrasonic sterilization, microwave sterilization, radiations, and chemical disinfectants
18
sterilization are currently being used according to the property of medium. Mechanisms
19
and characteristics of these common sterilization methods are shown in Table 1.
20
Because heating the medium, usually by steam, is the most reliable and the easiest to
21
control on a large scale, it is the method of choice throughout the fermentation industry. 3
1
Spherical digester has been commonly used for sterilization of solid medium (SM) in an
2
industrial scale. The SM is heated to 121 °C and kept for at least 20 min by the injected
3
saturated steam in spherical digester, and then naturally cooled. The spherical digester
4
rotates to accelerate heat transfer during the whole process, in which the
5
microorganisms are killed by the moist heat. However, the main drawbacks of this
6
technique include long cycle time, incomplete discharge, and energy waste, which
7
results in high capital cost and low process efficiency (Zhang et al., 2007). Previous
8
study also pointed out that heat processing, especially for a long time, has an adverse
9
effect on nutrients since thermal degradation of nutrients can and does occur (Lund,
10
1988). Therefore, the lengthy time, degraded nutrients, and energy waste are the main
11
problems of traditional thermal sterilization of SM.
12
According to the Arrhenius equation, rate constant of chemical degradation is less
13
influenced by temperature than that of microbial inactivation due to the fact that the
14
activation energy of chemical degradation is lower than that of microbial inactivation
15
(Mann et al., 2001; Mei et al., 1999). Therefore, the sterilization process using a high
16
temperature and short time strategy could make it possible to minimize the breakdown
17
of nutrients while kill the microorganisms. Mann et al. (2001) studied the thermal
18
sterilization of heat-sensitive products. It was suggested that the levels of degradation
19
can be substantially reduced by employing high-temperature short-time sterilization.
20
The work of Kjellstrand et al. also suggested that high-temperature short-time strategy
21
should be used for heat sterilization to minimize the development of the breakdown
4
1
products (Kjellstrand et al., 1995). Steam explosion (SE) is one of the most efficient
2
treatment methods for lignocellulosic biomass and has been developed into commercial
3
scale (Chen & Liu, 2007). Solid materials in SE reactor are heated by high-pressure
4
saturated steam for a relatively short time (normally 2-8 min). Until reaching the desired
5
residence time the reaction system was depressurized instantaneously. This is a typical
6
high-temperature short-time process. In SE process, there are high-temperature steam
7
heating step and explosion step (Liu et al., 2013c). Besides the high-temperature heating,
8
the structure change of materials by physical collision in explosion step may also help
9
to destroy the microbial cells.
10
In this study, we aim to exploit a novel and potential sterilization strategy,
11
high-temperature and short-time steam explosion (SE) process, for solid medium.
12
Effects of SE temperature and time on sterilization performance were studied and the
13
SE conditions for complete sterilization were determined. Kinetics of SE sterilization
14
was established and the action of explosion step in SE was investigated. Nutrients
15
content in medium was considered as an important metric to evaluate the sterilization
16
performance. Additionally, fermentation performance was compared between SE and
17
conventional thermal sterilization (121 °C, 20 min) to examine the feasibility of SE
18
sterilization. Finally, sterilization efficiency and energy utilization were analyzed from
19
the point of industrial scale.
5
1
2. Materials and methods
2
2.1. Experimental materials
3
The SM prepared for sterilization consisted of 200 g wheat bran, 20 g soybean
4
meal, 5 g glucose, 10 g CaCO3 and 200 mL of 2.5% KH2PO4, 1.25% (NH4)2SO4, 0.05%
5
MnSO4 inorganic salt solution. The initial moisture content of the SM was adjusted to
6
60% (w/w). Chemicals were purchased from Beijing Chemical works.
7
2.2. Steam explosion sterilization (SES)
8 9
Steam explosion sterilization (SES) was performed in a 2.0 L self-designed batch vessel which mainly consisted of a reactor chamber, a reception chamber and a steam
10
generator (Figure 1). During sterilization, the SM was top-loaded into the reactor
11
chamber and treated at constant saturated steam pressure until reaching the set residence
12
time. The reaction system was then terminated with a suddenly explosion by the ball
13
valve into the sterilized reception chamber. The materials were then prepared for
14
analysis. The SE severity factor can be calculated according to the following equation
15
(Liu et al., 2013d; Overend et al., 1987):
16
= ∗ exp (
.
)
(1)
17
Where R0 is the severity factor; t is the residence time, min; and T is the holding
18
temperature, °C. Table 2 lists the experimental conditions of SE and the corresponding
19
severity factor.
20
Conventional thermal sterilization (CTS) was performed as control. The SM with 6
1
same components was loaded into a 2.0 L Erlenmeyer flask and put into a vertical
2
autoclave (YXQ-LS-50A, Shanghai Boxun Industry & Commerce Co., Ltd). The
3
temperature and holding time were set to 121 °C for 20 min and 128 °C for 5 min,
4
respectively. All these experiments were conducted with two replicates.
5
2.3. Determination of viable cell number
6
5 g of SM after sterilization treatment was mixed with sterilized water (1:20, w/v)
7
in a 250 mL Erlenmeyer flask and shaken at 150 rpm for 30 min at 37 °C. Then, 100 µL
8
of the mixture was inoculated in duplicate in PDA and LB plate’s surface. After 36 h of
9
incubation at 37 °C, the number of colonies was determined and expressed as
10
colony-forming units (CFU).
11
2.4. Characterization of color-changing of sterilization indicator tape (SIT)
12
Two sterilization indicator tapes (purchased from 3M Company) with about 6 cm
13
length which were embedded in the SM were put into the SE reactor. Color of the heat
14
sensitive area would change if the SIT withstood moist heat by steam. It is known that
15
the gray value of the white is 255 while that is 0 of black in RGB images. The darker
16
the color is, the smaller the gray value is. Digital image processing was used to analyze
17
the color-changing of SIT. The sterilization indicator tapes before and after sterilization
18
treatment were photographed using a FUJIFILM FinePix Z20fd camera with same focal
19
length and under constant illumination. Images were acquired at a resolution 3648×
20
2736 pixels. The digital image processing was carried out by using the commercial 7
1
software ImageJ 1.44 (National Institutes of Health, USA) and MatLab 7.1 (MathWorks
2
Inc., USA). The schematic representation of image processing and analysis procedure
3
was illustrated in Figure 2A. The first step was threshold segmentation of the RGB image
4
of SIT after sterilization treatment according to the brightness by using ImageJ 1.44.
5
Through the segmentation, brightness of the heat sensitive area was retained while that
6
of background was changed to be 255. Once the threshold segmentation was performed,
7
the heat sensitive area and the background were distinguished significantly. Then the
8
image was converted into grayscale image. The average gray value of heat sensitive
9
area was calculated by automated protocol which was developed in MatLab 7.1 (Couri
10
et al., 2006; Duan et al., 2012).
11
2.5. Analysis of nutrients and inhibitors after sterilization
12
4 mL of the mixture in Section 2.3 was centrifuged at 8000 rpm for 10 min at room
13
temperature. The contents of glucose, xylose and inhibiting compounds such as acetic
14
acid, formic acid, furfural, and hydroxymethylfurfural (HMF) in the supernatants were
15
analyzed by high-performance liquid chromatography (HPLC; Agilent 1200, USA)
16
equipped with a refractive index detector. 10 µL samples were loaded on an Aminex
17
HPX-87H column (6.2 ×.250 mm, 5 µm), eluted with 0.5 mM H2SO4 solution at a
18
flow rate of 0.6 mL/min and operated at 55 °C. The content of each compound was
19
expressed as g/g dry medium (DM).
8
1
2
2.6. Fourier transform infrared spectroscopy (FTIR)
In order to identify and characterize the SM before and after sterilization, FTIR
3
spectra were studied between 4000 and 400 cm-1 using a FTIR-8400S spectrometer
4
(Shimadzu, Japan). Discs were prepared by mixing 2 mg of dried SM samples with 200
5
mg of KBr in an agate mortar and pressing 150 mg of the resultant mixture at 8 ton for 8
6
min. Discs were analyzed using 32 scans at a resolution of 4 cm-1. The characteristic
7
bands were assigned according to the literatures. In addition, a semi-quantitative
8
analysis of the FTIR spectra was carried out. In this analysis, the band at 1515 cm-1
9
(aromatic skeletal vibration) was considered as the reference band. Thus, the Ax/A1515
10
ratio represented the relative intensity of the specific band to the reference band at 1515
11
cm-1 (Casas et al., 2012).
12
2.7. Fermentation experiment
13
Bacillus subtilis PFK1302 used in this study was kindly provided by Hunan
14
PERFLY-Bio Co., Ltd., China. It was stored in LB medium slant at 4 °C. Bacillus
15
subtilis PFK1302 was precultivated in 100 mL liquid medium including 4% (w/w)
16
glucose, 1% (w/w) peptone, 1% (w/w) yeast extract, 1% (w/w) CaCO3 and 0.05% (w/w)
17
MgSO4 at 37 °C and 150 rpm for 24 h before inoculation.
18
25~40 g of SM after sterilization was cooled down and inoculated by adding 0.4
19
mL seed solution per gram of dry medium. Solid-state fermentation were carried out in
20
100 mL Erlenmeyer flasks and incubated at 37 °C for 72 h. At intervals of 24 h, 3~5 g
9
1
of SM were sampled to determine the bacteria amount and nutrients content similarly as
2
described in Sections 2.3 and 2.5.
3
3. Results and discussion
4
3.1. Evaluation of sterilization effect by SIT image processing
5
In order to evaluate the sterilization effect conveniently and choose a proper
6
parameter to predict the sterilization performance, the color-changing of SIT under
7
different SE conditions were investigated. SIT is exposed in steam moist heat in the SE
8
sterilization process. The greater the heat intensity, the more obvious is the
9
color-changing of SIT, which results in the smaller gray value of the heat sensitive area
10
in SIT. Therefore, the average gray value of the heat sensitive area in SIT could be
11
relevant to the sterilization performance. Figure 2B shows that the gray value decreased
12
obviously after either SES or CTS. In SES, the gray value decreased monotonically with
13
the increase of SE temperature at the same residence time. Results showed that the
14
complete sterilization of SES was obtained above 172 °C for 2 min and above 128 °C
15
for 5 min and 8 min, while the corresponding average gray value of the sensitive area in
16
SIT was 41.30, 59.06, and 58.71, respectively. It was noticed that the average gray
17
value under 172 °C for 2 min was obviously lower than that under 128 °C for 5 min and
18
8 min. This phenomenon may be attributed to that the color-changing of SIT is more
19
sensitive to the temperature than time. Nevertheless, when the SE residence time was 2
20
min, the average gray value of the sensitive area in SIT was not above 41.30 for
10
1
meaning complete sterilization. Correspondingly, the threshold value was 59.06 and
2
58.71 for complete sterilization when the SE residence time was 5 min and 8 min
3
respectively. Therefore, an effective parameter, average gray value of the sensitive area
4
in SIT, was successful to indicate SE sterilization effect conveniently and rapidly.
5
3.2. Determination of SE conditions for complete sterilization
6
3.2.1. Effects of SE temperature and explosion on sterilization efficiency
7
Effects of SE temperature and explosion on sterilization efficiency were
8
investigated. As shown in Table 2, the sterilization efficiency improved with the
9
increase of SE temperature at the same residence time. The reason for this result was
10
that coagulation and denaturation of proteins in microbes became serious at higher
11
temperature. Therefore, microbes were more likely to be killed, resulting in the better
12
sterilization efficiency at the higher temperature. It is interesting to note from number 7
13
and 15 in Table 2 that the SM was completely sterilized by SES with 128 °C for 5 min
14
while not by CTS with the same temperature and time. This phenomenon clearly
15
indicated that the explosion step in SE helped to destroy the microbial cells and thus
16
improved the sterilization efficiency. Overall, Table 2 shows that the complete
17
sterilization conditions by SES were above 172 °C for 2 min, and above 128 °C for 5
18
min and 8 min. Compared with the commonly used CTS condition (121 °C, 20 min),
19
the time of SES was shortened dramatically, showing the advantage of improving
20
sterilization efficiency by SES.
11
1
2
3.2.2. Effect of SE residence time on sterilization efficiency Residence time of SE is another key factor determining the sterilization effect. It
3
was found in Table 2 that to achieve the complete sterilization, the temperature needs to
4
reach 172 °C with the residence time of 2 min while that was 128 °C with the residence
5
time of 5 min. The results demonstrated that the required temperature for complete
6
sterilization decreased apparently with longer residence time. The Arrhenius equation
7
can be used to estimate the inactivation of microorganisms for heat sterilization process
8
(Mann et al., 2001).
9
k = A * exp (−
∆
)
(2)
10
Where k is the microorganisms inactivation rate constant, s-1; A is the Arrhenius
11
frequency factor, s-1; ∆ is the activation energy, J/mol; R is the universal gas constant,
12
J/(mol·K); and T is the absolute temperature, K. According to the Arrhenius equation,
13
higher sterilization temperature T resulted in larger microorganisms inactivation rate
14
constant k, requiring shorter residence time. In turn, longer residence time required
15
lower sterilization temperature, which was consistent with the results described above.
16
On the other hand, severity factor which combines temperature and residence time
17
is a concept to express the severity of a given treatment by SE. It was reported that
18
severity factor was a potential parameter for SE scale-up (Heitz et al., 1991; Iroba et al.,
19
2014; Overend et al., 1987). As seen from number 2 and 8 in Table 2, the temperature
20
and time combination of 134 °C * 5 min resulted in better sterilization effect than that of
21
148 °C * 2 min with almost the same SE severity factor R0. The possible reason was that
12
1
although the temperature was higher in group 2, the heat could not transfer evenly in the
2
medium within 2 min due to the low thermal conductivity of SM, which led to the
3
incomplete sterilization. While for group 8, despite the lower temperature, heat could
4
transfer uniformly during the longer residence time. Therefore, there were no blind
5
spots within the medium. The complete sterilization was therefore achieved. It also can be seen in number 7 and 11 from Table 2 that the sterilization effect of
6 7
group 11 with the temperature and time combination of 124 °C * 8 min was worse than
8
that of group 7 with 128 °C * 5 min, although the residence time was longer and the SE
9
severity factor was larger of group 11 than that of group 7. Number 11 and 12 showed
10
that temperature needed to reach 128 °C for complete sterilization with the residence
11
time of 8 min. These results implied that when the time was longer enough, it was no
12
longer the limiting factor for sterilization efficiency. Overall, combining the effects of
13
SE temperature and residence time on sterilization efficiency, it can be concluded that
14
using SE technique for SM sterilization was effective. SE conditions for complete
15
sterilization were temperature and time combination of 172 °C * 2 min and 128 °C * 5
16
min.
17
3.2.3. SE sterilization kinetics
18
The logarithm of microorganism survivors in SM after SE treatment at 128 °C was
19
plotted in Figure 3. It was shown that the inactivation curve exhibited a non-linear
20
behavior. Microorganism inactivation by SE progressed rapidly during pressure-hold
21
for 1 min. 5.1-log reduction was observed after SE treatment at 128 °C for 1 min, while
13
1
greater holding times had comparatively limited effect. There was no CFU observed
2
when the holding time reached to 5 min. Similar trends in inactivation have been
3
observed in other work involving high pressure and temperature treatments of
4
Clostridium sporogenes spores in ground beef and Bacillus stearothermophilus spores
5
in egg patties (Rajan et al., 2006; Zhu et al., 2008). The nonlinear survivor curve may
6
indicate that SE has multiple targets of action on microbial cells. The observed tailing
7
phenomenon could be attributed to cell damage by the instant pressure release. Overall,
8
it was noticed that the survival rate of SE treatments at 128 °C for more than 1 min were
9
all lower than 0.001, which was generally acceptable in sterilization evaluation. Rajan et
10
al. (2006) reported that only 1.5-log reduction was observed under the conventional
11
thermal treatment of Bacillus stearothermophilus spores in egg patties at 121 °C for 15
12
min. Therefore, it was indicated that using SE treatment for SM sterilization could result
13
in a much quicker processing and better sterilization effect.
14
3.3. Nutrients contents before and after sterilization
15
Heat processing also has a detrimental effect on nutrients since thermal
16
degradation of nutrients can and does occur (Lund, 1988). The effects of SES and CTS
17
on nutrients contents in medium were investigated. As shown in Figure 4, both glucose
18
and xylose contents decreased apparently after CTS than those of raw medium,
19
suggesting that CTS was adverse for nutrients. It was noted that the glucose and xylose
20
contents after CTS at 128 °C for 5 min were higher than those after CTS at 121 °C for
14
1
20 min, which confirmed that the high-temperature short-time heat process decreased
2
the nutrients breakdown. While for SES, the effects on nutrients contents were
3
complex. With the same residence time of SE, glucose and xylose contents increased
4
initially and then decreased with the temperature increase. This phenomenon implied
5
that SE may affect nutrients contents on two aspects: degrading polysaccharides into
6
glucose and xylose and degrading glucose and xylose into small molecules. Firstly, the
7
polysaccharides were degraded more and more intensely as SE severity increased thus
8
to release more glucose and xylose. However, when the SE severity reached a certain
9
value, the degradation of glucose and xylose began to increase drastically, leading to
10
the reduction of glucose and xylose contents in medium. Nevertheless, results clearly
11
showed that SES improved glucose and xylose contents in medium, especially
12
compared with CTS. It is interesting to note that the glucose and xylose contents after
13
SES at 128 °C for 5 min increased by 80.0% and 58.8% respectively than those after
14
CTS with the same temperature and time. This result revealed that the instant pressure
15
relief in SE helped to release glucose and xylose from the SM efficiently. From the
16
perspective of nutrients and efficiency, combination of temperature and residence time
17
of 128 °C * 5 min was chosen as the preferable SE condition for sterilization, under
18
which the glucose and xylose contents were 2.57 and 1.93 times of those after the
19
effective CTS condition (121 °C, 20 min). The result clearly indicated that SES
20
improved the SM nutrition.
21
In order to reveal the effect of SES on glucose release explicitly, the medium
15
1
containing only wheat bran and soybean meal with moisture of 60% (w/w) were
2
employed for SE treatment. Figure 5 shows that SES obviously converted glucan into
3
glucose in the medium. At the residence time of 2 min, the glucose content increased
4
firstly and then began to decrease with the temperature increase, of which the trends
5
coincided with the results described above. The possible reason was that the released
6
glucose was further degraded under the higher SE severity. Additionally, it was noticed
7
that the CTS (121 °C, 20 min) could also lead to glucan conversion to glucose, but this
8
effect was negligible compared with SES. Overall, SES increased nutrients contents in
9
medium, which should be beneficial to the subsequent fermentation.
10
Previous works have also suggested that inhibiting compounds, such as weak acids,
11
furan derivatives may generate during the steam treatment of the lignocelluloses
12
(Palmqvist & Hahn-Hagerdal, 2000; Wang & Chen, 2011). These compounds are
13
inhibitory to microorganisms and limit the fermentation performance. Negro et al.
14
observed that inhibitors generated in the liquid fractions of steam exploded olive tree
15
pruning, of which the concentrations of acetic acid, formic acid, furfural and HMF
16
ranged from 2.7-3.5 g/L, 0.1-0.4 g/L, 0.7-1.4 g/L and 0.1-0.3 g/L respectively under the
17
SE conditions of 175 °C and 195 °C for 10 min. The liquid fractions were then
18
subjected to fermentation with the xylose fermenting microorganism S. stipitis for
19
ethanol production. However, neither ethanol nor growth was observed, indicating that
20
the presence of toxic compounds inhibited both growth and fermentation of
21
microorganisms (Negro et al., 2014). While in the present work, the inhibitors were not
16
1
detected, which was beneficial to the subsequent fermentation process. The possible
2
reason was that the SE severity for sterilization in the present work was much lower
3
than that for pretreatment. Therefore, the generation reactions of inhibitors were not
4
stimulated.
5
3.4. FTIR analysis of solid medium before and after sterilization
6
FTIR, which is a quite convenient and frequently used analysis technique for
7
structural characterization, was applied in this study to investigate the structural changes
8
of SM before and after sterilization. As shown in Table 3, the spectra presented peaks at
9
3342 cm-1 (OH), 1658 cm-1 (CO, CN), 1541 cm-1 (CN, NH), 1421 cm-1 (CH2) and 1103
10
cm-1 (OH), which indicated the presence of cellulose (3342 cm-1 and 1421 cm-1),
11
polysaccharides (3342 cm-1 and 1103 cm-1) and proteins (1658 cm-1and 1541 cm-1)
12
(Wang et al., 2012). A semi-quantitative analysis of the FTIR spectra was established
13
and the band at 1515 cm-1 (aromatic skeletal stretching) was used as a reference band to
14
estimate the relative intensities of other bands (Nada et al., 1998; Wang & Chen, 2014).
15
Table 3 shows that some bands underwent detectable relative intensity changes as a
16
result of different sterilization methods. The relative intensity A3342/A1515 of band
17
attributed to the hydroxyl groups in polysaccharides decreased from 1.4652 of raw SM
18
to 1.2673 of SM after SES, which indicated that SES degraded polysaccharides and may
19
help to release glucose. Whereas the A3342/A1515 value of SM after CTS was 1.4616,
20
which was little lower than that of raw SM. The information showed that CTS can also
17
1
help to degrade polysaccharides, but the effect was little to neglect, which was
2
consistent with the results described in Figure 5. Moreover, the relative intensities of the
3
1421 cm−1 band originating from CH2 bending vibrations of cellulose and 1103 cm−1
4
band attributed to the C-OH skeletal vibration decreased more obviously after SES,
5
which confirmed the polysaccharides degradation by SES again. Both SES and CTS
6
reduced the intensities of the 1658 cm-1 and 1541 cm-1 bands attributed to the C=O
7
stretching, C-N stretching and N-H bending vibrations respectively of amide groups in
8
proteins or protein-like compounds. The information indicated that SES and CTS could
9
break down the long chain protein into the short, of which the effect by SES was more
10
obvious. This can be confirmed by the relatively lower absorbance of C-N stretching
11
vibration band at 1155 cm−1 in SM after SES compared with that after CTS. This effect
12
may make that microbes utilized amino acids in SM more effectively after SES. In
13
addition, SES obviously reduced the intensity of the 1242 cm-1 band attributed to the
14
C-O stretching band (guaiacyl units), which indicated that the relative content of
15
guaiacyl lignin units after SES had significant changes compared with that after CTS.
16
SES also reduced the intensity of peak at 1034 cm−1 attributed to the C-O-C stretching
17
typical of glucan and xylan significantly. The modification may correspond to the
18
degradation of glucan and xylan, which facilitated the release of glucose and xylose.
19
Thus, these results indicated that SES helped to disrupt the structure of polysaccharides
20
more efficiently than CTS, which contributed to the nutrients improvement in SM. This
21
information also corresponded to the data in Figure 4 that the glucose and xylose
18
1
contents increased in SM after SES than those after CTS.
2
3.5. Comparison of fermentation performance on SES and CTS medium
3
Fermentation performance is the key metric to evaluate the SES and CTS. As
4
shown in Figure 6, Bacillus subtilis fermented on SES medium grew fast after the
5
fermentation time of 24 h, and the maximum number reached 2.66 * 1011/g DM at 48 h
6
and decreased thereafter. While for CTS medium, the bacteria number increased slowly
7
during the fermentation process and reached 6.94 * 10 10/g DM at 48 h. It was reported
8
that the optimum fermentation period for Bacillus subtilis growth was 48 h by Fu et al.
9
(2010) and Joshi et al. (2008). The number of Bacillus subtilis fermented on CTS
10
medium reached the maximum of 6.67 * 1010/g DM at 48 h (Fu et al., 2010), which was
11
in accordance with the result in the present study. Therefore, the bacteria number on
12
SES medium was 2.83 times larger than that of CTS medium, which implied that SES
13
improved the fermentation performance apparently. Figure 6 also shows that the glucose
14
content of SES medium was 2.28 times of that of CTS medium at the fermentation time
15
of 24 h. Then it was decreased due to the consumption by Bacillus subtilis growth, and
16
the value was 1.92 times of that of CTS medium at 48 h. Finally, the glucose content of
17
SES medium was 1.08 times of that of CTS medium when the fermentation time
18
reached 72 h. In view of these above results, it was found that the glucose content of
19
SES medium was higher than that of CTS medium during the fermentation process.
20
That is, the medium after SES was more nutritional for Bacillus subtilis fermentation,
19
1
which should be an important reason for the enhanced Bacillus subtilis growth on SES
2
medium than that on CTS medium. Therefore, the results demonstrated that SES
3
facilitated the fermentability of SM and improved SSF performance efficiently.
4
3.6. Comparison of production efficiency between SES and CTS on large scale
5
Sterilization time and energy consumption affect the production efficiency and
6
economic cost, especially for industrialization. The sterilization time and energy
7
utilization of CTS for SM on large scale, spherical digester, and SES were analyzed and
8
compared. Table 4 shows the process flow of the conventional spherical digester
9
sterilization and SES. The significant difference of these two processes occurred on the
10
following two aspects. First, there was no cooling stage for SES. In spherical digester
11
sterilization, pressure inside the spherical digester was about 0.11 MPa (gauge pressure)
12
after the temperature maintenance stage. Therefore, the spherical digester should be
13
cooled for 30 min before discharge for security. It is noted that the cooling operation not
14
only consumes time, but also wastes the energy for heating. As for SES, the high
15
pressure was utilized to transport the materials aseptically and help to destroy the
16
microbe cells. After the instant pressure relief, the temperature of the moist air inside
17
the SE reactor was still high, which saved energy for heating in the next batch operation.
18
Besides, the time for temperature maintenance was shortened by 75% of SES than that
19
of spherical digester due to the enhanced sterilization performance. It was indicated
20
from Table 4 that the time needed for a batch operation of spherical digester sterilization
20
1
was 63 min while the time was 19 min of SES. That means 69.8% of time was saved by
2
SES, suggesting that the utilization efficiency of equipment obviously increased in SES
3
process. Therefore, SES could not only save energy efficiently, but also reduce
4
operation time dramatically. This is conductive to improving industrial production
5
efficiency and reducing the economic cost.
6
4. Conclusions
7
This study clearly showed the feasibility of SE technology for SM sterilization.
8
Owing to the higher temperature and physical collision effect, SES shortened
9
sterilization time by 69.8%. Meanwhile, it was worth noting that the glucose and xylose
10
contents in SM after SES were 2.57 and 1.93 times of those after CTS (121 °C, 20 min),
11
which suggested that SES improved the fermentability of SM. Bacillus subtilis
12
fermentation productivity on SES medium increased by 2.83 times than that on CTS
13
medium. Therefore, SE technology could be a new path in further SM sterilization,
14
which should promote the economy of solid-state fermentation.
15
Acknowledgements
16
This work was financially supported by the National Basic Research Program of
17
China (973 Project, No. 2011CB707401), the National High Technology Research and
18
Development Program (863 Program, 2012AA021302) and Open Funding Project of
19
the State Key Laboratory of Biochemical Engineering (No. 2013KF-01).
21
1
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33
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35 36 24
1
Figure captions
2
Figure 1 Schematic diagram of the experimental apparatus for steam explosion
3
sterilization.
4
Figure 2 (A) Schematic representation of the image processing procedure. SIT,
5
sterilization indicator tape. (B) Average gray value of the sensitive area in SIT before
6
and after sterilization. CTS, conventional thermal sterilization; SES, steam explosion
7
sterilization.
8
Figure 3 Survival curve of microorganisms in solid medium after steam explosion
9
treatment at 128 °C. The blue horizontal line represents the survival rate of 0.001, below
10
which is acceptable for sterilization.
11
Figure 4 Effects of steam explosion sterilization (SES) on nutrients contents in medium
12
compared with conventional thermal sterilization (CTS).
13
Figure 5 Effects of steam explosion sterilization on glucose generation in wheat bran
14
and soybean meal. CTS, conventional thermal sterilization; SES, steam explosion
15
sterilization.
16
Figure 6 Fermentation kinetics on solid medium after steam explosion sterilization
17
(SES, 128 °C, 5 min) and conventional thermal sterilization (CTS, 121 °C, 20 min).
18
25
1
Table 1 Mechanisms and characteristics of different sterilization methods Sterilization methods Dry heat sterilization
Mechanisms
Coagulation of proteins thus destroy microorganisms by hot air Moist heat Denaturation of proteins in sterilizuxiaation microorganisms by steam
Ultrasonic sterilization
Microwave sterilization
Radiation sterilization
Killing microorganisms by radiation pressure, ultrasonic pressure, heat effect, cavitation effect and chemical effect Denaturation of protein by thermal effect and changing cell membrane permeability by nonthermal effect Penetrate microorganism cells by radiations
Advantages and disadvantages References Simple operation; no corrosion; lengthy time (about 1 h); nutrients degradation Large latent heat; strong penetrating power; low operation cost; lengthy time for solid materials; nutrients degradation Convenient operation; no pollution; high cost
(Halpern et al., 2014) (Mei et al., 1999)
(Wang, 2003)
Short time; convenient (Jeng et al., operation; non uniform action; 1987) harmful to human body
Simple operation; limited (Rai et al., application area; harmful to 2013) human body Chemical Oxidation of microorganisms Applicable to materials that (Halpern et disinfectants or damaging cells by cannot be heated; pollution to al., 2014) sterilization chemical disinfectants materials Steam explosion Deactivation of Short time; improving nutrition This study sterilization microorganisms by the high of solid medium; energy saving; temperature steam; destroy applicable to solid materials cells by the instant pressure relief 2
26
1
Table 2 Effects of temperature and residence time of steam explosion on sterilization
2
performance Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14b 15c 16d
3
a
Residence time (min) 2 2 2 2 2 5 5 5 5 5 8 8 8 20 5 −
Temperature (°C)
LgR0
135 148 158 165 172 124 128 134 137 141 124 128 134 121 128 −
1.33 1.71 2.01 2.21 2.42 1.41 1.52 1.70 1.79 1.91 1.61 1.73 1.90 − − −
+++ means lawn; ++ means CFU ≧30 but lawn was not formed; + means 30 > CFU > 0; ND means not determined (CFU = 0).
4
Colony-forming units (CFU)a ++ ++ ++ + ND + ND ND ND ND + ND ND ND + +++
5
b
Conventional thermal sterilization (CTS) at 121 °C for 20 min.
6
c
CTS at 128 °C for 5 min.
7
d
Medium without sterilization treatment.
27
1
Table 3 FTIR semi-quantitative analysis of raw solid medium (SM), SM after
2
conventional thermal sterilization (CTS, 121 °C, 20 min) and SM after steam explosion
3
sterilization (SES, 128 °C, 5 min) Bond (cm-1) 3342 1658
1541
1515
1421 1242
1155
1103 1034
4
a
Ax/A1515a Assignment
Raw SM
SM after CTS (121 °C, 20 min)
SM after SES (128 °C, 5 min)
1.4652
1.4616
1.2673
1.3930
1.2732
1.2095
0.9937
0.9828
0.9627
1.0000
1.0000
1.0000
1.4704
1.3843
1.3788
1.1528
1.1129
1.0379
1.6630
1.5930
1.4803
1.8662
1.7187
1.5758
2.1208
2.0341
1.8460
OH stretching and hydrogen bonds (Wang et al., 2012) CO stretching of amide groups in proteins or protein-like compounds, i.e., amide I (Wang et al., 2012) CN stretching and NH bending vibrations of amide groups in proteins or protein-like compounds, i.e., amide II (Wang et al., 2012) Aromatic skeletal vibration (Nada et al., 1998; Wang & Chen, 2014) CH2 bending vibrations of cellulose (Wang et al., 2012) CO stretching in the acetyl and phenolic groups (Liu et al., 2013d; Verma et al., 2012) CN stretching vibration of the protein fractions (Wang & Chen, 2009) OH skeletal vibration (Wang et al., 2012; Wang & Chen, 2009) COC stretching typical of glucan and xylan (Liu et al., 2013d)
Relative intensity of the specific band to the reference band at 1515 cm-1 .
5
28
1
Table 4 Processes and time consumption of conventional spherical digester sterilization
2
and novel steam explosion sterilization (SES) in a batch operation (data in this table
3
with the unit of min) Feedstock Steam heating Conventional spherical digester sterilization Steam explosion sterilization
Temperature Cooling Discharge maintenance
Total
5
5 (heating to 121 °C)
20
30
3
63
5
6 (heating to 128 °C)
5
0
3
19
4 5
29
1
2
Figure 1 Schematic diagram of the experimental apparatus for steam explosion
3
sterilization.
4
30
1
2 3
Figure 2 (A) Schematic representation of the image processing procedure. SIT,
4
sterilization indicator tape. (B) Average gray value of the sensitive area in SIT before
5
and after sterilization. CTS, conventional thermal sterilization; SES, steam explosion
6
sterilization. 31
1 2
Figure 3 Survival curve of microorganisms in solid medium after steam explosion
3
treatment at 128 °C. The blue horizontal line represents the survival rate of 0.001, below
4
which is acceptable for sterilization.
5 6 7
32
1 2
Figure 4 Effects of steam explosion sterilization (SES) on nutrients contents in medium
3
compared with conventional thermal sterilization (CTS).
4
5 6
33
1 2
Figure 5 Effects of steam explosion sterilization on glucose generation in wheat bran
3
and soybean meal. CTS, conventional thermal sterilization; SES, steam explosion
4
sterilization.
5 6
34
1 2
Figure 6 Fermentation kinetics on solid medium after steam explosion sterilization
3
(SES, 128 °C, 5 min) and conventional thermal sterilization (CTS, 121 °C, 20 min).
4
35
Highlights
1
2
A novel SE sterilization improves SSF performance.
3
SE conditions for complete sterilization are 172 °C * 2 min and 128 °C * 5 min.
4
Instant pressure relief in SE helps to destroy microbial cells and release glucose.
5
SE sterilization improves SM nutrition and increases SSF productivity by 2.83
6
times.
7
This work would benefit for improving economy of SM sterilization and further
8
SSF.
9
36
