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Stimulatory effect of in-situ detoxification on bioethanol production by rice straw
Accepted Manuscript Stimulatory effect of in-situ detoxification on bioethanol production by rice straw
Qiuzhuo Zhang, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal PII:
S0360-5442(17)31093-9
DOI:
10.1016/j.energy.2017.06.099
Reference:
EGY 11107
To appear in:
Energy
Received Date:
22 February 2017
Revised Date:
10 May 2017
Accepted Date:
18 June 2017
Please cite this article as: Qiuzhuo Zhang, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal, Stimulatory effect of in-situ detoxification on bioethanol production by rice straw, Energy (2017), doi: 10.1016/j.energy.2017.06.099
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In-situ detoxification
Hydrolysis
Alkaline
Trichoderma reesei
Available hydrolysates
Pretreatment
Degradation
Reducing sugars
Rice straw Ferulic acid
Escherichia sp. HHQ-1
Consortia degrading system
Graphic Abstract
Bioethanol
ACCEPTED MANUSCRIPT 1
Stimulatory effect of in-situ detoxification on bioethanol production by rice
2
straw
3
Qiuzhuo Zhang*, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal
4
Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration, School of Ecological and
5
Environmental Sciences, East China Normal University, 200241 Shanghai, China
6 7
Abstract: An effective ferulic acid degrading bacterium, Escherichia sp. HHQ-1, was
8
added to alkali-pretreated rice straw degrading system to in-situ detoxify the ferulic
9
acid inhibitors existed in the hydrolysates. It was shown that the production of
10
reducing sugars in Escherichia sp. HHQ-1-Trichoderma reesei consortia degrading
11
system could achieve 221.33 mg·L-1 at 60 h, which was 7.84% higher than that in
12
single degrading system. All the three main cellulases were more stable in consortia
13
degrading system, and the activity of β-glucosidase was 4.23 fold higher than that in
14
single degrading system. Besides, it was supposed that cell membrane was more
15
integrally protected in consortia degrading system. Scanning Electron Microscope
16
(SEM), X-ray Diffraction (XRD) and Fourier Transform Infrared Spectrometer
17
(FTIR) were used to observe the morphological changes of rice straw biomass. It was
18
indicated that in-situ detoxification could severely destroy basic tissue of rice straw,
19
dramatically decrease crystallinity index of crystalline region and effectively degrade
20
refractory-degraded lignin parts of lignocellulose. Compared to the single degrading
21
system, the consortia degrading system could in-situ detoxify the main inhibitors
22
which existed in pretreated rice straw hydrolysates, realize simultaneous pretreatment
23
and detoxification, thus increasing the bioethanol yield and reducing the cost of
24
bioethanol production.
25
Keywords: consortia system; in-situ detoxification; rice straw; bioethanol
26
List of abbreviations:
27
CICC: China Center of Industrial Culture Collection; CrI: Crystallinity index; DNS method: 3, 5-dinitrosalicylic
28
acid colorimetry; ECNU: East China Normal University; FA: Ferulic Acid (4-hydroxy-3-methoxycinnamic acid);
29
FTIR: Fourier Transform Infrared Spectrometer; GC/MS: Gas Chromatography-Mass Spectromete; LB: Luria-
30
Bertani medium; OD: Optical Density; PDA: Potato Dextrose Agar medium; SEM: Scanning Electron 1
ACCEPTED MANUSCRIPT 31
Microscope; UV: Ultraviolet and Visible Spectrophotometer; XRD: X-ray Diffraction
32
1. Introduction
33
Bioethanol, an alternative liquid fuel to overcome energy shortage problem, is a
34
promising near-term production, which has been developed extensively for more than
35
two decades (Yu et al., 2016; Domínguez et al., 2017; Khatiwada and Silveira, 2017).
36
Among renewable sources, lignocellulosic biomass is one of the most attractive and
37
main potential raw materials for bioethanol production because of its easy availability,
38
low price and high sugar content (Barros-Rios et al., 2016; Zhao et al., 2016).
39
Lignocellulosic biomass has a complex structure composed mainly of cellulose,
40
hemicellulose and lignin (Sindhu et al., 2016). These components interconnected well
41
through non-covalent and covalent bonds into a highly organized network that may
42
restrict enzyme accessibility and thereby reducing the efficiency of decomposing
43
enzymes (Cripwell et al., 2015).
44
Pretreatment is an indispensible step in lignocelluloses fermentation process
45
(Merali et al., 2016; Licari et al., 2016; Yang and Rosentrater, 2017). A well-designed
46
pretreatment method could help us to overcome recalcitrance from the rigid biomass
47
structure, which is beneficial for promoting enzymatic hydrolysis and realizing low-
48
cost lignocellulosic bioethanol (Cai et al., 2016; Dongen et al., 2011; Baral and Shah,
49
2017). Among the numerous pretreatment methods, alkaline pretreatment has
50
emerged as one of the most viable options primarily due to its lower energy
51
consumption, less sugar degradation, fewer furan derivatives and caustic salts loss
52
(Cai et al., 2016; Li et al., 2015; Akpinar and Usal, 2015; Hideno, 2017). More
53
importantly, it could selectively remove lignin without degrading carbohydrates, and
54
increase porosity and internal surface area of biomass, thereby enhancing enzymatic
55
hydrolysis process (Kim et al., 2016; Zhang and Cai, 2008). Nevertheless, various
56
kinds of inhibitors, especially ferulic acids (4-hydroxy-3-methoxycinnamic acid, FA),
57
were released by cleaving the ester linkages with polysaccharides and the ether
58
linkages with lignin after pretreatment (Li et al., 2015; Jiang et al., 2016; Rouches et
59
al., 2016). Alkaline pretreatment was even used to separate the lignin component from
60
lignocellulose materials and extract ferulic acid (Torres et al., 2009). 2
ACCEPTED MANUSCRIPT 61
As one of the most common lignocellulose-derived microbial inhibitory
62
compounds, FA possesses a benzene ring, methoxy group, hydroxyl group, and
63
double bond within its side chain (Liu et al., 2016; Pérez-Rodríguez et al., 2016).
64
Previous researches showed that FA exerted negative effects on the growth,
65
metabolism and product formation of microorganism cells in the fermentation process
66
at very low concentrations (Huang et al., 2012; Lee et al., 2012). Thus, it is important
67
to eliminate the FA inhibitor in hydrolysates in the pretreatment process to ensure an
68
effective and smoothly pretreatment procedure.
69
There are many detoxification strategies to counteract inhibition problems,
70
whereas a separate process step is required using traditional detoxification methods
71
(Kapoor et al., 2015; Jönsson and Martín, 2016; Saravanakumar et al., 2016; Yu and
72
Christopher, 2017). In-situ detoxification was paid much attention nowadays and was
73
evaluated to be an effective detoxification method by a combination with various
74
kinds of pretreatment process, including steam-exploded pretreatment, γ-irradiation
75
pretreatment, dilute acid pretreatment, etc. (Liu et al., 2016; Zhu et al., 2016; Yu et
76
al., 2011). However, attention has seldom been directed to the combination of alkaline
77
pretreatment.
78
An effective ferulic acid degradation bacterium Escherichia sp. HHQ-1 was
79
preserved in our lab. To detoxify ferulic acid inhibitor in the hydrolysates and
80
enhance bioethanol production, HHQ-1 was added in-situ into alkali-pretreated
81
lignocellulose degrading process in the present study, where both cellulose degrading
82
microbe and ferulic acid degrading bacteria worked together. Meanwhile, the
83
mechanisms of the stimulatory effect of ferulic acid degrading bacteria on bioethanol
84
production were investigated, which could therefore indicate us to choose a suitable
85
pretreatment and detoxification method for enhancing second generation bioethanol
86
production.
87
2. Materials and methods
88
2.1 Materials
89
Rice straw was obtained from Wujing Town, Shanghai, China. It was washed 4-5
90
times with tap water to remove extraneous matters, and then cut to 3 cm. The chopped 3
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clean rice straw was dried in oven at 70 ℃ until constant weight, followed by
92
pulverized below 40 meshes and stored at room temperature till further use. The
93
initial composition of rice straw was determined to be 36.1% cellulose, 24.7%
94
hemicellulose, 16.4% lignin and 22.8% ashes.
95
2.2 Microorganism
96
HHQ-1, an effective ferulic acid degrading bacterial isolate, which was identified
97
as Escherichia sp., was screened from soils in Minhang Campus, East China Normal
98
University (ECNU) and preserved in our lab. Then, it was cultivated in liquid Luria-
99
Bertani medium (LB) at 30℃ with a velocity of 160 r·min-1 for 24 h. The diluted
100
inoculum with OD600 value at 2 was preserved as seed fermentation broth for further
101
use.
102
Trichoderma reesei was bought from China Center of Industrial Culture
103
Collection (CICC) and grown on Potato Dextrose Agar medium (PDA). The spores of
104
Trichoderma reesei were cultivated at 30℃ for 60 h (achieve stationary phase) in a
105
shaking bed of 200 r·min-1. Trichoderma reesei was formed to globular after 2-3
106
days’ growth in liquid medium, thus the relationship between spore’s amount and
107
absorbance is needed to investigate to ensure the accurate inoculums quantity.
108
Preparation of Trichoderma reesei inoculums was followed by our previous research
109
(Hou et al., 2017).
110
2.3 Building microbial degrading systems
111
The smashed rice straw was fist pretreated by 2% NaOH at 85℃ for 1 h before
112
enzymatic hydrolysis (Zhang and Cai, 2008). Subsequently, 3 mL prepared inoculum
113
of Trichoderma reesei, defined as single degrading system, was added for the
114
degradation of alkali-pretreated rice straw. Meanwhile, 1 mL prepared HHQ-1 seed
115
fermentation broth was added in-situ to the single degrading system, which could
116
ensure in-situ detoxification of ferulic acid inhibitors in the hydrolysates. Thus, the
117
Trichoderma reesei and Escherichia sp. HHQ-1 co-degradation system (consortia
118
system) was built.
119
2.4 Analytical methods
120
2.4.1 Degradation rate of ferulic acid 4
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Two gram alkali-pretreated rice straw was added to 100 mL Escherichia sp.
122
HHQ-1 single degrading system directly, and it was fermented for 20 h at 30℃ in a
123
shaking bed under 160 r·min-1. 50 mL fermentation broth was extracted by 20 mL
124
dichloromethane, and the substratum liquid was then centrifuged at 8000 r·min-1 for
125
10 min. After the supernatant was concentrated to 0.5 mL by heating, it was analyzed
126
by GC/MS (Agilent 7890A-5975C) for the content of residue ferulic acid which was
127
degraded by HHQ-1. The column used for GC/MS was HP-5MS capillary column
128
(30m×0.25mm×0.25μm). Helium was used as carrier gas and was held at a constant
129
flow of 1 mL·min-1. The oven temperature was ramped from 80 to 280℃ with the
130
heating rate of 10℃·min-1 and was held at 280℃ for 1 min. The inlet temperature
131
was kept constant at 250℃, and the temperature of detector was kept at 300℃.
132
Hydrogen flow was 30 mL·min-1 and air flow was 400 mL·min-1. Standard curves
133
were obtained by comparing MS peak area ratios relative to various fixed
134
concentrations of ferulic acid. From the standard curves, the residue amount of ferulic
135
acid existed in rice straw hydrolysates were calculated. The degradation rate of ferulic
136
acid was calculated by the following equation:
137 138
Degradation rate of ferulic acid (%) Initial concentration of ferulic acid × initial volume ‒ residue amount of ferulic acid = Initial concentration of ferulic acid × initial volume
2.4.2 Production of reducing sugars
139
Two gram alkali-pretreated rice straw was added to 100 mL Trichoderma reesei-
140
Escherichia sp. HHQ-1 consortia degrading system directly, and it was fermented at
141
30℃ in a shaking bed with a rotate speed of 160 r·min-1. 10 mL fermentation broth
142
was centrifuged at 8000 r·min-1 for 10 min, and the liquid supernatant was then
143
filtered by 0.45 µm membrane filter. Ultimately, the supernatant was used to
144
determine their reducing sugars content by 3, 5-dinitrosalicylic acid colorimetry (DNS
145
method) (Miller, 1959).
146
2.4.3 Determination of cellulases
147
Endoglucanases (E.C.3.2.1.4), exoglucanases (E.C.3.2.1.91) and β-glucosidase
148
(E.C.3.2.1.21) are three main components of cellulases. The activities of
149
endoglucanases and β-glucosidase were determined according to the standard 5
ACCEPTED MANUSCRIPT 150
procedure recommended by the Commission Biotechnology, IUPAC (Ghose, 1987),
151
whereas the activity of exoglucanases was measured following the method described
152
by Ooshima et al. (1990). As for endoglucanases and β-glucosidase, one unit of
153
enzyme was defined as the amount of enzyme capable of producing 1μmol of
154
reducing sugars in 1 min. In terms of the activity of exoglucanases, one unit of
155
enzyme was recognized as the amount of enzyme capable of producing 2 μmol of
156
reducing sugars in 1 min.
157
2.4.4 OD550 and Conductivity measurement
158
Two gram alkali-pretreated rice straw was added to 100 mL Trichoderma reesei-
159
Escherichia sp. HHQ-1 consortia degrading system directly, and it was fermented at
160
30℃ in a shaking bed with a rotation speed of 160 r·min-1. Half part of the
161
fermentation broth was used for Optical Density (OD550) determination by Ultraviolet
162
and Visible Spectrophotometer (UV-2550, Shimadzu, JPN), and the residue half part
163
of the fermentation broth was retained for conductivity determination. The residue
164
fermentation broth was first centrifuged at 5000 r·min-1 for 10 min, and 10 mL
165
supernatant was then mixed with 100 mL deionized water. The conductivity of the
166
mixture was determined by conductivity meter (DDS-307A, China) at room
167
temperature.
168
2.4.5 Statistical analysis
169
All the experiments were performed in triplicates, and the mean values were
170
represented and used ultimately. Error bars on graphs showed the standard deviation.
171
The data were analyzed by Microsoft Excel 2010.
172
2.5 Morphology analysis
173
SEM (S4800, HITACHI, JPN), XRD (AXS-D8, BRUKER, GER) and FTIR
174
(Nicolet iS5, Thermo Fisher Scientific, USA) were used to investigate the
175
morphology and structural features of rice straw in microbial degrading system.
176
3. Results and discussion
177
3.1 The degradation of ferulic acid by adding HHQ-1
178
The degradation rate of ferulic acid in different initial concentrations after adding
179
Escherichia sp. HHQ-1 was shown in Fig.1. It was indicated that the degradation rate 6
ACCEPTED MANUSCRIPT 180
of ferulic acid was elevated as initial concentration of ferulic acid increased. The
181
maximum degradation rate could achieve 62.9% by adding HHQ-1 when initial
182
concentration of ferulic acid was 1000 mg·L-1. Compared to previous report, HHQ-1
183
did not possess the strongest ability for ferulic acid degradation; however, it could
184
tolerate the highest ferulic acid concentration (Xie et al., 2015).
185
Thus, it was supposed that the addition of HHQ-1 to rice straw degrading system
186
could degrade ferulic acid inhibitor in the hydrolysates therefore increasing reducing
187
sugar production.
188
Fig. 1 Degradation rate of ferulic acid by adding HHQ-1. The bars denote the standard deviation (S.D.)
189
3.2 The influence of reducing sugars production by adding HHQ-1
190
Fig. 2 Production of reducing sugars in two different lignocellulose degrading systems. The bars denote the
191
standard deviation (S.D.)
192
HHQ-1 inoculums were added in-situ into alkaline-pretreated rice straw
193
degrading system to build microbial consortia degrading system (followed by part
194
2.4.2), and the production of reducing sugars were shown in Fig. 2.
195
It was shown that the production of reducing sugars in consortia degrading system
196
was less than that of single degrading system before 36 h as there was inhibiting
197
effect of HHQ-1 on lignocellulose degrading system during initial stage. However,
198
with the growth of HHQ-1, the reducing sugars produced by consortia degrading
199
system exceeded single degrading system, which verified our assumption. HHQ-1
200
existed in the consortia degrading system might remove ferulic acid inhibitor from the
201
hydrolysate with additional advantage of restoring reducing sugars content in the
202
hydrolysate at the same time. The production of reducing sugars in consortia
203
degrading system could achieve 221.33 mg·L-1 at 60 h, which was 7.84% higher than
204
that in single degrading system.
205
3.3 Composition of cellulases in different lignocellulose degrading systems
206
Fig .3 Enzyme activities in different lignocellulose degrading systems (a) The enzyme activity of
207
exoglucanase; (b) The enzyme activity of endoglucanase; (c) The enzyme activity of β-glucanase. The bars
208
denote the standard deviation (S.D.)
209
Efficient cellulose hydrolysis requires the cooperative action of endoglucanases,
210
exoglucanases and β-glucosidase (Gottschalk et al., 2010). Activities of the three 7
ACCEPTED MANUSCRIPT 211
main cellulases in single degrading system and consortia degrading system were
212
shown in Fig.3 (a)-(c), respectively.
213
It was showed that activities of the three important cellulases in consortia
214
degrading system were increased after 48 h in the presence of Escherichia sp. HHQ-1.
215
The enhancement of enzymatic activities in the consortia degrading system was lag 12
216
h than the increase of reducing sugars. Meanwhile, compared to the single degrading
217
system, all the three main cellulases were more stable in consortia degrading system.
218
Stable and effective enzymes are of paramount importance, as the high cost and
219
instability of enzymes restricts their use in large scale applications for the conversion
220
of lignocellulosic materials.
221
Besides, as rate-limiting enzyme in rice straw hydrolysis and fermentation
222
process, the activity and proportion of β-glucosidase was highly increased in the
223
consortia system. It could achieve 6.10 U·mL-1 in consortia degrading system, which
224
was 4.23 fold than that in single degrading system. The extremely high β-glucosidase
225
load could reduce the accumulation of cellobiose, thus increasing reducing sugar
226
production and facilitating lignocellulose degrading process.
227
3.4 Conductivity in different lignocellulose degrading systems
228
Fig .4 Conductivity in different lignocellulose degrading systems. The bars denote the standard deviation
229
(S.D.)
230
After preparing fermentation broth and inoculums followed by part 2.4.4, the
231
OD550 value of microbes and the conductivities of fermentation solution in different
232
lignocellulose degrading systems were shown in Fig. 4. It was obvious that a negative
233
correlation relationship existed between conductivity and OD550 in both degrading
234
systems.
235
OD550 in consortia degrading system was much higher than that in single
236
degrading system in the first stage, especially in 60 h. The result indicated that adding
237
HHQ-1 to the degrading system could reduce ferulic acid inhibitor in the hydrolysates
238
successfully, thus ensuring the subsequently fermentation process proceeded
239
smoothly. This result was coordinated with observations of reducing sugars
240
production in hydrolysates (Fig. 2). Meanwhile, accompanied by the rapid growth of 8
ACCEPTED MANUSCRIPT 241
Trichoderma reesei and Escherichia sp. HHQ-1 in degrading systems, inorganic salts
242
in the solutions were largely consumed, which led to the tremendous reduction of
243
conductivity.
244
After 60 h, the conductivities of fermentation solution and OD550 of microbes in
245
different lignocellulose degrading systems converted to another stage. As the
246
microbes declined, OD550 value in both degrading systems decreased rapidly whilst
247
the intracellular metabolites were leached out, thus making the conductivities in
248
fermentation solution sharply increased (Bryant et al., 2011). Besides, the inhibitors
249
existed in hydrolysates could destroy cell membrane, which further enhanced
250
conductivities in fermentation solutions. The increase of fiber fines in amorphous
251
region could also increase the conductivities in fermentation broth (Khalil et al.,
252
2017). Although two different kinds of microbes were co-existed in the consortia
253
degrading system, the conductivities in the fermentation solution were maintained
254
comparative value than that in the single degrading system. Compared to the single
255
degrading system, the effectively removal of inhibitors in the consortia degrading
256
system could protect the cell membrane relatively integrated (Bharadwaj et al., 2011).
257
It should be mentioned that the hydrophobicity of cells and Zeta potential of
258
fermentation solutions were important indexes for the inhibitory effect of different
259
compounds (Du et al., 2010), which are deserved to be investigated in future.
260
3.5 Morphology and structural analysis in different lignocellulose degrading
261
system
262
Since both pretreatment and in situ detoxification by Escherichia sp. HHQ-1
263
could facilitate rice straw degrading process, it became of interest to examine the
264
morphological changes of rice straw in different conditions. Morphology and
265
structural changes of rice straw obtained under different conditions were investigated
266
by means of SEM, XRD and FTIR.
267
3.5.1 SEM analysis of rice straw in different degrading systems
268
The SEM micrographs of rice straw before and after pretreatment were shown in
269
Fig. 5 (a) and Fig. 5 (b), respectively, and the SEM micrograph of rice straw in single
270
degrading system and consortia degrading system were shown in Fig.5 (c) and Fig.5 9
ACCEPTED MANUSCRIPT 271
(d), respectively.
272
Fig. 5 SEM micrographs of rice straw in different lignocellulose degrading systems (a) Before alkaline
273
pretreatment; (b) After alkaline pretreatment; (c) Single degrading system; (d) Consortia degrading system
274
SEM images visually indicated that untreated rice straw displayed a continuous
275
surface and rigid and highly ordered fibrils, while the fibrils after alkaline
276
pretreatment exhibited considerable numbers of heterogeneous layer with pores and
277
showed a sieve like structure. The micro fibrils were separated from initial connected
278
structure and fully exposed, thus increasing the external surface area and porosity of
279
the rice straw, which was coordinate with our previous study (Zhang and Cai, 2008;
280
Hou et al., 2017).
281
After degrading by microbes in both single degrading system and consortia
282
degrading system, basic tissue of rice straw was further severely destroyed. The
283
tissues of vascular bundle were swelled, and the epidermal layers of lignocellulose
284
was dissociated and fractured. It is distinct to see that microbes were absorbed on the
285
surface of rice straw sample. The obvious histological changes of rice straw after
286
adding microbes could provide more adsorption sites on rice straw surface, and
287
enhance the effective attack of microbes to cellulose portion, thus facilitating rice
288
straw degrading process (Ma et al., 2015).
289
3.5.2 XRD analysis of rice straw in different degrading systems
290
XRD provides information related to the crystal and amorphous parts of
291
cellulose (Udeh and Erkurt, 2017). XRD spectra of rice straw in different degrading
292
systems were exhibited in Fig. 6. Crystallinity index (CrI) is an important
293
characteristic that affects enzymatic saccharification of cellulose. The CrI value was
294
calculated by MDI-JADE 5.0 software (Table 1), followed by Segal et al. (1954).
295
Fig. 6 XRD spectra of rice straw in different lignocellulose degrading systems
296
Table 1
The diffraction peak and crystallinity index in different degrading systems
297
All XRD spectra of rice straw samples in different degrading systems exhibited
298
the similar shape and the difference among them was the changes of diffraction peak
299
intensity. The most obvious characteristic peaks of cellulose, which was the peak
300
around 22° (2θ, crystalline region, cellulose I) and 16° (2θ, amorphous region, 10
ACCEPTED MANUSCRIPT 301
cellulose II), was observed in all samples. Therefore, two types of cellulose (cellulose
302
I and cellulose II) coexisted in untreated, pretreated and degraded rice straw biomass
303
(Phitsuwan et al., 2016).
304
The adsorption intensity of rice straw changed obviously after alkaline
305
pretreatment, and the area of crystalline region increased. The CrI value increased
306
from 0.397 to 0.418 after alkaline pretreatment, which might be due to the removal of
307
parts of lignin and amorphous material by alkalinity, leading to relatively high
308
cellulose CrI (Xin et al., 2015; Udeh and Erkurt, 2017; Wang et al., 2016).
309
Meanwhile, alkaline pretreatments changed the cellulose crystal structure of rice
310
straw, thus benefiting the subsequent enzymatic hydrolysis (Jin et al., 2016).
311
The intensity of crystalline region of rice straw was obviously decreased after
312
degrading by microbes, which indicated that the cellulose portion was severely
313
degraded. Microbes could commendably degrade the crystalline region of rice straw,
314
thus facilitating the subsequent fermentation process and increasing bioethanol
315
production. Compared to the single degrading system, the intensity of crystalline
316
region declined more distinctly in the consortia degrading system, which manifested
317
that lignocellulosic biomass was degraded more thorough in the consortia degrading
318
system. This result again manifested the efficient in-situ detoxification effect by
319
HHQ-1.
320
It is worth noting that there was a new strong peak around 32° (2θ, the
321
characteristic peak of SiO2) appeared after degrading by microbes, which is because
322
silicate compounds existed in rice straw biomass was decomposed by microbes at the
323
same time. The interesting phenomenon need to be further investigated in future.
324
3.5.3 FTIR analysis of rice straw in different degrading systems
325
The FTIR spectra of rice straw in different degrading conditions were shown in
326
Fig.7. The characteristic peaks in FTIR spectra were illustrated in Table 2.
327
Fig. 7 FTIR spectra of rice straw in different lignocellulose degrading systems
328
Table 2
Characteristic peaks in FTIR spectra of rice straw
329
The peaks around 3408 cm-1, which is recognized as main infrared sensitive
330
groups of lignocellulose, represents stretching vibration and overlapping of O-H. The 11
ACCEPTED MANUSCRIPT 331
band strengthened after alkaline pretreatment, indicating that alkaline broke the
332
hydrogen bonds in the cellulose to some extent by the formation of hydrogen bonds
333
with cellulose (Hou et al., 2017; Zhang et al., 2016). The peak at 1060 cm-1 represents
334
C-O stretching of cellulose, hemicellulose and lignin or to C–O–C stretching in
335
cellulose and hemicellulose (Perrone et al., 2017). The band intensity was increased
336
after alkaline pretreatment, which again confirm the increase of cellulose relative
337
content. However, it is weaker in microbes degrading system (including the single
338
degrading system and consortia degrading system) than that in the untreated sample,
339
which manifested that hemicellulose and cellulose were partly degraded by microbes.
340
The peak at 2900 cm-1 stands for symmetric or dissymmetric stretching vibration
341
of C-H group, which is one of the characteristic peaks of cellulose. Compared to
342
others, the shape of this peak exhibited narrower in both single degrading system and
343
consortia degrading system, which might because reactions occurred between
344
microbes and other molecular groups. Meanwhile, there is a new peak around 2810
345
cm-1 in both microbial degrading systems, which indicated that aldehyde groups have
346
been formed.
347
The peak at 1637 cm-1 weakened after pretreatment and microbes degrading,
348
which manifested that the molecular geometry of lignin was damaged (Eliana et al.,
349
2014). The peaks at 1510 cm-1 and 1421 cm-1 stand for the vibrations of benzene ring,
350
which are the characteristic peak of lignin. The tremendous change of these peaks
351
further revealed that the lignin part of rice straw was degraded after both alkaline
352
pretreatment and microbes degrading. Several previous studies described that phenols
353
were one of the most abundance compounds in rice straw after alkaline pretreatment,
354
which is coincidence with our results (Karaki et al., 2016; Torre et al., 2008; Banerjee
355
et al., 2016).
356
Meanwhile, the peak at 897 cm-1, which is the characteristic peak of β-glucosidic
357
linkages amid monosaccharide units, was elevated in the alkali-pretreated rice straw
358
samples. This suggested exposure of rice straw fibers after pretreatment. The
359
phenomenon agreed well with Banerjee et al. (2016), and it also accordance with the
360
change of β-glucosidic activity value we determined as explained in Part 3.2. 12
ACCEPTED MANUSCRIPT 361
However, it became much weaker in microbes degrading systems. The breakage of β-
362
glucoside bond is a rate-limiting step in lignocellulose degrading process; therefore,
363
the weakened β-glucoside bond after microbes degrading could enormously promote
364
the efficiency of rice straw hydrolysis.
365
3.6 Comparison of single degrading system and consortia degrading system
366
By determining the production of reducing sugars, composition of cellulases,
367
conductivity of the fermentation broth, and analyzing the morphology and structure
368
change in both degrading systems, lots of merits were emerged in consortia degrading
369
system than that in single degrading system (shown in Table 3).
370
It was observed that the maximum production of reducing sugars in the consortia
371
degrading system could achieve 221.33 mg·L-1. This might be ascribed to the
372
detoxification by adding HHQ-1, which could degrade 62.9% ferulic acid in the
373
system. More stable of cellulases, higher activity of β-glucanase and relative
374
stationary conductivity also resulted in an effective detoxification in the consortia
375
degrading system. Moreover, there were obvious morphological and structure changes
376
in the consortia degrading system than that of single degrading system. These features
377
would make in-situ detoxification smoothly proceeded in the consortia degrading
378
system.
379
Detoxification process is necessary to remove the inhibitory and toxic
380
compounds before fermentation in the process of bioethanaol production; however, a
381
separate detoxification might increase the cost of bioethanol production and waste
382
time (Liu et al., 2016; Zhu et al., 2016). In our present research, the consortia
383
degrading system could rapidly degrade most parts of ferulic acid inhibitor in the
384
pretreated rice straw hydrolysates, therefore showing increased reducing sugars
385
production compared to the single degrading system. This in-situ detoxification could
386
make pretreatment and detoxification process proceeded simultaneously, thus
387
exhibited practical benefits for bioethanol production in future.
388
Table 3
The merits of using consortia degrading system over single degrading system
389 390
4. Conclusion 13
ACCEPTED MANUSCRIPT 391
In-situ detoxification by adding ferulic acid degrading bacteria to alkali-
392
pretreated rice straw degradation system was realized, which could purposefully
393
remove inhibitors in the hydrolysates, thus increasing reducing sugars production and
394
ensure bioethanol production preceded smoothly. The HHQ-1-Trichoderma reesei
395
consortia degrading system showed potential benefits for protecting the integrity of
396
microbial membrane, providing microbes for more adsorption sites in lignocellulose
397
material, strengthening activities of important cellulases, forming hydrogen bond,
398
decreasing crystallinity index and degrading lignin parts of rice straw effectively.
399
These might be the main mechanisms of the stimulatory effect of bioethanol
400
production by in-situ detoxification using microbial consortia degrading system. The
401
use of new microorganisms in in-situ detoxification process could play a key role for
402
the conversion of carbohydrates contained lignocellulosic biomass into fermentable
403
sugars, thus providing a viable route for bioethanol production.
404 405
Acknowledgment
406
This project is funded by National Natural Science Foundation of China (NSFC,
407
No. 31400513), Shanghai Science and Technology Committee (No. 17295810600)
408
and Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration
409
(SHUES2016B01). The authors would like to thank them for funding this work.
410 411
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583 584 585 586 587 588 589 590 591 592 593 594 595 596
20
ACCEPTED MANUSCRIPT
80 70
Degradation rate (%)
60 50 40 30 20 10 0 500
750
1000
1250
Initial concentration of ferulic acid (mgL-1) 597 598
Fig. 1 Degradation rate of ferulic acid by adding HHQ-1. The bars denote the standard deviation (S.D.)
The content of reducing sugars（ mgL-1（
350
Single degrading system Consortia degrading system
300 250 200 150 100 50 0 0
12
24
36
48
60
72
Time (h) 599 600
Fig. 2 Production of reducing sugars in two different lignocellulose degrading systems. The bars denote the
601
standard deviation (S.D.)
602 603 21
ACCEPTED MANUSCRIPT 604 10.0
10.0
Single degrading system Consortia degrading system
605
608 609
8.0
Enzyme activity (UmL-1)
607
Enzyme activity (UmL-1)
8.0
606
6.0
4.0
2.0
6.0
4.0
2.0
0.0
610
Single degrading system Consortia degrading system
0.0 12
24
36
48
60
72
12
24
Enzymatic time (h)
611
10.0
612
8.0
36
48
60
72
Enzymatic time (h)
Enzyme Activity (UmL-1)
Single degrading system Consortia degrading system
613 614 615 616
6.0
4.0
2.0
0.0 12
24
36
617
48
60
72
Enzymatic time (h)
618
Fig .3 Enzyme activities in different lignocellulose degrading systems (a) The enzyme activity of
619
exoglucanase; (b) The enzyme activity of endoglucanase; (c) The enzyme activity of β-glucanase. The bars
620
denote the standard deviation (S.D.)
621 2.4
Single degrading system Consortia degrading system
623
0.7
2.2
Conductivity (Mscm-1)
624 625 626 627 628
0.8
0.6
2.0
0.5 1.8 0.4 1.6
OD550
622
0.3
1.4
0.2
629 1.2
630
0.1 12
24
36
48
60
72
Time (h)
631 632
Fig .4 Conductivity in different lignocellulose degrading systems. The bars denote the standard deviation
633
(S.D.)
634 22
ACCEPTED MANUSCRIPT 635
636
(a)
(b)
(c)
(d)
637 638
Fig. 5 SEM micrographs of rice straw in different lignocellulose degrading systems (a) Before alkaline
639
pretreatment; (b) After alkaline pretreatment; (c) Single degrading system; (d) Consortia degrading system
640 4.0
641
Before pretreatment After alkaline pretreatment Single degrading system Consortia degrading system
3.6 3.2
643
2.8
644 645
Intensity (104)
642
646
649
2.0 1.6 1.2 0.8
647 648
2.4
0.4 12
15
18
21
24
27
30
33
36
39
42
45
2 Fig. 6 XRD spectra of rice straw in different lignocellulose degrading systems
650 105
651 652
654 655 656 657 658
100
Transmittance (%)
653
Before pretreatment After alkaline pretreatment Single degrading system Consortia degrading system
95
90
85
80 3500
3000
2500
2000
1500
1000
-1
Wave length (cm )
659 660
Fig. 7 FTIR spectra of rice straw in different lignocellulose degrading systems
661 23
500
ACCEPTED MANUSCRIPT
662
Table 1
The diffraction peak and crystallinity index in different degrading systems
Diffraction Peak
Substrate
Crystallinity Index
2θ=16°
2θ=22°
Untreated rice straw
20960
33721
0.397
After alkaline pretreatment
23778
40864
0.418
Single degrading system
17684
29110
0.393
Consortia degrading system
17063
27403
0.377
663 664 665
Table 2
Characteristic peaks in FTIR spectra of rice straw
Adsorption peak（cm-1）
Affiliation of characteristic peaks
3408
stretching vibration and overlapping of O-H
2900
symmetric or dissymmetric stretching vibration of C-H group
1637
stretching vibration of C=O (lignin)
1510
Characteristic group vibrations of benzene ring (lignin)
1421
Characteristic group vibrations of benzene ring (lignin)
1321
Characteristic group vibrations of C-O (lignin)
1200
Stretching vibration of CO-OR
1060
C-O stretching of hemicellulose and cellulose
897
C-H deformation of skeleton vibration of saccharides and cellulose
666 667 668 669
Table 3
The merits of using consortia degrading system over single degrading system
670 Items Maximum degradation rate of ferulic acid (%) Maximum production of reducing sugars
(mg·L-1)
Stability of cellulases Activity of β-glucanase
(U·mL-1)
OD550 value at 60 h Conductivity at 60 h
(MS·cm-1)
Morphology and structure analysis
Single degrading system
Consortia degrading system
0
62.9
205.24
221.33
Less stable
More stable
1.44
6.10
0.44
0.67
1.40
1.56
SEM
Seriously destroyed tissue
Seriously destroyed tissue
XRD
Higher CrI value (0.393)
Lower CrI value (0.377)
FTIR
No hydrogen bond
Forming hydrogen bond
671
24
ACCEPTED MANUSCRIPT Highlights:
In-situ detoxification was successfully realized for bioethanol production.
In-situ detoxification increased the production of reducing sugars effectively.
Mechanisms of the stimulatory effect of in-situ detoxification were investigated.
Morphology changes of rice straw were analyzed by SEM, XRD and FTIR.
Qiuzhuo Zhang, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal PII:
S0360-5442(17)31093-9
DOI:
10.1016/j.energy.2017.06.099
Reference:
EGY 11107
To appear in:
Energy
Received Date:
22 February 2017
Revised Date:
10 May 2017
Accepted Date:
18 June 2017
Please cite this article as: Qiuzhuo Zhang, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal, Stimulatory effect of in-situ detoxification on bioethanol production by rice straw, Energy (2017), doi: 10.1016/j.energy.2017.06.099
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In-situ detoxification
Hydrolysis
Alkaline
Trichoderma reesei
Available hydrolysates
Pretreatment
Degradation
Reducing sugars
Rice straw Ferulic acid
Escherichia sp. HHQ-1
Consortia degrading system
Graphic Abstract
Bioethanol
ACCEPTED MANUSCRIPT 1
Stimulatory effect of in-situ detoxification on bioethanol production by rice
2
straw
3
Qiuzhuo Zhang*, Huiqin Huang, Hui Han, Zhen Qiu, Varenyam Achal
4
Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration, School of Ecological and
5
Environmental Sciences, East China Normal University, 200241 Shanghai, China
6 7
Abstract: An effective ferulic acid degrading bacterium, Escherichia sp. HHQ-1, was
8
added to alkali-pretreated rice straw degrading system to in-situ detoxify the ferulic
9
acid inhibitors existed in the hydrolysates. It was shown that the production of
10
reducing sugars in Escherichia sp. HHQ-1-Trichoderma reesei consortia degrading
11
system could achieve 221.33 mg·L-1 at 60 h, which was 7.84% higher than that in
12
single degrading system. All the three main cellulases were more stable in consortia
13
degrading system, and the activity of β-glucosidase was 4.23 fold higher than that in
14
single degrading system. Besides, it was supposed that cell membrane was more
15
integrally protected in consortia degrading system. Scanning Electron Microscope
16
(SEM), X-ray Diffraction (XRD) and Fourier Transform Infrared Spectrometer
17
(FTIR) were used to observe the morphological changes of rice straw biomass. It was
18
indicated that in-situ detoxification could severely destroy basic tissue of rice straw,
19
dramatically decrease crystallinity index of crystalline region and effectively degrade
20
refractory-degraded lignin parts of lignocellulose. Compared to the single degrading
21
system, the consortia degrading system could in-situ detoxify the main inhibitors
22
which existed in pretreated rice straw hydrolysates, realize simultaneous pretreatment
23
and detoxification, thus increasing the bioethanol yield and reducing the cost of
24
bioethanol production.
25
Keywords: consortia system; in-situ detoxification; rice straw; bioethanol
26
List of abbreviations:
27
CICC: China Center of Industrial Culture Collection; CrI: Crystallinity index; DNS method: 3, 5-dinitrosalicylic
28
acid colorimetry; ECNU: East China Normal University; FA: Ferulic Acid (4-hydroxy-3-methoxycinnamic acid);
29
FTIR: Fourier Transform Infrared Spectrometer; GC/MS: Gas Chromatography-Mass Spectromete; LB: Luria-
30
Bertani medium; OD: Optical Density; PDA: Potato Dextrose Agar medium; SEM: Scanning Electron 1
ACCEPTED MANUSCRIPT 31
Microscope; UV: Ultraviolet and Visible Spectrophotometer; XRD: X-ray Diffraction
32
1. Introduction
33
Bioethanol, an alternative liquid fuel to overcome energy shortage problem, is a
34
promising near-term production, which has been developed extensively for more than
35
two decades (Yu et al., 2016; Domínguez et al., 2017; Khatiwada and Silveira, 2017).
36
Among renewable sources, lignocellulosic biomass is one of the most attractive and
37
main potential raw materials for bioethanol production because of its easy availability,
38
low price and high sugar content (Barros-Rios et al., 2016; Zhao et al., 2016).
39
Lignocellulosic biomass has a complex structure composed mainly of cellulose,
40
hemicellulose and lignin (Sindhu et al., 2016). These components interconnected well
41
through non-covalent and covalent bonds into a highly organized network that may
42
restrict enzyme accessibility and thereby reducing the efficiency of decomposing
43
enzymes (Cripwell et al., 2015).
44
Pretreatment is an indispensible step in lignocelluloses fermentation process
45
(Merali et al., 2016; Licari et al., 2016; Yang and Rosentrater, 2017). A well-designed
46
pretreatment method could help us to overcome recalcitrance from the rigid biomass
47
structure, which is beneficial for promoting enzymatic hydrolysis and realizing low-
48
cost lignocellulosic bioethanol (Cai et al., 2016; Dongen et al., 2011; Baral and Shah,
49
2017). Among the numerous pretreatment methods, alkaline pretreatment has
50
emerged as one of the most viable options primarily due to its lower energy
51
consumption, less sugar degradation, fewer furan derivatives and caustic salts loss
52
(Cai et al., 2016; Li et al., 2015; Akpinar and Usal, 2015; Hideno, 2017). More
53
importantly, it could selectively remove lignin without degrading carbohydrates, and
54
increase porosity and internal surface area of biomass, thereby enhancing enzymatic
55
hydrolysis process (Kim et al., 2016; Zhang and Cai, 2008). Nevertheless, various
56
kinds of inhibitors, especially ferulic acids (4-hydroxy-3-methoxycinnamic acid, FA),
57
were released by cleaving the ester linkages with polysaccharides and the ether
58
linkages with lignin after pretreatment (Li et al., 2015; Jiang et al., 2016; Rouches et
59
al., 2016). Alkaline pretreatment was even used to separate the lignin component from
60
lignocellulose materials and extract ferulic acid (Torres et al., 2009). 2
ACCEPTED MANUSCRIPT 61
As one of the most common lignocellulose-derived microbial inhibitory
62
compounds, FA possesses a benzene ring, methoxy group, hydroxyl group, and
63
double bond within its side chain (Liu et al., 2016; Pérez-Rodríguez et al., 2016).
64
Previous researches showed that FA exerted negative effects on the growth,
65
metabolism and product formation of microorganism cells in the fermentation process
66
at very low concentrations (Huang et al., 2012; Lee et al., 2012). Thus, it is important
67
to eliminate the FA inhibitor in hydrolysates in the pretreatment process to ensure an
68
effective and smoothly pretreatment procedure.
69
There are many detoxification strategies to counteract inhibition problems,
70
whereas a separate process step is required using traditional detoxification methods
71
(Kapoor et al., 2015; Jönsson and Martín, 2016; Saravanakumar et al., 2016; Yu and
72
Christopher, 2017). In-situ detoxification was paid much attention nowadays and was
73
evaluated to be an effective detoxification method by a combination with various
74
kinds of pretreatment process, including steam-exploded pretreatment, γ-irradiation
75
pretreatment, dilute acid pretreatment, etc. (Liu et al., 2016; Zhu et al., 2016; Yu et
76
al., 2011). However, attention has seldom been directed to the combination of alkaline
77
pretreatment.
78
An effective ferulic acid degradation bacterium Escherichia sp. HHQ-1 was
79
preserved in our lab. To detoxify ferulic acid inhibitor in the hydrolysates and
80
enhance bioethanol production, HHQ-1 was added in-situ into alkali-pretreated
81
lignocellulose degrading process in the present study, where both cellulose degrading
82
microbe and ferulic acid degrading bacteria worked together. Meanwhile, the
83
mechanisms of the stimulatory effect of ferulic acid degrading bacteria on bioethanol
84
production were investigated, which could therefore indicate us to choose a suitable
85
pretreatment and detoxification method for enhancing second generation bioethanol
86
production.
87
2. Materials and methods
88
2.1 Materials
89
Rice straw was obtained from Wujing Town, Shanghai, China. It was washed 4-5
90
times with tap water to remove extraneous matters, and then cut to 3 cm. The chopped 3
ACCEPTED MANUSCRIPT 91
clean rice straw was dried in oven at 70 ℃ until constant weight, followed by
92
pulverized below 40 meshes and stored at room temperature till further use. The
93
initial composition of rice straw was determined to be 36.1% cellulose, 24.7%
94
hemicellulose, 16.4% lignin and 22.8% ashes.
95
2.2 Microorganism
96
HHQ-1, an effective ferulic acid degrading bacterial isolate, which was identified
97
as Escherichia sp., was screened from soils in Minhang Campus, East China Normal
98
University (ECNU) and preserved in our lab. Then, it was cultivated in liquid Luria-
99
Bertani medium (LB) at 30℃ with a velocity of 160 r·min-1 for 24 h. The diluted
100
inoculum with OD600 value at 2 was preserved as seed fermentation broth for further
101
use.
102
Trichoderma reesei was bought from China Center of Industrial Culture
103
Collection (CICC) and grown on Potato Dextrose Agar medium (PDA). The spores of
104
Trichoderma reesei were cultivated at 30℃ for 60 h (achieve stationary phase) in a
105
shaking bed of 200 r·min-1. Trichoderma reesei was formed to globular after 2-3
106
days’ growth in liquid medium, thus the relationship between spore’s amount and
107
absorbance is needed to investigate to ensure the accurate inoculums quantity.
108
Preparation of Trichoderma reesei inoculums was followed by our previous research
109
(Hou et al., 2017).
110
2.3 Building microbial degrading systems
111
The smashed rice straw was fist pretreated by 2% NaOH at 85℃ for 1 h before
112
enzymatic hydrolysis (Zhang and Cai, 2008). Subsequently, 3 mL prepared inoculum
113
of Trichoderma reesei, defined as single degrading system, was added for the
114
degradation of alkali-pretreated rice straw. Meanwhile, 1 mL prepared HHQ-1 seed
115
fermentation broth was added in-situ to the single degrading system, which could
116
ensure in-situ detoxification of ferulic acid inhibitors in the hydrolysates. Thus, the
117
Trichoderma reesei and Escherichia sp. HHQ-1 co-degradation system (consortia
118
system) was built.
119
2.4 Analytical methods
120
2.4.1 Degradation rate of ferulic acid 4
ACCEPTED MANUSCRIPT 121
Two gram alkali-pretreated rice straw was added to 100 mL Escherichia sp.
122
HHQ-1 single degrading system directly, and it was fermented for 20 h at 30℃ in a
123
shaking bed under 160 r·min-1. 50 mL fermentation broth was extracted by 20 mL
124
dichloromethane, and the substratum liquid was then centrifuged at 8000 r·min-1 for
125
10 min. After the supernatant was concentrated to 0.5 mL by heating, it was analyzed
126
by GC/MS (Agilent 7890A-5975C) for the content of residue ferulic acid which was
127
degraded by HHQ-1. The column used for GC/MS was HP-5MS capillary column
128
(30m×0.25mm×0.25μm). Helium was used as carrier gas and was held at a constant
129
flow of 1 mL·min-1. The oven temperature was ramped from 80 to 280℃ with the
130
heating rate of 10℃·min-1 and was held at 280℃ for 1 min. The inlet temperature
131
was kept constant at 250℃, and the temperature of detector was kept at 300℃.
132
Hydrogen flow was 30 mL·min-1 and air flow was 400 mL·min-1. Standard curves
133
were obtained by comparing MS peak area ratios relative to various fixed
134
concentrations of ferulic acid. From the standard curves, the residue amount of ferulic
135
acid existed in rice straw hydrolysates were calculated. The degradation rate of ferulic
136
acid was calculated by the following equation:
137 138
Degradation rate of ferulic acid (%) Initial concentration of ferulic acid × initial volume ‒ residue amount of ferulic acid = Initial concentration of ferulic acid × initial volume
2.4.2 Production of reducing sugars
139
Two gram alkali-pretreated rice straw was added to 100 mL Trichoderma reesei-
140
Escherichia sp. HHQ-1 consortia degrading system directly, and it was fermented at
141
30℃ in a shaking bed with a rotate speed of 160 r·min-1. 10 mL fermentation broth
142
was centrifuged at 8000 r·min-1 for 10 min, and the liquid supernatant was then
143
filtered by 0.45 µm membrane filter. Ultimately, the supernatant was used to
144
determine their reducing sugars content by 3, 5-dinitrosalicylic acid colorimetry (DNS
145
method) (Miller, 1959).
146
2.4.3 Determination of cellulases
147
Endoglucanases (E.C.3.2.1.4), exoglucanases (E.C.3.2.1.91) and β-glucosidase
148
(E.C.3.2.1.21) are three main components of cellulases. The activities of
149
endoglucanases and β-glucosidase were determined according to the standard 5
ACCEPTED MANUSCRIPT 150
procedure recommended by the Commission Biotechnology, IUPAC (Ghose, 1987),
151
whereas the activity of exoglucanases was measured following the method described
152
by Ooshima et al. (1990). As for endoglucanases and β-glucosidase, one unit of
153
enzyme was defined as the amount of enzyme capable of producing 1μmol of
154
reducing sugars in 1 min. In terms of the activity of exoglucanases, one unit of
155
enzyme was recognized as the amount of enzyme capable of producing 2 μmol of
156
reducing sugars in 1 min.
157
2.4.4 OD550 and Conductivity measurement
158
Two gram alkali-pretreated rice straw was added to 100 mL Trichoderma reesei-
159
Escherichia sp. HHQ-1 consortia degrading system directly, and it was fermented at
160
30℃ in a shaking bed with a rotation speed of 160 r·min-1. Half part of the
161
fermentation broth was used for Optical Density (OD550) determination by Ultraviolet
162
and Visible Spectrophotometer (UV-2550, Shimadzu, JPN), and the residue half part
163
of the fermentation broth was retained for conductivity determination. The residue
164
fermentation broth was first centrifuged at 5000 r·min-1 for 10 min, and 10 mL
165
supernatant was then mixed with 100 mL deionized water. The conductivity of the
166
mixture was determined by conductivity meter (DDS-307A, China) at room
167
temperature.
168
2.4.5 Statistical analysis
169
All the experiments were performed in triplicates, and the mean values were
170
represented and used ultimately. Error bars on graphs showed the standard deviation.
171
The data were analyzed by Microsoft Excel 2010.
172
2.5 Morphology analysis
173
SEM (S4800, HITACHI, JPN), XRD (AXS-D8, BRUKER, GER) and FTIR
174
(Nicolet iS5, Thermo Fisher Scientific, USA) were used to investigate the
175
morphology and structural features of rice straw in microbial degrading system.
176
3. Results and discussion
177
3.1 The degradation of ferulic acid by adding HHQ-1
178
The degradation rate of ferulic acid in different initial concentrations after adding
179
Escherichia sp. HHQ-1 was shown in Fig.1. It was indicated that the degradation rate 6
ACCEPTED MANUSCRIPT 180
of ferulic acid was elevated as initial concentration of ferulic acid increased. The
181
maximum degradation rate could achieve 62.9% by adding HHQ-1 when initial
182
concentration of ferulic acid was 1000 mg·L-1. Compared to previous report, HHQ-1
183
did not possess the strongest ability for ferulic acid degradation; however, it could
184
tolerate the highest ferulic acid concentration (Xie et al., 2015).
185
Thus, it was supposed that the addition of HHQ-1 to rice straw degrading system
186
could degrade ferulic acid inhibitor in the hydrolysates therefore increasing reducing
187
sugar production.
188
Fig. 1 Degradation rate of ferulic acid by adding HHQ-1. The bars denote the standard deviation (S.D.)
189
3.2 The influence of reducing sugars production by adding HHQ-1
190
Fig. 2 Production of reducing sugars in two different lignocellulose degrading systems. The bars denote the
191
standard deviation (S.D.)
192
HHQ-1 inoculums were added in-situ into alkaline-pretreated rice straw
193
degrading system to build microbial consortia degrading system (followed by part
194
2.4.2), and the production of reducing sugars were shown in Fig. 2.
195
It was shown that the production of reducing sugars in consortia degrading system
196
was less than that of single degrading system before 36 h as there was inhibiting
197
effect of HHQ-1 on lignocellulose degrading system during initial stage. However,
198
with the growth of HHQ-1, the reducing sugars produced by consortia degrading
199
system exceeded single degrading system, which verified our assumption. HHQ-1
200
existed in the consortia degrading system might remove ferulic acid inhibitor from the
201
hydrolysate with additional advantage of restoring reducing sugars content in the
202
hydrolysate at the same time. The production of reducing sugars in consortia
203
degrading system could achieve 221.33 mg·L-1 at 60 h, which was 7.84% higher than
204
that in single degrading system.
205
3.3 Composition of cellulases in different lignocellulose degrading systems
206
Fig .3 Enzyme activities in different lignocellulose degrading systems (a) The enzyme activity of
207
exoglucanase; (b) The enzyme activity of endoglucanase; (c) The enzyme activity of β-glucanase. The bars
208
denote the standard deviation (S.D.)
209
Efficient cellulose hydrolysis requires the cooperative action of endoglucanases,
210
exoglucanases and β-glucosidase (Gottschalk et al., 2010). Activities of the three 7
ACCEPTED MANUSCRIPT 211
main cellulases in single degrading system and consortia degrading system were
212
shown in Fig.3 (a)-(c), respectively.
213
It was showed that activities of the three important cellulases in consortia
214
degrading system were increased after 48 h in the presence of Escherichia sp. HHQ-1.
215
The enhancement of enzymatic activities in the consortia degrading system was lag 12
216
h than the increase of reducing sugars. Meanwhile, compared to the single degrading
217
system, all the three main cellulases were more stable in consortia degrading system.
218
Stable and effective enzymes are of paramount importance, as the high cost and
219
instability of enzymes restricts their use in large scale applications for the conversion
220
of lignocellulosic materials.
221
Besides, as rate-limiting enzyme in rice straw hydrolysis and fermentation
222
process, the activity and proportion of β-glucosidase was highly increased in the
223
consortia system. It could achieve 6.10 U·mL-1 in consortia degrading system, which
224
was 4.23 fold than that in single degrading system. The extremely high β-glucosidase
225
load could reduce the accumulation of cellobiose, thus increasing reducing sugar
226
production and facilitating lignocellulose degrading process.
227
3.4 Conductivity in different lignocellulose degrading systems
228
Fig .4 Conductivity in different lignocellulose degrading systems. The bars denote the standard deviation
229
(S.D.)
230
After preparing fermentation broth and inoculums followed by part 2.4.4, the
231
OD550 value of microbes and the conductivities of fermentation solution in different
232
lignocellulose degrading systems were shown in Fig. 4. It was obvious that a negative
233
correlation relationship existed between conductivity and OD550 in both degrading
234
systems.
235
OD550 in consortia degrading system was much higher than that in single
236
degrading system in the first stage, especially in 60 h. The result indicated that adding
237
HHQ-1 to the degrading system could reduce ferulic acid inhibitor in the hydrolysates
238
successfully, thus ensuring the subsequently fermentation process proceeded
239
smoothly. This result was coordinated with observations of reducing sugars
240
production in hydrolysates (Fig. 2). Meanwhile, accompanied by the rapid growth of 8
ACCEPTED MANUSCRIPT 241
Trichoderma reesei and Escherichia sp. HHQ-1 in degrading systems, inorganic salts
242
in the solutions were largely consumed, which led to the tremendous reduction of
243
conductivity.
244
After 60 h, the conductivities of fermentation solution and OD550 of microbes in
245
different lignocellulose degrading systems converted to another stage. As the
246
microbes declined, OD550 value in both degrading systems decreased rapidly whilst
247
the intracellular metabolites were leached out, thus making the conductivities in
248
fermentation solution sharply increased (Bryant et al., 2011). Besides, the inhibitors
249
existed in hydrolysates could destroy cell membrane, which further enhanced
250
conductivities in fermentation solutions. The increase of fiber fines in amorphous
251
region could also increase the conductivities in fermentation broth (Khalil et al.,
252
2017). Although two different kinds of microbes were co-existed in the consortia
253
degrading system, the conductivities in the fermentation solution were maintained
254
comparative value than that in the single degrading system. Compared to the single
255
degrading system, the effectively removal of inhibitors in the consortia degrading
256
system could protect the cell membrane relatively integrated (Bharadwaj et al., 2011).
257
It should be mentioned that the hydrophobicity of cells and Zeta potential of
258
fermentation solutions were important indexes for the inhibitory effect of different
259
compounds (Du et al., 2010), which are deserved to be investigated in future.
260
3.5 Morphology and structural analysis in different lignocellulose degrading
261
system
262
Since both pretreatment and in situ detoxification by Escherichia sp. HHQ-1
263
could facilitate rice straw degrading process, it became of interest to examine the
264
morphological changes of rice straw in different conditions. Morphology and
265
structural changes of rice straw obtained under different conditions were investigated
266
by means of SEM, XRD and FTIR.
267
3.5.1 SEM analysis of rice straw in different degrading systems
268
The SEM micrographs of rice straw before and after pretreatment were shown in
269
Fig. 5 (a) and Fig. 5 (b), respectively, and the SEM micrograph of rice straw in single
270
degrading system and consortia degrading system were shown in Fig.5 (c) and Fig.5 9
ACCEPTED MANUSCRIPT 271
(d), respectively.
272
Fig. 5 SEM micrographs of rice straw in different lignocellulose degrading systems (a) Before alkaline
273
pretreatment; (b) After alkaline pretreatment; (c) Single degrading system; (d) Consortia degrading system
274
SEM images visually indicated that untreated rice straw displayed a continuous
275
surface and rigid and highly ordered fibrils, while the fibrils after alkaline
276
pretreatment exhibited considerable numbers of heterogeneous layer with pores and
277
showed a sieve like structure. The micro fibrils were separated from initial connected
278
structure and fully exposed, thus increasing the external surface area and porosity of
279
the rice straw, which was coordinate with our previous study (Zhang and Cai, 2008;
280
Hou et al., 2017).
281
After degrading by microbes in both single degrading system and consortia
282
degrading system, basic tissue of rice straw was further severely destroyed. The
283
tissues of vascular bundle were swelled, and the epidermal layers of lignocellulose
284
was dissociated and fractured. It is distinct to see that microbes were absorbed on the
285
surface of rice straw sample. The obvious histological changes of rice straw after
286
adding microbes could provide more adsorption sites on rice straw surface, and
287
enhance the effective attack of microbes to cellulose portion, thus facilitating rice
288
straw degrading process (Ma et al., 2015).
289
3.5.2 XRD analysis of rice straw in different degrading systems
290
XRD provides information related to the crystal and amorphous parts of
291
cellulose (Udeh and Erkurt, 2017). XRD spectra of rice straw in different degrading
292
systems were exhibited in Fig. 6. Crystallinity index (CrI) is an important
293
characteristic that affects enzymatic saccharification of cellulose. The CrI value was
294
calculated by MDI-JADE 5.0 software (Table 1), followed by Segal et al. (1954).
295
Fig. 6 XRD spectra of rice straw in different lignocellulose degrading systems
296
Table 1
The diffraction peak and crystallinity index in different degrading systems
297
All XRD spectra of rice straw samples in different degrading systems exhibited
298
the similar shape and the difference among them was the changes of diffraction peak
299
intensity. The most obvious characteristic peaks of cellulose, which was the peak
300
around 22° (2θ, crystalline region, cellulose I) and 16° (2θ, amorphous region, 10
ACCEPTED MANUSCRIPT 301
cellulose II), was observed in all samples. Therefore, two types of cellulose (cellulose
302
I and cellulose II) coexisted in untreated, pretreated and degraded rice straw biomass
303
(Phitsuwan et al., 2016).
304
The adsorption intensity of rice straw changed obviously after alkaline
305
pretreatment, and the area of crystalline region increased. The CrI value increased
306
from 0.397 to 0.418 after alkaline pretreatment, which might be due to the removal of
307
parts of lignin and amorphous material by alkalinity, leading to relatively high
308
cellulose CrI (Xin et al., 2015; Udeh and Erkurt, 2017; Wang et al., 2016).
309
Meanwhile, alkaline pretreatments changed the cellulose crystal structure of rice
310
straw, thus benefiting the subsequent enzymatic hydrolysis (Jin et al., 2016).
311
The intensity of crystalline region of rice straw was obviously decreased after
312
degrading by microbes, which indicated that the cellulose portion was severely
313
degraded. Microbes could commendably degrade the crystalline region of rice straw,
314
thus facilitating the subsequent fermentation process and increasing bioethanol
315
production. Compared to the single degrading system, the intensity of crystalline
316
region declined more distinctly in the consortia degrading system, which manifested
317
that lignocellulosic biomass was degraded more thorough in the consortia degrading
318
system. This result again manifested the efficient in-situ detoxification effect by
319
HHQ-1.
320
It is worth noting that there was a new strong peak around 32° (2θ, the
321
characteristic peak of SiO2) appeared after degrading by microbes, which is because
322
silicate compounds existed in rice straw biomass was decomposed by microbes at the
323
same time. The interesting phenomenon need to be further investigated in future.
324
3.5.3 FTIR analysis of rice straw in different degrading systems
325
The FTIR spectra of rice straw in different degrading conditions were shown in
326
Fig.7. The characteristic peaks in FTIR spectra were illustrated in Table 2.
327
Fig. 7 FTIR spectra of rice straw in different lignocellulose degrading systems
328
Table 2
Characteristic peaks in FTIR spectra of rice straw
329
The peaks around 3408 cm-1, which is recognized as main infrared sensitive
330
groups of lignocellulose, represents stretching vibration and overlapping of O-H. The 11
ACCEPTED MANUSCRIPT 331
band strengthened after alkaline pretreatment, indicating that alkaline broke the
332
hydrogen bonds in the cellulose to some extent by the formation of hydrogen bonds
333
with cellulose (Hou et al., 2017; Zhang et al., 2016). The peak at 1060 cm-1 represents
334
C-O stretching of cellulose, hemicellulose and lignin or to C–O–C stretching in
335
cellulose and hemicellulose (Perrone et al., 2017). The band intensity was increased
336
after alkaline pretreatment, which again confirm the increase of cellulose relative
337
content. However, it is weaker in microbes degrading system (including the single
338
degrading system and consortia degrading system) than that in the untreated sample,
339
which manifested that hemicellulose and cellulose were partly degraded by microbes.
340
The peak at 2900 cm-1 stands for symmetric or dissymmetric stretching vibration
341
of C-H group, which is one of the characteristic peaks of cellulose. Compared to
342
others, the shape of this peak exhibited narrower in both single degrading system and
343
consortia degrading system, which might because reactions occurred between
344
microbes and other molecular groups. Meanwhile, there is a new peak around 2810
345
cm-1 in both microbial degrading systems, which indicated that aldehyde groups have
346
been formed.
347
The peak at 1637 cm-1 weakened after pretreatment and microbes degrading,
348
which manifested that the molecular geometry of lignin was damaged (Eliana et al.,
349
2014). The peaks at 1510 cm-1 and 1421 cm-1 stand for the vibrations of benzene ring,
350
which are the characteristic peak of lignin. The tremendous change of these peaks
351
further revealed that the lignin part of rice straw was degraded after both alkaline
352
pretreatment and microbes degrading. Several previous studies described that phenols
353
were one of the most abundance compounds in rice straw after alkaline pretreatment,
354
which is coincidence with our results (Karaki et al., 2016; Torre et al., 2008; Banerjee
355
et al., 2016).
356
Meanwhile, the peak at 897 cm-1, which is the characteristic peak of β-glucosidic
357
linkages amid monosaccharide units, was elevated in the alkali-pretreated rice straw
358
samples. This suggested exposure of rice straw fibers after pretreatment. The
359
phenomenon agreed well with Banerjee et al. (2016), and it also accordance with the
360
change of β-glucosidic activity value we determined as explained in Part 3.2. 12
ACCEPTED MANUSCRIPT 361
However, it became much weaker in microbes degrading systems. The breakage of β-
362
glucoside bond is a rate-limiting step in lignocellulose degrading process; therefore,
363
the weakened β-glucoside bond after microbes degrading could enormously promote
364
the efficiency of rice straw hydrolysis.
365
3.6 Comparison of single degrading system and consortia degrading system
366
By determining the production of reducing sugars, composition of cellulases,
367
conductivity of the fermentation broth, and analyzing the morphology and structure
368
change in both degrading systems, lots of merits were emerged in consortia degrading
369
system than that in single degrading system (shown in Table 3).
370
It was observed that the maximum production of reducing sugars in the consortia
371
degrading system could achieve 221.33 mg·L-1. This might be ascribed to the
372
detoxification by adding HHQ-1, which could degrade 62.9% ferulic acid in the
373
system. More stable of cellulases, higher activity of β-glucanase and relative
374
stationary conductivity also resulted in an effective detoxification in the consortia
375
degrading system. Moreover, there were obvious morphological and structure changes
376
in the consortia degrading system than that of single degrading system. These features
377
would make in-situ detoxification smoothly proceeded in the consortia degrading
378
system.
379
Detoxification process is necessary to remove the inhibitory and toxic
380
compounds before fermentation in the process of bioethanaol production; however, a
381
separate detoxification might increase the cost of bioethanol production and waste
382
time (Liu et al., 2016; Zhu et al., 2016). In our present research, the consortia
383
degrading system could rapidly degrade most parts of ferulic acid inhibitor in the
384
pretreated rice straw hydrolysates, therefore showing increased reducing sugars
385
production compared to the single degrading system. This in-situ detoxification could
386
make pretreatment and detoxification process proceeded simultaneously, thus
387
exhibited practical benefits for bioethanol production in future.
388
Table 3
The merits of using consortia degrading system over single degrading system
389 390
4. Conclusion 13
ACCEPTED MANUSCRIPT 391
In-situ detoxification by adding ferulic acid degrading bacteria to alkali-
392
pretreated rice straw degradation system was realized, which could purposefully
393
remove inhibitors in the hydrolysates, thus increasing reducing sugars production and
394
ensure bioethanol production preceded smoothly. The HHQ-1-Trichoderma reesei
395
consortia degrading system showed potential benefits for protecting the integrity of
396
microbial membrane, providing microbes for more adsorption sites in lignocellulose
397
material, strengthening activities of important cellulases, forming hydrogen bond,
398
decreasing crystallinity index and degrading lignin parts of rice straw effectively.
399
These might be the main mechanisms of the stimulatory effect of bioethanol
400
production by in-situ detoxification using microbial consortia degrading system. The
401
use of new microorganisms in in-situ detoxification process could play a key role for
402
the conversion of carbohydrates contained lignocellulosic biomass into fermentable
403
sugars, thus providing a viable route for bioethanol production.
404 405
Acknowledgment
406
This project is funded by National Natural Science Foundation of China (NSFC,
407
No. 31400513), Shanghai Science and Technology Committee (No. 17295810600)
408
and Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration
409
(SHUES2016B01). The authors would like to thank them for funding this work.
410 411
References
412
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583 584 585 586 587 588 589 590 591 592 593 594 595 596
20
ACCEPTED MANUSCRIPT
80 70
Degradation rate (%)
60 50 40 30 20 10 0 500
750
1000
1250
Initial concentration of ferulic acid (mgL-1) 597 598
Fig. 1 Degradation rate of ferulic acid by adding HHQ-1. The bars denote the standard deviation (S.D.)
The content of reducing sugars（ mgL-1（
350
Single degrading system Consortia degrading system
300 250 200 150 100 50 0 0
12
24
36
48
60
72
Time (h) 599 600
Fig. 2 Production of reducing sugars in two different lignocellulose degrading systems. The bars denote the
601
standard deviation (S.D.)
602 603 21
ACCEPTED MANUSCRIPT 604 10.0
10.0
Single degrading system Consortia degrading system
605
608 609
8.0
Enzyme activity (UmL-1)
607
Enzyme activity (UmL-1)
8.0
606
6.0
4.0
2.0
6.0
4.0
2.0
0.0
610
Single degrading system Consortia degrading system
0.0 12
24
36
48
60
72
12
24
Enzymatic time (h)
611
10.0
612
8.0
36
48
60
72
Enzymatic time (h)
Enzyme Activity (UmL-1)
Single degrading system Consortia degrading system
613 614 615 616
6.0
4.0
2.0
0.0 12
24
36
617
48
60
72
Enzymatic time (h)
618
Fig .3 Enzyme activities in different lignocellulose degrading systems (a) The enzyme activity of
619
exoglucanase; (b) The enzyme activity of endoglucanase; (c) The enzyme activity of β-glucanase. The bars
620
denote the standard deviation (S.D.)
621 2.4
Single degrading system Consortia degrading system
623
0.7
2.2
Conductivity (Mscm-1)
624 625 626 627 628
0.8
0.6
2.0
0.5 1.8 0.4 1.6
OD550
622
0.3
1.4
0.2
629 1.2
630
0.1 12
24
36
48
60
72
Time (h)
631 632
Fig .4 Conductivity in different lignocellulose degrading systems. The bars denote the standard deviation
633
(S.D.)
634 22
ACCEPTED MANUSCRIPT 635
636
(a)
(b)
(c)
(d)
637 638
Fig. 5 SEM micrographs of rice straw in different lignocellulose degrading systems (a) Before alkaline
639
pretreatment; (b) After alkaline pretreatment; (c) Single degrading system; (d) Consortia degrading system
640 4.0
641
Before pretreatment After alkaline pretreatment Single degrading system Consortia degrading system
3.6 3.2
643
2.8
644 645
Intensity (104)
642
646
649
2.0 1.6 1.2 0.8
647 648
2.4
0.4 12
15
18
21
24
27
30
33
36
39
42
45
2 Fig. 6 XRD spectra of rice straw in different lignocellulose degrading systems
650 105
651 652
654 655 656 657 658
100
Transmittance (%)
653
Before pretreatment After alkaline pretreatment Single degrading system Consortia degrading system
95
90
85
80 3500
3000
2500
2000
1500
1000
-1
Wave length (cm )
659 660
Fig. 7 FTIR spectra of rice straw in different lignocellulose degrading systems
661 23
500
ACCEPTED MANUSCRIPT
662
Table 1
The diffraction peak and crystallinity index in different degrading systems
Diffraction Peak
Substrate
Crystallinity Index
2θ=16°
2θ=22°
Untreated rice straw
20960
33721
0.397
After alkaline pretreatment
23778
40864
0.418
Single degrading system
17684
29110
0.393
Consortia degrading system
17063
27403
0.377
663 664 665
Table 2
Characteristic peaks in FTIR spectra of rice straw
Adsorption peak（cm-1）
Affiliation of characteristic peaks
3408
stretching vibration and overlapping of O-H
2900
symmetric or dissymmetric stretching vibration of C-H group
1637
stretching vibration of C=O (lignin)
1510
Characteristic group vibrations of benzene ring (lignin)
1421
Characteristic group vibrations of benzene ring (lignin)
1321
Characteristic group vibrations of C-O (lignin)
1200
Stretching vibration of CO-OR
1060
C-O stretching of hemicellulose and cellulose
897
C-H deformation of skeleton vibration of saccharides and cellulose
666 667 668 669
Table 3
The merits of using consortia degrading system over single degrading system
670 Items Maximum degradation rate of ferulic acid (%) Maximum production of reducing sugars
(mg·L-1)
Stability of cellulases Activity of β-glucanase
(U·mL-1)
OD550 value at 60 h Conductivity at 60 h
(MS·cm-1)
Morphology and structure analysis
Single degrading system
Consortia degrading system
0
62.9
205.24
221.33
Less stable
More stable
1.44
6.10
0.44
0.67
1.40
1.56
SEM
Seriously destroyed tissue
Seriously destroyed tissue
XRD
Higher CrI value (0.393)
Lower CrI value (0.377)
FTIR
No hydrogen bond
Forming hydrogen bond
671
24
ACCEPTED MANUSCRIPT Highlights:
In-situ detoxification was successfully realized for bioethanol production.
In-situ detoxification increased the production of reducing sugars effectively.
Mechanisms of the stimulatory effect of in-situ detoxification were investigated.
Morphology changes of rice straw were analyzed by SEM, XRD and FTIR.