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Application of engineered yeast strain fermentation for oligogalacturonides production from pectin-rich waste biomass
Journal Pre-proofs Application of engineered yeast strain fermentation for oligogalacturonides production from pectin-rich waste biomass Guojun Yang, Haidong Tan, Shuguang Li, Meng Zhang, Jia Che, Kuikui Li, Wei Chen, Heng Yin PII: DOI: Reference:
S0960-8524(19)31874-7 https://doi.org/10.1016/j.biortech.2019.122645 BITE 122645
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
11 October 2019 14 December 2019 16 December 2019
Please cite this article as: Yang, G., Tan, H., Li, S., Zhang, M., Che, J., Li, K., Chen, W., Yin, H., Application of engineered yeast strain fermentation for oligogalacturonides production from pectin-rich waste biomass, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122645
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1
Application of engineered yeast strain fermentation for oligogalacturonides
2
production from pectin-rich waste biomass
3
Guojun Yanga, b, c, Haidong Tana, Shuguang Lia, Meng Zhang a, Jia Che a, Kuikui Li a,
4
Wei Chena, Heng Yina, b, *
5
a
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Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical
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Physics, Chinese Academy of Sciences, Dalian 116023, China
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b
University of Chinese Academy of Sciences, Beijing 100049, China
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c
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China
Dalian Engineering Research Center for Carbohydrate Agricultural Preparations,
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* Corresponding author at: Liaoning Provincial Key Laboratory of Carbohydrates,
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Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023,
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China.
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E-mail address: [email protected] (H. Yin).
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Abstract Citrus wastes disposal is a problem faced by many juice plants due to high disposal
24
costs. However, the citrus peel wastes (CPW) biomass, as bulk bioresources from the
25
agro-industrial waste, is a good source of pectin. Present study aimed to utilize these
26
CPW biomass by engineered yeast strain fermentation with an inexpensive method to
27
produce oligogalacturonides (OGs). The results showed that the engineered yeast strain
28
fermentation can produce significant amounts of OGs with the degree of polymerization
29
ranged from 2–7 from the CPW bioresources. Under the optimized conditions using the
30
response surface methodology, the best OGs yield were 26.1%. The present work is the
31
first to use the engineered yeast strain for direct CPW biomass fermentation produced
32
the OGs. We thereby paved a new way to utilize the pectin-rich bioresources.
33
Keywords: Citrus peel waste, Engineered strain fermentation, Enzymatic hydrolysis,
34
Oligogalacturonides
35 36 37 38 39 40 41 42
43 44
1. Introduction Citrus fruits production around the world in the fiscal year 2017/18 have reached
45
92 million metric tons (USDA, 2018; USDA, 2019). Citrus peel waste (CPW)
46
accounted for about half of the total weight of fresh fruit after industrial processing
47
(Choi et al., 2015), and estimated to be more than 40 million tons worldwide (Sharma et
48
al., 2017). These CPW biomass have serious impact on the environment as it’s rich in
49
fermentable sugars and some other nutrients, and must be treated carefully before
50
disposal (Choi et al., 2015; Mahato et al., 2018). However, the disposal of these wastes
51
has been becoming much more expensive due to land limitations, labor and
52
transportation costs (Lin et al., 2013).
53
Many attempts have been made to recover the value-added products such as
54
biofuel, polyphenols, dietary fiber and animal feed from these renewable bioresource
55
(Mahato et al., 2018; Sharma et al., 2017). Among them pectin extraction is considered
56
to be the most reasonable ways due to high pectin content in agro-industrial waste,
57
representing 15–25% of the dry CPW, i.e., 6–10 million tons pectin per year (Banerjee
58
et al., 2016; USDA, 2018; USDA, 2019). In traditional industry, however, pectin extract
59
was conducted in an acid hydrolysis process at an elevated temperature (100–120 °C),
60
generating large volumes of acidified wastewater and consuming a lot of energy and
61
high costs in this process (Banerjee et al., 2018; Gonzalez-Rivera et al., 2016; Sharma et
62
al., 2017). Meanwhile, pectin market demand is low in comparison with the world
63
availability of agro-industrial waste (Martinez et al., 2010). To address above
64
challenges, seeking an alternative environmentally friendly method to recycling by-
65
products from these CPW is imperative.
66
Pectin, a most complex and heterogeneous of plant cell-wall polysaccharide and
67
abundant in CPW and other vegetable and fruit processing wastes, is mainly composed
68
of homogalacturonan (HG) which is a long linear homogeneous-chain polymer of a-1,4-
69
glycoside-linked D-galacturonic acid (GalA) (Banerjee et al., 2018; Putnik et al., 2017;
70
Wang et al., 2019 ).
71
There is an increased interest on producing and commercialization of
72
oligogalacturonides (OGs) by physical, chemical or enzymatic methods due to
73
bioactivity (Gullon et al., 2013). OGs exerts a series of human health effects such as
74
prebiotic properties, reduction in glycemic and cholesterol levels, relief of constipation,
75
promoting mineral absorption, anticancer, anti-inflammatory, antioxidant and
76
antiobesity (Embaby et al., 2016; Gullon et al., 2013; Tan et al., 2018). Moreover, OGs,
77
as well-known defense elicitors in plants, has become a research hotspot of plant
78
defenses recently, which plays an important role in plant disease defense and regulate
79
plants growth and development such as damage-associated molecular patterns (DAMPs)
80
to stimulate plant immunity (Benedetti et al., 2017; Benedetti et al., 2018; Ferrari et al.,
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2013; Jeandet, 2017; Selim et al., 2017).
82
Commercially, pectin is extracted from CPW (pectin: 15–25%), apple pomace
83
(pectin: 10–15%), sunflower plate (pectin: 15–25%), and sugar beet (pectin: 10–20%)
84
(Banerjee et al., 2016). Generally, OGs are produced via partial depolymerization of
85
pectin by enzymatic, acidic and hydrothermal methods, which is limited by high costs
86
and/or increasing environmental pollution issues (Embaby et al., 2016). Alternatively,
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OGs can also be prepared from pretreated pectin-rich wastes biomass by enzymatic
88
hydrolysis, acid hydrolysis, hydrothermal processing or physical degradation (Gomez et
89
al., 2016a). Exploitation of efficient and economical methods for oligogalacturonides
90
(OGs) production from renewable CPW bioresources has received special attention
91
owing to its biological function and high value-added (Babbar et al., 2016; Martinez et
92
al., 2010; Zhang et al., 2018b). But so far, preparation of OGs from CPW biomass
93
requires chemical or physical pretreatment of these biomass to extracted pectin, which
94
generally are multi-step processes, high costs, long time and also cause environmental
95
problems.
96
Therefore, finding novel ways that to utilize these CPW biomass for the OGs
97
production which is environmentally-friendly, low costs and simple process will have
98
broad application potential. Pectin-rich agro-industrial waste derived from vegetable
99
and fruit processing was an ideal raw material for fermentation (Protzko et al., 2018).
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Due to high GalA contents in CPW biomass, however, the fermentation broth is at low
101
pH which restrict contaminating microbes’ growth and also limit production hosts. But
102
studies find that pectin-rich wastes could be fermented by yeast and fungi due to their
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tolerance to acidic environments and apply in industrial processes (Protzko et al., 2018).
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Meanwhile, aspergillus from fungi is used for acid pectinases production in scalable
105
bioprocesses upon their preference for pectin-rich substrates (Embaby et al., 2016).
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Pectin-rich agro-industrial waste could be efficiently hydrolyzed by commercial
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pectinases. Moreover, yeast exhibit high growth rates and short fermentation times.
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Thus, engineering Pichia Pastoris contained one highly efficient pectinase gene as a
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fermentation host for utilization theses pectin-rich wastes for OGs production would
110
add value without environmental pollution and high disposal costs.
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In this study, a simple, efficient and rapid OGs preparation directly from CPW
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biomass media was developed by engineered yeast strain fermentation method. The
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results demonstrated that using this engineered strain for direct bioconversion of the
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pectin-rich waste biomass into OGs with DP ranged from DP 2 to DP 7. Together, using
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the engineered strain as fermentation and enzyme-producing host for comprehensive
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utilization of CPW biomass provide a new method for OGs preparation.
117
2. Materials and methods
118
2.1 Materials and Medium
119
The OGs standards (DP 2–DP 7) for quantitative analysis of OGs mixtures are
120
prepared by our own laboratory. The Aspergillus niger 1805 strain was isolated from
121
rotten mandarin peel, identified and stored in our laboratory. The gene (GAQ40478.1)
122
encoding endo-polygalacturonase (AnPG28A) from A. niger 1805 was constructed as a
123
soluble secreted expression protein in P. pastoris X-33. Yeast extract peptone dextrose
124
medium (YPDZ) and buffered glycerol-complex medium (BMGY) were prepared for
125
the active and amplified culture of the engineered yeast strain, respectively. All reagents
126
were analytical grade and commercially available.
127
2.2 Substrates pretreatment
128
Mandarin (Citrus reticulate Blanco) and Gannan navel orange (Citrus sinensis
129
Osbeck) produced in Gannan, Jiangxi Province and Sichuan province, respectively,
130
were purchased from the local fruit market in Dalian, Liaoning, China, subsequently
131
washed these CPW thoroughly. The peels waste was sterilized by UV for 30 min and
132
then freeze-dried. The dried peels were ground into powder using a food grinder and
133
then passed through 100–mesh sieve. A fine powder of average particle size < 150 μm
134
was obtained. The CPW powder was packaged in a polyethylene bag, then kept in
135
desiccator until use.
136
2.3 Shake-flask fermentation for OGs production
137
The reaction condition of the engineered recombinant strain fermentation was
138
optimized for OGs production using mandarin or orange-peel powder as the sole
139
substrates. The activated single colony of engineered yeast strain P. pastoris X-33 was
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cultured in BMGY medium until the OD600 reached 2.0 under the optimal culture
141
conditions (28 °C, 200 rpm). During the continuous fermentative induction stage,
142
different volumes of the seed culture were transferred into the CPW powder liquid
143
medium (substrate concentration: 1%–11% (w/v), 50 mM buffer (acetate buffer from
144
3.0 to 5.0, phosphate buffer from 6.0 to 8.0), 0.0004 % biotin, 0.5 % methanol) and the
145
final culture volume was up to 50 ml in a baffled flask and cultured for 5 days under the
146
above conditions. 0.5 % (v/v) methanol was added per day to induce the AnPG28A
147
expressed. Concisely, 1.0 mL of the fermentation broth was collected every day for
148
monitoring OGs composition and yield by HPAEC-PAD. Then, boiling of the
149
fermentation broth was performed for 10 minutes leading the enzyme to lose activity
150
totally. Then, the hydrolysate was separated by centrifugation (12000 × g for 10 min)
151
and stored at 4 °C until analyzed.
152
2.4. Analytical methods
153
2.4.1 Particle size analysis for the CPW powder
154
The particle size of the CPW powder were determined by the HELOS laser
155
diffraction particle size analyzer (SYMPATEC GmbH, Germany). The particle size
156
value of X50 is corresponding to the cumulative distribution percentage reaching 50 %
157
of total volume shown by the cumulative undersize (%) curves.
158
2.4.2 Uronic acids determination
159
Refer to the previous method, the GalA content in the CPW powder was
160
determined (Blumenkrantz & Asboe-Hansen, 1973).
161
2.4.3. Scanning electron microscopy (SEM) analysis
162
Fermentation broths was separated by centrifugation (10000×g for 5 min) to
163
harvest the hydrolyzed granular residues from the precipitate and nanofiber particle
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from the fermentation supernatant, and then freeze-dried for morphological structure
165
characterizations. In order to assess the effect of fermentation treatment, SEM (EOL
166
JSM-7800F) was used to characterize the differences in morphological structure and
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nanostructured fiber particles released by pectin degraded of the CPW powder before
168
and after the engineered strain fermentation treatment.
169 170
2.4.4. OGs analysis from the fermentation products Qualitative and quantitative analysis of OGs produced by the engineered yeast
171
strain P. pastoris X-33 was conducted by HPAEC-PAD method using a PA100
172
analytical column (4 × 250 mm) mounted on Dionex ICS-3000 Ion Chromatography
173
system. First, the samples for HPAEC-PAD analysis need to be filtered with a 0.22 um
174
hydrophilic membrane.
175
The samples eluted at a flow rate of 0.3 ml/min using the following protocol: 0–10
176
min, equilibrate the columns with 90% Eluent A (50 mM NaOH) and 10% Eluent B (50
177
mM NaOH, 1 M NaAc); 10–45 min, separate OGs with eluent A from 90 to 20% and
178
eluent B from 10 to 80%; 45–55 min, clean the column with 100% eluent B.
179
The monosaccharide composition in fermentation broth was analyzed using
180
HPAEC-PAD method as precious described (Voiniciuc et al., 2016). The samples (0.25
181
mL) was enclosed in a glass tube and 0.25 mL deionized water was incubated in boiling
182
water for 15 min. Then, addition of 2.0 mL 2 mol L−1 trifluoroacetic acid (TFA) and
183
tightly sealed the glass tube, and subsequently the samples were hydrolyzed at 121 °C
184
for 2 h. The hydrolyzed sample solution was cooled down and TFA was removed by
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rotary evaporation, and then were dissolved in 1.0 mL ultrapure water, followed by
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filtration through 0.22 μm membrane and 10 μL was injected in each analysis by
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HPAEC-PAD.
188 189
The monosaccharide composition of the acid hydrolysates of the pectins were quantitatively and qualitatively gauged by HPAEC-PAD using CarboPac PA20 Guard
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and Analytical columns. The samples eluted at a flow rate of 0.4 ml/min using the
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following protocol: 0−18 min, separate neutral sugars with 10 mM NaOH; 18−25.5 min,
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separate uronic acids with 70% 730 mM NaOH; 25.5−30.5 min, rinse the column with
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730 mM NaOH; 30.5−42 min, equilibrate the columns with 10 mM NaOH.
194
2.5. Optimization of fermentation process by response surface methodology
195
To achieve higher OGs yield, response surface methodology (RSM) was applied
196
which is more efficient and easier methods to predict optimal variable conditions and
197
corresponding response values (Baskar et al., 2018; Li et al., 2019). The pH, inoculum
198
size, and substance concentration are the main variables affecting the OGs content. To
199
evaluate the interactions of the three variables and further optimized, the Box-Behnken
200
design (BBD) was adopted with the pH (A), inoculum size (B), and substance
201
concentration (C) as the variables. According to the BBD results, the variable value
202
ranges were set to 4.0–6.0, 5%–25%, and 1–9%, respectively, each at three degrees
203
(lower, middle and higher level). The details of these three selected process parameters
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are shown in Figure 1.
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3. Results and Discussion
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3.1. Construction of the engineered yeast strain
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Aspergillus species is the main source of pectinases in most industrial applications
209
(Hassan et al., 2019). Therefore, it is critical to efficiently heterologous express the
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endo-polygalacturonase gene derived from Aspergillus sp. in a suitable host for the
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industrial scale production of OGs from pectin. As the expression host of recombinant
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protein，it has demonstrated that P. pastoris is suitable for basic scientific research and
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industrial application (Liu et al., 2014). So, P. pastoris X-33 was selected as host strain
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for constructing the engineered yeast strain.
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In our previous study, the recombinant protein AnPG28A from A. niger was
216
successfully expressed in above engineered strain and could efficiently produce the high
217
purity AnPG28A within 96 h without further purification. The AnPG28A exhibited
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much more efficient degradation capability toward pectin and polygalacturonic acid,
219
showed the greatest activity at pH 5.0 °C and 30 °C, and applying this recombinant
220
enzyme to prepare high-purity OG standards (DP 2–DP 7). The crude enzyme activity
221
of recombinant protein AnPG28A towards low-methoxyl pectin was 5571 U/mg that
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was significantly higher than the other purified pectinase activities (Martins et al.,
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2007). This implied that the engineered yeast strain P. pastoris X-33 carrying the
224
optimized and overexpressed AnPG28A possessed remarkable potential application in
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the pectin-rich waste biomass hydrolysis. In the following pre-experiment, we found
226
that the engineered yeast strain P. pastoris X-33 exhibited efficient degradation towards
227
the fresh pectin-rich wastes or fruit in a short period of time. Thus, in this study, we
228
took the engineered yeast strain P. pastoris X-33 and CPW biomass as fermentation
229
hosts and fermentation broth, respectively, and optimized the reaction system to prepare
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OGs.
231
3.2. Grinding pretreatment of the citrus peel waste
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A fine powder of particle size < 350 μm was obtained from the grinded CPW
233
biomass. The particle size distribution of the mandarin and orange peel powder were
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distributed in a range from 1.50 to 333.65 𝜇m and from 1.50 to 234.15 𝜇m, respectively.
235
Since orange peel waste gave significantly higher cellulose and hemicellulose contents
236
compared with mandarin peel waste, indicating that pectin in orange peel waste was
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more tightly crosslinked with cellulose and hemicellulose. Thus, compared to mandarin
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peel waste, the orange peel waste was much more difficult to grind into smaller particles
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and degradation (Table 2).
240
Indeed, the microbial enzymatic hydrolysis of substrate particles depends greatly
241
on the chemical composition, accessible area and the particle size of the biomass
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particles (Hendriks & Zeeman, 2009). Some research has found that the smaller the
243
substrate particles, the easier to degrade due to increasing the surface area of the particle
244
and thus improving the accessibility between substance and enzyme, and then improve
245
enzymatic hydrolysis efficiency (Alvira et al., 2010; Silva et al., 2012).
246
3.3. Composition analysis of citrus peel waste
247
The GalA content was 27.3 % and 25.6 % for the mandarin and orange peel waste
248
powder, respectively. As waste biomass feedstocks, it is beneficial for OGs production.
249
Meanwhile, as shown in Table 3, the CPW were also rich in fermentable sugars (Choi et
250
al., 2015), which was conductive to engineered strain fermentation (Protzko et al.,
251
2018). In addition，CPW contained some micronutrients such as trace metals (Rivas et
252
al., 2008), are also rich in carbon and nitrogen. They can be directly used as substrates
253 254
for engineered strain fermentation (Pathak et al., 2017). The citrus peel is a complex intertwisted mesh of glycans which pectin, cellulose
255
and hemicellulose are interwoven to form a network structure (Adetunji et al., 2017;
256
Dick-Perez et al., 2011; Marcus et al., 2008), and making pectin difficult to close to
257
degrading enzymes. Therefore, in order to efficiently degrade pectin in CPW, grinding
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CPW into powder is the most important step to break down the structure of the citrus
259
peel (John et al., 2017; Kumar et al., 2009), thus improving the pectin accessibility to
260
enzymes and achieving the purpose of preparing OGs.
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3.4. Characterization of the substrates morphological structure changes
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The micrograph of CPW powder showed that the peel powder was compact,
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ordered fibers and smooth surface, suggesting that the cellulose–hemicellulose and
264
pectin were interwoven into a tight network. The structural disruption and increased the
265
surface area was observed after one-step fermentation treated the CPW powder,
266
presented rough and porous structure which were attributed to the pectin removal. So,
267
there were significant structural changes after one step fermentation combined with
268
enzymatic hydrolysis, and a similar result was also observed by multistep treatment to
269
remove pectin from mandarin peel waste (Hiasa et al., 2014). Moreover, part of
270
cellulose and hemicellulose fragment attached to the pectin were released into the
271
fermentation broth, forming uniform spherical cellulose nanofiber particles. Further
272
characterization of the cellulose nanofiber particles may be required to determine its
273
physicochemical property and the application potential.
274 275
3.5. OGs compositional analysis of fermented broth For producing OGs and removing the soluble fermentable sugars in the CPW, the
276
engineered yeast strain P. pastoris X-33 was applied for fermentation using CPW
277
directly as substrate. First, the engineered yeast strain was cultured in YPD medium
278
under optimal conditions. Then, the seed liquid was transferred to the CPW medium as
279
a feed ratio 15 (v/v) in 250 ml flasks. Subsequently, the CPW biomass with 7%
280
(mandarin peel powder) and 5% (orange peel powder) were added to the culture to
281
perform the OGs production at 28 °C and pH 5.0. In this one-step engineered strain
282
fermentation method, OGs production was combined with AnPG28A secretion
283
expression.
284
Under the above conditions, the engineered strain exhibited good degraded
285
properties towards to the CPW biomass. During the fermentation process, the OGs
286
content was reached the highest after 72 h with the maximum OGs content of 3.6 and
287
1.9 mg/ml for mandarin and orange peel powder, respectively (Fig. 1). Along with the
288
engineered strain growth, the fermentable sugars were gradually consumed as carbon
289
source (Li et al., 2019), and thus as a new carbon source the GalA and OGs were
290
consumed and rapidly decreased after 96 h fermentation. In this period, the OGs content
291
from mandarin peel was much higher than that orange peel after 72 h fermentation (Fig.
292
1). This was probably because there was a higher content of D-limonene in the orange
293
peel waste (1.70%) compared with mandarin peel waste (0.21%) which was considered
294
to be a microbial fermentation inhibitor (Wilkins et al., 2007), and pectin accessible area
295
and substrate concentration difference were also affect the degradation efficiency of the
296
CPW biomass. The OGs yield derived from the CPW biomass was 19 and 15 % for
297
mandarin and orange peel powder after 72 h fermentation, respectively.
298
The composition of the CPW biomass mainly consisted of crude protein, water
299
insoluble material, carbohydrate, and total ash (Martinez et al., 2010; Sharma et al.,
300
2017). The undegraded water-insoluble material, crude protein, and ash could be
301
removed by centrifugation after boil. After fermentation for 72 h, the GalA content was
302
62.5 and 50.5% from mandarin and orange peel waste broth, respectively.
303
Carbohydrates mainly present in CPW fermentation broth are GalA, Glucose, Galactose
304
and Arabinose. The fermentable sugars could be gradually consumed in the
305
fermentation process (Li et al., 2019). Additionally, the engineered yeast strain contains
306
only one endo-polygalacturonase which only acts on the HG part of pectin, so that the
307
main oligosaccharides produced by CPW fermentation are OGs.
308
To further determine the DP distribution and quantitative analysis of the OGs, the
309
fermentable products was subjected to HPAEC-PAD analysis. The DP distribution of
310
the OGs in the fermentation broth was ranging from 2 to 7 for mandarin peel and
311
ranging from 2 to 5 for orange peel, respectively. From mandarin peel waste
312
fermentation broth, the main products were OGs with DP 2–3 and high-molecular
313
weight fragments DP 7 (Fig. 2). The orange peel waste fermentation extracted products
314
yielded mostly OGs with DP 2–3 with low formation of DP 4 and DP 5 (Fig. 2).
315
These results indicated that the engineering yeast strain was suitable for OGs
316
production, directly using CPW as fermentation substrate. In this study, the one-step
317
engineered yeast fermentation method coupled enzyme-induced expression with CPW
318
degradation during biomass fermentation which has been shown to reduce costs, save
319
time, environment-friendly and simplify bioprocesses. Therefore, the one-step
320
bioprocess strategy in the present study had a brilliant applied prospect in the CPW
321
biomass utilizing for OGs production due to its unpolluted environment and cost
322
savings. Moreover, as a material basis for scientific and applied research, the cellulose
323
and hemicellulose contained in CPW biomass were pretreated by engineered yeast
324
fermentation which was feasible and environmentally friendly method compared with
325
the chemical treatment method (Alemdar and Sain, 2008; Sahoo et al., 2018).
326
3.6. Optimization of citrus peel waste fermentation conditions for OGs production
327
Since the one-step engineered strain fermentation method gave a good result, so
328
further optimized the experiment for OGs production. Using the RSM to optimize
329
process parameters and predict response value save time, and also provide information
330
about interactions between parameters (Sathendra et al., 2019; Yao et al., 2015).
331
Seventeen groups of experiments were designed with the OGs content (Y) from
332
mandarin and orange peel waste fermentation and enzymatic hydrolysis as the response
333
values.
334
Through multiple regression analysis, the regression model for OGs content in the
335
fermentation broth is given by Eq. (1) and Eq. (2) for mandarin and orange peel
336
substrate, respectively.
337 338 339 340 341
YMP (OGs mg/mL) = -57.7+13.9A+0.266B+6.72C-0.0220AB-0.341AC +0.00645BC-1.12A2-0.00297B2-0.361C2 (1) YOP (OGs mg/mL) = -9.49+3.37A+0.176B+0.367C+0.00964AB+0.0172AC +0.00492BC-0.351A2-0.00657B2-0.0489C2 (2) According to ANOVA for RSM, the model with P < 0.05 is considered statistically
342
significant (Liu et al., 2018). The regression analysis for mandarin and orange peel
343
substrate revealed the regression coefficient (R2) was 0.910 and 0.857, and the ‘‘Model
344
F-value’’ of 7.89 and 4.67 indicated that the model is significant, and 0.63% and 2.72%
345
of the total change cannot be explained by the model for mandarin and orange peel
346
substrate, respectively. The model with the signal to noise ratio greater than 4 can be
347
used to predict the OGs content from CPW (Liu et al., 2018).
348
The effect of the variables on the OGs content can be better understood by
349
examining the three-dimensional response surface plots in Fig. 3, which shows the
350
interaction effect of important process variable combinations on OGs content. For
351
mandarin peel waste fermentation, the OGs content increased with the increase in
352
substrate concentration and pH, and reached the highest value around their middle level.
353
With a further increasing in substrate concentration and pH, the OGs content began to
354
decrease (Fig. 3a and 3e). However, the OGs content was increased with increase in
355
inoculum size as observed in Fig. 3c. In addition, for orange peel waste fermentation,
356
substrate concentration, inoculum size and pH has decreased OGs content when their
357
increased around middle level to higher level and decreased around middle level to
358 359
lower level (Fig. 3b, 3d and 3f). The RSM analysis for mandarin and orange peel substrate showed that the optimal
360
conditions were a pH of 5.17 and 5.04, inoculum size of 29.5% and 20.5%, and
361
substrate concentration of 6.9% and 4.7% for a theoretical maximum OGs production
362
which yielded a predicted maximum OGs production of 4.53 and 1.94 mg/mL after 72 h
363
fermentation, respectively. To validate the adequacy of RSM responses, the verification
364
experiment was carried out in triplicates under above conditions. The results showed
365
that the OGs content was 4.49±0.48 and 1.99±0.13 mg/mL which corresponds to 26.1
366
and 15.7% of OGs yield for mandarin and orange peel substrate, respectively.
367
Therefore, the results from the validated experiment were in good agreement with the
368
predicted value which proved the adequacy of the model in predicting the response
369
together with the optimization of fermentation parameters.
370
The results suggest that OGs yield from the mandarin peel waste was significantly
371
higher than OGs production from enzymatic hydrolysis, acid hydrolysis or
372
hydrothermal treatments of CPW biomass (Babbar et al., 2016; Gomez et al., 2013;
373
Gomez et al., 2016b; Sabajanes et al., 2012), and with the difference that the CPW
374
biomass need not pretreated by physical, chemical or mechanical treatments to extract
375
pectin and remove soluble material in this study. Therefore, the use of engineered yeast
376
strain fermentation has distinct advantages, such as low costs, environment-friendly,
377
simple process and high hydrolysis efficiency.
378
In this study, we developed one-step fermentation method for oligogalacturonides
379
(OGs) production directly from CPW biomass. To our knowledge, this is the first time
380
to produce OGs using CPW as a direct substrate using one-step engineered yeast strains
381
fermentation method which is an environmentally friendly, simplify process and cost-
382
effective technique for OGs production. The study provides new strategies for the
383
development and utilization of CPW resources for OGs production and has great
384
potential for CPW recycling utilization.
385
4. Conclusions
386
In this study, one-step fermentation method for OGs production, as an innovative
387
green and novel strategy, have been successfully developed. The DP distribution of the
388
OGs produced in the fermentation broth were DP 2–7 and DP 2–5 for mandarin and
389
orange peel waste, respectively. With RSM, a maximal OGs yield of 26.1 % was
390
obtained from mandarin peel waste. As a new process, this one-step fermentation
391
method using the CPW bioresources as the direct substrates has great significance for
392
CPW biomass utilization due to its rapid, low cost, effective, eco-friendly and easily
393
bioprocess.
394
Acknowledgements
395
This work was supported by the National Key R&D Program of China, China
396
(Grant No. 2017YFD0200900 and 2017YFD0200902), National Natural Science
397
Foundation of China (Grant No. 31670803), Liaoning Revitalization Talents Program,
398
China (Grant No. XLYC1807041) and DICP, China (Grant No. ZZBS201704 and
399
BioChE-X201801).
400 401
Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper.
402
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556 557 558 559 560 561 562 563 564 565 566 567
568 569 570
Figure Captions
571
Fig. 1. OGs production during the fermentation treatment from the CPW biomass.
572 573
Fig. 2. OGs composition analysis after 72 h fermentation treatment.
574 575
Fig. 3. Response contour plot for the optimization of OGs production from CPW
576
biomass. a & b: pH and inoculum size (%); c & d: pH and substrate concentration (%);
577
e & f substrate concentration (%) and inoculum size (%). a, c and e represent mandarin
578
peel waste; b, d and e orange peel waste.
579 580 581 582 583 584 585 586 587 588
589 590 591
Table 1 The three variables selected for the optimization of OGs production using one-
592
step fermentation by the engineered yeast strain. Range and levels Symbols
Independent variables Lower (−1)
Middle (0)
Higher (+1)
Mandarin peel waste A
pH
4
5
6
B
Inoculum size, % (v/v)
5
15
25
5
7
9
Substrate C concentration, % (w/v) Orange peel waste A
pH
4
5
6
B
Inoculum size, % (v/v)
5
15
25
1
5
9
Substrate C concentration, % (w/v)
593 594 595 596 597
598 599 600
Table 2 Particle size distribution of the citrus peels powder substrate. Substrate source
X50,3
SMD (um)
VMD (um)
Mandarin peel powder (MP)
63.79±0.26
23.14±0.18
85.17±1.27
Ornge peel powder (OP)
37.56±0.95
16.52±0.28
49.68±1.89
601
X50 stands for the median particle size; SMD stands for surface mean diameter; VMD stands for
602
volume surface mean diameter.
603 604 605 606 607 608 609 610 611 612 613 614 615
616 617 618
Table 3 Fermentable sugars (FS) and total sugars in citrus wastes. Sucrose
Glucose
Fructose
FS
Total sugars
CPW %, DW (dry weight)
619 620 621 622 623 624 625 626 627 628 629 630 631 632
MP
7.4 ± 0.2
39.4 ± 1.1
10.3 ± 0.8
57.1 ± 0.6
71.9 ± 0.9
OP
5.6 ± 0.2
35.5 ± 0.5
12.1 ± 0.4
53.2 ± 0.4
68.2 ± 0.5
633 634 635
636 637 638 639 640 641 642 643 644 645 646 647
Fig. 1.
648 649 650
651 652 653 654 655 656 657 658 659 660 661 662 663 664
Fig. 2.
665 666 667
668 669 670 671 672 673 674 675
Fig. 3.
676
677 678
Highlights
679
● First production of oligogalacturonides (OGs) directly from citrus peel wastes.
680
● An innovative green and novel fermentation strategy has been developed.
681
● After fermentation for 72 h, the DP of OGs was ranging from 2 to 7.
682 683
Declaration of interests
684 685
☒ The authors declare that they have no known competing financial interests or personal
686
relationships that could have appeared to influence the work reported in this paper.
687 688
☐The authors declare the following financial interests/personal relationships which
689
may be considered as potential competing interests:
690 691 692 693 694 695
696
Sample CRediT author statement
697
Guojun Yang: Investigation, Writing- Original draft preparation, Haidong Tan:
698
Supervision, Shuguang Li: Resources, Meng Zhang: Visualization, Jia Che: Resources,
699
Kuikui Li: Formal analysis, Wei Chen: Data curation, Heng Yin: Writing- Reviewing and
700
Editing, Project administration, Funding acquisition.
701
S0960-8524(19)31874-7 https://doi.org/10.1016/j.biortech.2019.122645 BITE 122645
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
11 October 2019 14 December 2019 16 December 2019
Please cite this article as: Yang, G., Tan, H., Li, S., Zhang, M., Che, J., Li, K., Chen, W., Yin, H., Application of engineered yeast strain fermentation for oligogalacturonides production from pectin-rich waste biomass, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122645
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1
Application of engineered yeast strain fermentation for oligogalacturonides
2
production from pectin-rich waste biomass
3
Guojun Yanga, b, c, Haidong Tana, Shuguang Lia, Meng Zhang a, Jia Che a, Kuikui Li a,
4
Wei Chena, Heng Yina, b, *
5
a
6
Liaoning Provincial Key Laboratory of Carbohydrates, Dalian Institute of Chemical
7
Physics, Chinese Academy of Sciences, Dalian 116023, China
8
b
University of Chinese Academy of Sciences, Beijing 100049, China
9
c
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China
Dalian Engineering Research Center for Carbohydrate Agricultural Preparations,
10
* Corresponding author at: Liaoning Provincial Key Laboratory of Carbohydrates,
11
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023,
12
China.
13
E-mail address: [email protected] (H. Yin).
14 15 16 17 18 19 20 21
22 23
Abstract Citrus wastes disposal is a problem faced by many juice plants due to high disposal
24
costs. However, the citrus peel wastes (CPW) biomass, as bulk bioresources from the
25
agro-industrial waste, is a good source of pectin. Present study aimed to utilize these
26
CPW biomass by engineered yeast strain fermentation with an inexpensive method to
27
produce oligogalacturonides (OGs). The results showed that the engineered yeast strain
28
fermentation can produce significant amounts of OGs with the degree of polymerization
29
ranged from 2–7 from the CPW bioresources. Under the optimized conditions using the
30
response surface methodology, the best OGs yield were 26.1%. The present work is the
31
first to use the engineered yeast strain for direct CPW biomass fermentation produced
32
the OGs. We thereby paved a new way to utilize the pectin-rich bioresources.
33
Keywords: Citrus peel waste, Engineered strain fermentation, Enzymatic hydrolysis,
34
Oligogalacturonides
35 36 37 38 39 40 41 42
43 44
1. Introduction Citrus fruits production around the world in the fiscal year 2017/18 have reached
45
92 million metric tons (USDA, 2018; USDA, 2019). Citrus peel waste (CPW)
46
accounted for about half of the total weight of fresh fruit after industrial processing
47
(Choi et al., 2015), and estimated to be more than 40 million tons worldwide (Sharma et
48
al., 2017). These CPW biomass have serious impact on the environment as it’s rich in
49
fermentable sugars and some other nutrients, and must be treated carefully before
50
disposal (Choi et al., 2015; Mahato et al., 2018). However, the disposal of these wastes
51
has been becoming much more expensive due to land limitations, labor and
52
transportation costs (Lin et al., 2013).
53
Many attempts have been made to recover the value-added products such as
54
biofuel, polyphenols, dietary fiber and animal feed from these renewable bioresource
55
(Mahato et al., 2018; Sharma et al., 2017). Among them pectin extraction is considered
56
to be the most reasonable ways due to high pectin content in agro-industrial waste,
57
representing 15–25% of the dry CPW, i.e., 6–10 million tons pectin per year (Banerjee
58
et al., 2016; USDA, 2018; USDA, 2019). In traditional industry, however, pectin extract
59
was conducted in an acid hydrolysis process at an elevated temperature (100–120 °C),
60
generating large volumes of acidified wastewater and consuming a lot of energy and
61
high costs in this process (Banerjee et al., 2018; Gonzalez-Rivera et al., 2016; Sharma et
62
al., 2017). Meanwhile, pectin market demand is low in comparison with the world
63
availability of agro-industrial waste (Martinez et al., 2010). To address above
64
challenges, seeking an alternative environmentally friendly method to recycling by-
65
products from these CPW is imperative.
66
Pectin, a most complex and heterogeneous of plant cell-wall polysaccharide and
67
abundant in CPW and other vegetable and fruit processing wastes, is mainly composed
68
of homogalacturonan (HG) which is a long linear homogeneous-chain polymer of a-1,4-
69
glycoside-linked D-galacturonic acid (GalA) (Banerjee et al., 2018; Putnik et al., 2017;
70
Wang et al., 2019 ).
71
There is an increased interest on producing and commercialization of
72
oligogalacturonides (OGs) by physical, chemical or enzymatic methods due to
73
bioactivity (Gullon et al., 2013). OGs exerts a series of human health effects such as
74
prebiotic properties, reduction in glycemic and cholesterol levels, relief of constipation,
75
promoting mineral absorption, anticancer, anti-inflammatory, antioxidant and
76
antiobesity (Embaby et al., 2016; Gullon et al., 2013; Tan et al., 2018). Moreover, OGs,
77
as well-known defense elicitors in plants, has become a research hotspot of plant
78
defenses recently, which plays an important role in plant disease defense and regulate
79
plants growth and development such as damage-associated molecular patterns (DAMPs)
80
to stimulate plant immunity (Benedetti et al., 2017; Benedetti et al., 2018; Ferrari et al.,
81
2013; Jeandet, 2017; Selim et al., 2017).
82
Commercially, pectin is extracted from CPW (pectin: 15–25%), apple pomace
83
(pectin: 10–15%), sunflower plate (pectin: 15–25%), and sugar beet (pectin: 10–20%)
84
(Banerjee et al., 2016). Generally, OGs are produced via partial depolymerization of
85
pectin by enzymatic, acidic and hydrothermal methods, which is limited by high costs
86
and/or increasing environmental pollution issues (Embaby et al., 2016). Alternatively,
87
OGs can also be prepared from pretreated pectin-rich wastes biomass by enzymatic
88
hydrolysis, acid hydrolysis, hydrothermal processing or physical degradation (Gomez et
89
al., 2016a). Exploitation of efficient and economical methods for oligogalacturonides
90
(OGs) production from renewable CPW bioresources has received special attention
91
owing to its biological function and high value-added (Babbar et al., 2016; Martinez et
92
al., 2010; Zhang et al., 2018b). But so far, preparation of OGs from CPW biomass
93
requires chemical or physical pretreatment of these biomass to extracted pectin, which
94
generally are multi-step processes, high costs, long time and also cause environmental
95
problems.
96
Therefore, finding novel ways that to utilize these CPW biomass for the OGs
97
production which is environmentally-friendly, low costs and simple process will have
98
broad application potential. Pectin-rich agro-industrial waste derived from vegetable
99
and fruit processing was an ideal raw material for fermentation (Protzko et al., 2018).
100
Due to high GalA contents in CPW biomass, however, the fermentation broth is at low
101
pH which restrict contaminating microbes’ growth and also limit production hosts. But
102
studies find that pectin-rich wastes could be fermented by yeast and fungi due to their
103
tolerance to acidic environments and apply in industrial processes (Protzko et al., 2018).
104
Meanwhile, aspergillus from fungi is used for acid pectinases production in scalable
105
bioprocesses upon their preference for pectin-rich substrates (Embaby et al., 2016).
106
Pectin-rich agro-industrial waste could be efficiently hydrolyzed by commercial
107
pectinases. Moreover, yeast exhibit high growth rates and short fermentation times.
108
Thus, engineering Pichia Pastoris contained one highly efficient pectinase gene as a
109
fermentation host for utilization theses pectin-rich wastes for OGs production would
110
add value without environmental pollution and high disposal costs.
111
In this study, a simple, efficient and rapid OGs preparation directly from CPW
112
biomass media was developed by engineered yeast strain fermentation method. The
113
results demonstrated that using this engineered strain for direct bioconversion of the
114
pectin-rich waste biomass into OGs with DP ranged from DP 2 to DP 7. Together, using
115
the engineered strain as fermentation and enzyme-producing host for comprehensive
116
utilization of CPW biomass provide a new method for OGs preparation.
117
2. Materials and methods
118
2.1 Materials and Medium
119
The OGs standards (DP 2–DP 7) for quantitative analysis of OGs mixtures are
120
prepared by our own laboratory. The Aspergillus niger 1805 strain was isolated from
121
rotten mandarin peel, identified and stored in our laboratory. The gene (GAQ40478.1)
122
encoding endo-polygalacturonase (AnPG28A) from A. niger 1805 was constructed as a
123
soluble secreted expression protein in P. pastoris X-33. Yeast extract peptone dextrose
124
medium (YPDZ) and buffered glycerol-complex medium (BMGY) were prepared for
125
the active and amplified culture of the engineered yeast strain, respectively. All reagents
126
were analytical grade and commercially available.
127
2.2 Substrates pretreatment
128
Mandarin (Citrus reticulate Blanco) and Gannan navel orange (Citrus sinensis
129
Osbeck) produced in Gannan, Jiangxi Province and Sichuan province, respectively,
130
were purchased from the local fruit market in Dalian, Liaoning, China, subsequently
131
washed these CPW thoroughly. The peels waste was sterilized by UV for 30 min and
132
then freeze-dried. The dried peels were ground into powder using a food grinder and
133
then passed through 100–mesh sieve. A fine powder of average particle size < 150 μm
134
was obtained. The CPW powder was packaged in a polyethylene bag, then kept in
135
desiccator until use.
136
2.3 Shake-flask fermentation for OGs production
137
The reaction condition of the engineered recombinant strain fermentation was
138
optimized for OGs production using mandarin or orange-peel powder as the sole
139
substrates. The activated single colony of engineered yeast strain P. pastoris X-33 was
140
cultured in BMGY medium until the OD600 reached 2.0 under the optimal culture
141
conditions (28 °C, 200 rpm). During the continuous fermentative induction stage,
142
different volumes of the seed culture were transferred into the CPW powder liquid
143
medium (substrate concentration: 1%–11% (w/v), 50 mM buffer (acetate buffer from
144
3.0 to 5.0, phosphate buffer from 6.0 to 8.0), 0.0004 % biotin, 0.5 % methanol) and the
145
final culture volume was up to 50 ml in a baffled flask and cultured for 5 days under the
146
above conditions. 0.5 % (v/v) methanol was added per day to induce the AnPG28A
147
expressed. Concisely, 1.0 mL of the fermentation broth was collected every day for
148
monitoring OGs composition and yield by HPAEC-PAD. Then, boiling of the
149
fermentation broth was performed for 10 minutes leading the enzyme to lose activity
150
totally. Then, the hydrolysate was separated by centrifugation (12000 × g for 10 min)
151
and stored at 4 °C until analyzed.
152
2.4. Analytical methods
153
2.4.1 Particle size analysis for the CPW powder
154
The particle size of the CPW powder were determined by the HELOS laser
155
diffraction particle size analyzer (SYMPATEC GmbH, Germany). The particle size
156
value of X50 is corresponding to the cumulative distribution percentage reaching 50 %
157
of total volume shown by the cumulative undersize (%) curves.
158
2.4.2 Uronic acids determination
159
Refer to the previous method, the GalA content in the CPW powder was
160
determined (Blumenkrantz & Asboe-Hansen, 1973).
161
2.4.3. Scanning electron microscopy (SEM) analysis
162
Fermentation broths was separated by centrifugation (10000×g for 5 min) to
163
harvest the hydrolyzed granular residues from the precipitate and nanofiber particle
164
from the fermentation supernatant, and then freeze-dried for morphological structure
165
characterizations. In order to assess the effect of fermentation treatment, SEM (EOL
166
JSM-7800F) was used to characterize the differences in morphological structure and
167
nanostructured fiber particles released by pectin degraded of the CPW powder before
168
and after the engineered strain fermentation treatment.
169 170
2.4.4. OGs analysis from the fermentation products Qualitative and quantitative analysis of OGs produced by the engineered yeast
171
strain P. pastoris X-33 was conducted by HPAEC-PAD method using a PA100
172
analytical column (4 × 250 mm) mounted on Dionex ICS-3000 Ion Chromatography
173
system. First, the samples for HPAEC-PAD analysis need to be filtered with a 0.22 um
174
hydrophilic membrane.
175
The samples eluted at a flow rate of 0.3 ml/min using the following protocol: 0–10
176
min, equilibrate the columns with 90% Eluent A (50 mM NaOH) and 10% Eluent B (50
177
mM NaOH, 1 M NaAc); 10–45 min, separate OGs with eluent A from 90 to 20% and
178
eluent B from 10 to 80%; 45–55 min, clean the column with 100% eluent B.
179
The monosaccharide composition in fermentation broth was analyzed using
180
HPAEC-PAD method as precious described (Voiniciuc et al., 2016). The samples (0.25
181
mL) was enclosed in a glass tube and 0.25 mL deionized water was incubated in boiling
182
water for 15 min. Then, addition of 2.0 mL 2 mol L−1 trifluoroacetic acid (TFA) and
183
tightly sealed the glass tube, and subsequently the samples were hydrolyzed at 121 °C
184
for 2 h. The hydrolyzed sample solution was cooled down and TFA was removed by
185
rotary evaporation, and then were dissolved in 1.0 mL ultrapure water, followed by
186
filtration through 0.22 μm membrane and 10 μL was injected in each analysis by
187
HPAEC-PAD.
188 189
The monosaccharide composition of the acid hydrolysates of the pectins were quantitatively and qualitatively gauged by HPAEC-PAD using CarboPac PA20 Guard
190
and Analytical columns. The samples eluted at a flow rate of 0.4 ml/min using the
191
following protocol: 0−18 min, separate neutral sugars with 10 mM NaOH; 18−25.5 min,
192
separate uronic acids with 70% 730 mM NaOH; 25.5−30.5 min, rinse the column with
193
730 mM NaOH; 30.5−42 min, equilibrate the columns with 10 mM NaOH.
194
2.5. Optimization of fermentation process by response surface methodology
195
To achieve higher OGs yield, response surface methodology (RSM) was applied
196
which is more efficient and easier methods to predict optimal variable conditions and
197
corresponding response values (Baskar et al., 2018; Li et al., 2019). The pH, inoculum
198
size, and substance concentration are the main variables affecting the OGs content. To
199
evaluate the interactions of the three variables and further optimized, the Box-Behnken
200
design (BBD) was adopted with the pH (A), inoculum size (B), and substance
201
concentration (C) as the variables. According to the BBD results, the variable value
202
ranges were set to 4.0–6.0, 5%–25%, and 1–9%, respectively, each at three degrees
203
(lower, middle and higher level). The details of these three selected process parameters
204
are shown in Figure 1.
205 206
3. Results and Discussion
207
3.1. Construction of the engineered yeast strain
208
Aspergillus species is the main source of pectinases in most industrial applications
209
(Hassan et al., 2019). Therefore, it is critical to efficiently heterologous express the
210
endo-polygalacturonase gene derived from Aspergillus sp. in a suitable host for the
211
industrial scale production of OGs from pectin. As the expression host of recombinant
212
protein，it has demonstrated that P. pastoris is suitable for basic scientific research and
213
industrial application (Liu et al., 2014). So, P. pastoris X-33 was selected as host strain
214
for constructing the engineered yeast strain.
215
In our previous study, the recombinant protein AnPG28A from A. niger was
216
successfully expressed in above engineered strain and could efficiently produce the high
217
purity AnPG28A within 96 h without further purification. The AnPG28A exhibited
218
much more efficient degradation capability toward pectin and polygalacturonic acid,
219
showed the greatest activity at pH 5.0 °C and 30 °C, and applying this recombinant
220
enzyme to prepare high-purity OG standards (DP 2–DP 7). The crude enzyme activity
221
of recombinant protein AnPG28A towards low-methoxyl pectin was 5571 U/mg that
222
was significantly higher than the other purified pectinase activities (Martins et al.,
223
2007). This implied that the engineered yeast strain P. pastoris X-33 carrying the
224
optimized and overexpressed AnPG28A possessed remarkable potential application in
225
the pectin-rich waste biomass hydrolysis. In the following pre-experiment, we found
226
that the engineered yeast strain P. pastoris X-33 exhibited efficient degradation towards
227
the fresh pectin-rich wastes or fruit in a short period of time. Thus, in this study, we
228
took the engineered yeast strain P. pastoris X-33 and CPW biomass as fermentation
229
hosts and fermentation broth, respectively, and optimized the reaction system to prepare
230
OGs.
231
3.2. Grinding pretreatment of the citrus peel waste
232
A fine powder of particle size < 350 μm was obtained from the grinded CPW
233
biomass. The particle size distribution of the mandarin and orange peel powder were
234
distributed in a range from 1.50 to 333.65 𝜇m and from 1.50 to 234.15 𝜇m, respectively.
235
Since orange peel waste gave significantly higher cellulose and hemicellulose contents
236
compared with mandarin peel waste, indicating that pectin in orange peel waste was
237
more tightly crosslinked with cellulose and hemicellulose. Thus, compared to mandarin
238
peel waste, the orange peel waste was much more difficult to grind into smaller particles
239
and degradation (Table 2).
240
Indeed, the microbial enzymatic hydrolysis of substrate particles depends greatly
241
on the chemical composition, accessible area and the particle size of the biomass
242
particles (Hendriks & Zeeman, 2009). Some research has found that the smaller the
243
substrate particles, the easier to degrade due to increasing the surface area of the particle
244
and thus improving the accessibility between substance and enzyme, and then improve
245
enzymatic hydrolysis efficiency (Alvira et al., 2010; Silva et al., 2012).
246
3.3. Composition analysis of citrus peel waste
247
The GalA content was 27.3 % and 25.6 % for the mandarin and orange peel waste
248
powder, respectively. As waste biomass feedstocks, it is beneficial for OGs production.
249
Meanwhile, as shown in Table 3, the CPW were also rich in fermentable sugars (Choi et
250
al., 2015), which was conductive to engineered strain fermentation (Protzko et al.,
251
2018). In addition，CPW contained some micronutrients such as trace metals (Rivas et
252
al., 2008), are also rich in carbon and nitrogen. They can be directly used as substrates
253 254
for engineered strain fermentation (Pathak et al., 2017). The citrus peel is a complex intertwisted mesh of glycans which pectin, cellulose
255
and hemicellulose are interwoven to form a network structure (Adetunji et al., 2017;
256
Dick-Perez et al., 2011; Marcus et al., 2008), and making pectin difficult to close to
257
degrading enzymes. Therefore, in order to efficiently degrade pectin in CPW, grinding
258
CPW into powder is the most important step to break down the structure of the citrus
259
peel (John et al., 2017; Kumar et al., 2009), thus improving the pectin accessibility to
260
enzymes and achieving the purpose of preparing OGs.
261
3.4. Characterization of the substrates morphological structure changes
262
The micrograph of CPW powder showed that the peel powder was compact,
263
ordered fibers and smooth surface, suggesting that the cellulose–hemicellulose and
264
pectin were interwoven into a tight network. The structural disruption and increased the
265
surface area was observed after one-step fermentation treated the CPW powder,
266
presented rough and porous structure which were attributed to the pectin removal. So,
267
there were significant structural changes after one step fermentation combined with
268
enzymatic hydrolysis, and a similar result was also observed by multistep treatment to
269
remove pectin from mandarin peel waste (Hiasa et al., 2014). Moreover, part of
270
cellulose and hemicellulose fragment attached to the pectin were released into the
271
fermentation broth, forming uniform spherical cellulose nanofiber particles. Further
272
characterization of the cellulose nanofiber particles may be required to determine its
273
physicochemical property and the application potential.
274 275
3.5. OGs compositional analysis of fermented broth For producing OGs and removing the soluble fermentable sugars in the CPW, the
276
engineered yeast strain P. pastoris X-33 was applied for fermentation using CPW
277
directly as substrate. First, the engineered yeast strain was cultured in YPD medium
278
under optimal conditions. Then, the seed liquid was transferred to the CPW medium as
279
a feed ratio 15 (v/v) in 250 ml flasks. Subsequently, the CPW biomass with 7%
280
(mandarin peel powder) and 5% (orange peel powder) were added to the culture to
281
perform the OGs production at 28 °C and pH 5.0. In this one-step engineered strain
282
fermentation method, OGs production was combined with AnPG28A secretion
283
expression.
284
Under the above conditions, the engineered strain exhibited good degraded
285
properties towards to the CPW biomass. During the fermentation process, the OGs
286
content was reached the highest after 72 h with the maximum OGs content of 3.6 and
287
1.9 mg/ml for mandarin and orange peel powder, respectively (Fig. 1). Along with the
288
engineered strain growth, the fermentable sugars were gradually consumed as carbon
289
source (Li et al., 2019), and thus as a new carbon source the GalA and OGs were
290
consumed and rapidly decreased after 96 h fermentation. In this period, the OGs content
291
from mandarin peel was much higher than that orange peel after 72 h fermentation (Fig.
292
1). This was probably because there was a higher content of D-limonene in the orange
293
peel waste (1.70%) compared with mandarin peel waste (0.21%) which was considered
294
to be a microbial fermentation inhibitor (Wilkins et al., 2007), and pectin accessible area
295
and substrate concentration difference were also affect the degradation efficiency of the
296
CPW biomass. The OGs yield derived from the CPW biomass was 19 and 15 % for
297
mandarin and orange peel powder after 72 h fermentation, respectively.
298
The composition of the CPW biomass mainly consisted of crude protein, water
299
insoluble material, carbohydrate, and total ash (Martinez et al., 2010; Sharma et al.,
300
2017). The undegraded water-insoluble material, crude protein, and ash could be
301
removed by centrifugation after boil. After fermentation for 72 h, the GalA content was
302
62.5 and 50.5% from mandarin and orange peel waste broth, respectively.
303
Carbohydrates mainly present in CPW fermentation broth are GalA, Glucose, Galactose
304
and Arabinose. The fermentable sugars could be gradually consumed in the
305
fermentation process (Li et al., 2019). Additionally, the engineered yeast strain contains
306
only one endo-polygalacturonase which only acts on the HG part of pectin, so that the
307
main oligosaccharides produced by CPW fermentation are OGs.
308
To further determine the DP distribution and quantitative analysis of the OGs, the
309
fermentable products was subjected to HPAEC-PAD analysis. The DP distribution of
310
the OGs in the fermentation broth was ranging from 2 to 7 for mandarin peel and
311
ranging from 2 to 5 for orange peel, respectively. From mandarin peel waste
312
fermentation broth, the main products were OGs with DP 2–3 and high-molecular
313
weight fragments DP 7 (Fig. 2). The orange peel waste fermentation extracted products
314
yielded mostly OGs with DP 2–3 with low formation of DP 4 and DP 5 (Fig. 2).
315
These results indicated that the engineering yeast strain was suitable for OGs
316
production, directly using CPW as fermentation substrate. In this study, the one-step
317
engineered yeast fermentation method coupled enzyme-induced expression with CPW
318
degradation during biomass fermentation which has been shown to reduce costs, save
319
time, environment-friendly and simplify bioprocesses. Therefore, the one-step
320
bioprocess strategy in the present study had a brilliant applied prospect in the CPW
321
biomass utilizing for OGs production due to its unpolluted environment and cost
322
savings. Moreover, as a material basis for scientific and applied research, the cellulose
323
and hemicellulose contained in CPW biomass were pretreated by engineered yeast
324
fermentation which was feasible and environmentally friendly method compared with
325
the chemical treatment method (Alemdar and Sain, 2008; Sahoo et al., 2018).
326
3.6. Optimization of citrus peel waste fermentation conditions for OGs production
327
Since the one-step engineered strain fermentation method gave a good result, so
328
further optimized the experiment for OGs production. Using the RSM to optimize
329
process parameters and predict response value save time, and also provide information
330
about interactions between parameters (Sathendra et al., 2019; Yao et al., 2015).
331
Seventeen groups of experiments were designed with the OGs content (Y) from
332
mandarin and orange peel waste fermentation and enzymatic hydrolysis as the response
333
values.
334
Through multiple regression analysis, the regression model for OGs content in the
335
fermentation broth is given by Eq. (1) and Eq. (2) for mandarin and orange peel
336
substrate, respectively.
337 338 339 340 341
YMP (OGs mg/mL) = -57.7+13.9A+0.266B+6.72C-0.0220AB-0.341AC +0.00645BC-1.12A2-0.00297B2-0.361C2 (1) YOP (OGs mg/mL) = -9.49+3.37A+0.176B+0.367C+0.00964AB+0.0172AC +0.00492BC-0.351A2-0.00657B2-0.0489C2 (2) According to ANOVA for RSM, the model with P < 0.05 is considered statistically
342
significant (Liu et al., 2018). The regression analysis for mandarin and orange peel
343
substrate revealed the regression coefficient (R2) was 0.910 and 0.857, and the ‘‘Model
344
F-value’’ of 7.89 and 4.67 indicated that the model is significant, and 0.63% and 2.72%
345
of the total change cannot be explained by the model for mandarin and orange peel
346
substrate, respectively. The model with the signal to noise ratio greater than 4 can be
347
used to predict the OGs content from CPW (Liu et al., 2018).
348
The effect of the variables on the OGs content can be better understood by
349
examining the three-dimensional response surface plots in Fig. 3, which shows the
350
interaction effect of important process variable combinations on OGs content. For
351
mandarin peel waste fermentation, the OGs content increased with the increase in
352
substrate concentration and pH, and reached the highest value around their middle level.
353
With a further increasing in substrate concentration and pH, the OGs content began to
354
decrease (Fig. 3a and 3e). However, the OGs content was increased with increase in
355
inoculum size as observed in Fig. 3c. In addition, for orange peel waste fermentation,
356
substrate concentration, inoculum size and pH has decreased OGs content when their
357
increased around middle level to higher level and decreased around middle level to
358 359
lower level (Fig. 3b, 3d and 3f). The RSM analysis for mandarin and orange peel substrate showed that the optimal
360
conditions were a pH of 5.17 and 5.04, inoculum size of 29.5% and 20.5%, and
361
substrate concentration of 6.9% and 4.7% for a theoretical maximum OGs production
362
which yielded a predicted maximum OGs production of 4.53 and 1.94 mg/mL after 72 h
363
fermentation, respectively. To validate the adequacy of RSM responses, the verification
364
experiment was carried out in triplicates under above conditions. The results showed
365
that the OGs content was 4.49±0.48 and 1.99±0.13 mg/mL which corresponds to 26.1
366
and 15.7% of OGs yield for mandarin and orange peel substrate, respectively.
367
Therefore, the results from the validated experiment were in good agreement with the
368
predicted value which proved the adequacy of the model in predicting the response
369
together with the optimization of fermentation parameters.
370
The results suggest that OGs yield from the mandarin peel waste was significantly
371
higher than OGs production from enzymatic hydrolysis, acid hydrolysis or
372
hydrothermal treatments of CPW biomass (Babbar et al., 2016; Gomez et al., 2013;
373
Gomez et al., 2016b; Sabajanes et al., 2012), and with the difference that the CPW
374
biomass need not pretreated by physical, chemical or mechanical treatments to extract
375
pectin and remove soluble material in this study. Therefore, the use of engineered yeast
376
strain fermentation has distinct advantages, such as low costs, environment-friendly,
377
simple process and high hydrolysis efficiency.
378
In this study, we developed one-step fermentation method for oligogalacturonides
379
(OGs) production directly from CPW biomass. To our knowledge, this is the first time
380
to produce OGs using CPW as a direct substrate using one-step engineered yeast strains
381
fermentation method which is an environmentally friendly, simplify process and cost-
382
effective technique for OGs production. The study provides new strategies for the
383
development and utilization of CPW resources for OGs production and has great
384
potential for CPW recycling utilization.
385
4. Conclusions
386
In this study, one-step fermentation method for OGs production, as an innovative
387
green and novel strategy, have been successfully developed. The DP distribution of the
388
OGs produced in the fermentation broth were DP 2–7 and DP 2–5 for mandarin and
389
orange peel waste, respectively. With RSM, a maximal OGs yield of 26.1 % was
390
obtained from mandarin peel waste. As a new process, this one-step fermentation
391
method using the CPW bioresources as the direct substrates has great significance for
392
CPW biomass utilization due to its rapid, low cost, effective, eco-friendly and easily
393
bioprocess.
394
Acknowledgements
395
This work was supported by the National Key R&D Program of China, China
396
(Grant No. 2017YFD0200900 and 2017YFD0200902), National Natural Science
397
Foundation of China (Grant No. 31670803), Liaoning Revitalization Talents Program,
398
China (Grant No. XLYC1807041) and DICP, China (Grant No. ZZBS201704 and
399
BioChE-X201801).
400 401
Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper.
402
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568 569 570
Figure Captions
571
Fig. 1. OGs production during the fermentation treatment from the CPW biomass.
572 573
Fig. 2. OGs composition analysis after 72 h fermentation treatment.
574 575
Fig. 3. Response contour plot for the optimization of OGs production from CPW
576
biomass. a & b: pH and inoculum size (%); c & d: pH and substrate concentration (%);
577
e & f substrate concentration (%) and inoculum size (%). a, c and e represent mandarin
578
peel waste; b, d and e orange peel waste.
579 580 581 582 583 584 585 586 587 588
589 590 591
Table 1 The three variables selected for the optimization of OGs production using one-
592
step fermentation by the engineered yeast strain. Range and levels Symbols
Independent variables Lower (−1)
Middle (0)
Higher (+1)
Mandarin peel waste A
pH
4
5
6
B
Inoculum size, % (v/v)
5
15
25
5
7
9
Substrate C concentration, % (w/v) Orange peel waste A
pH
4
5
6
B
Inoculum size, % (v/v)
5
15
25
1
5
9
Substrate C concentration, % (w/v)
593 594 595 596 597
598 599 600
Table 2 Particle size distribution of the citrus peels powder substrate. Substrate source
X50,3
SMD (um)
VMD (um)
Mandarin peel powder (MP)
63.79±0.26
23.14±0.18
85.17±1.27
Ornge peel powder (OP)
37.56±0.95
16.52±0.28
49.68±1.89
601
X50 stands for the median particle size; SMD stands for surface mean diameter; VMD stands for
602
volume surface mean diameter.
603 604 605 606 607 608 609 610 611 612 613 614 615
616 617 618
Table 3 Fermentable sugars (FS) and total sugars in citrus wastes. Sucrose
Glucose
Fructose
FS
Total sugars
CPW %, DW (dry weight)
619 620 621 622 623 624 625 626 627 628 629 630 631 632
MP
7.4 ± 0.2
39.4 ± 1.1
10.3 ± 0.8
57.1 ± 0.6
71.9 ± 0.9
OP
5.6 ± 0.2
35.5 ± 0.5
12.1 ± 0.4
53.2 ± 0.4
68.2 ± 0.5
633 634 635
636 637 638 639 640 641 642 643 644 645 646 647
Fig. 1.
648 649 650
651 652 653 654 655 656 657 658 659 660 661 662 663 664
Fig. 2.
665 666 667
668 669 670 671 672 673 674 675
Fig. 3.
676
677 678
Highlights
679
● First production of oligogalacturonides (OGs) directly from citrus peel wastes.
680
● An innovative green and novel fermentation strategy has been developed.
681
● After fermentation for 72 h, the DP of OGs was ranging from 2 to 7.
682 683
Declaration of interests
684 685
☒ The authors declare that they have no known competing financial interests or personal
686
relationships that could have appeared to influence the work reported in this paper.
687 688
☐The authors declare the following financial interests/personal relationships which
689
may be considered as potential competing interests:
690 691 692 693 694 695
696
Sample CRediT author statement
697
Guojun Yang: Investigation, Writing- Original draft preparation, Haidong Tan:
698
Supervision, Shuguang Li: Resources, Meng Zhang: Visualization, Jia Che: Resources,
699
Kuikui Li: Formal analysis, Wei Chen: Data curation, Heng Yin: Writing- Reviewing and
700
Editing, Project administration, Funding acquisition.
701