Bioresource Technology 267 (2018) 793–796

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Impact of lignin content on alkaline-sulfite pretreatment of Hybrid T

Pennisetum
⁎
Jingfeng Wang1, Xixun Hao1, Ming Yang, Yujie Qin, Lili Jia, Jie Chu, Junhua Zhang
College of Forestry, Northwest A&F University, No. 3 Taicheng Road, Yangling 712100, Shaanxi, China

A R T I C LE I N FO A B S T R A C T

Keywords: This work focuses to investigate the impact of lignin content on chemical compositions, crystallinity, surface
Hybrid Pennisetum characterizations, cellulase adsorption profiles and hydrolysability of Hybrid Pennisetum (HP) after alkaline
Lignin sulfite pretreatment (ASP). For the HP with lower lignin content, the increase of the cellulose content by ASP was
Alkaline sulfite pretreatment more obvious than raw HP. ASP decreased total lignin content and surface lignin content of HP substrates. HP
Surface characterizations
with lower lignin content (e.g., ∼15%) is suitable for ASP, because a pretty perfect glucose yield (91%) was
Enzymatic hydrolysis
obtained using a low dosage of enzyme loadings (5 FPU of cellulases/g dry matter). The study provides a po-
tential strategy to efficiently produce platform sugars from HP with reduced lignin content, indicating the im-
portance of reduction HP lignin content properly by breeding or transgenesis programs. The work could also help
elucidate the mechanism of alkaline sulfite pretreatment for efficient production of fermentable sugars from
lignocelluloses.

1. Introduction
The general ideas of pretreatment are to alter or remove hemi-
celluloses and/or lignin, increase surface area and decrease the crys-
tallinity of cellulose (CrI) (Jørgensen et al., 2007; Huang et al., 2018).
After pretreatments, the hydrolysis of cellulose in lignocelluloses by
cellulases, which are composed of endoglucanases, cellobiohydrolases,
and β-glucosidase, can be largely improved. Alkaline-sulfite pretreat-
ment (ASP) has been used to pretreat sugarcane bagasse with 5% NaOH
and 10% Na2SO3 at 140 °C–160 °C for 30–60 min and high lignin re-
moval (77%) and high glucose yield (up to 80%) is noticed (Tavares
et al., 2018). The main advantages of ASP are strong delignifiability,
high retention of polysaccharides and partial sulfonation of lignin
(Mendes et al., 2011, 2013; Mesquita et al., 2016).
As a physical barrier, lignin is reported to encapsulate and confine
cellulose, and negatively impacts enzymatic digestibility. Enhanced
digestibility in less lignified immature grasses when compared with
mature lignified plants has previously been demonstrated (Grabber
et al., 2002). The reduction of the original lignin content in alfalfa
plants proved to be an efficient way to increase its enzymatic digest-
ibility (Chen and Dixon, 2007). Several breeding or transgenesis pro-
grams concentrated on providing plants with reduced initial lignin
content have been reported in the literature (Lam et al., 2003; Chen and
Dixon, 2007; Mendes et al., 2011). But the lignin presence could retard
some deleterious changes from occurring, such as increases in the re-
lative cellulose Iβ, cellulose CrI, and cellulose crystallite dimension
during dilute acid pretreatment (Foston and Ragauskas, 2010; Sun
et al., 2014). However, the impact of initial lignin on effectiveness of
pretreatment (e.g. the changes of chemical compositions and surface
characterization, the increase of glucose yield) is less reported. In-
vestigating the effect of lignin content on biomass hydrolysability could
provide a new insight to exploit the genetically modified low-lignin
plants for production of biofuels (Sun et al., 2014).
Earlier studies about ASP mainly concentrated on the increase of
sugars yield instead of surface characterizations of biomass and ad-
sorption characteristics of cellulases on substrates, which are all re-
levant performance indicators of enzymatic hydrolysis (Mendes et al.,
2011; Lai et al., 2017; Li et al., 2017). Additionally, the literatures
about the effects of lignin content on effectiveness of ASP are quite
limited. In order to investigate the changes of these indices, Hybrid
Pennisetum substrates with controlled lignin contents were prepared
and then pretreated by alkaline sulfite.

⁎
Corresponding author.
E-mail address: junhuazhang@nwafu.edu.cn (J. Zhang).
1
Equally contributed by both authors in this article.

https://doi.org/10.1016/j.biortech.2018.07.049
Received 6 May 2018; Received in revised form 8 July 2018; Accepted 9 July 2018
Available online 10 July 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
J. Wang et al. Bioresource Technology 267 (2018) 793–796

2. Materials and methods 2.6. Enzymatic hydrolysis

2.1. Materials Hydrolysis of the HP samples by cellulase preparation (CEL) was

performed in the same system as previous reported (Yang et al., 2017).
Hybrid Pennisetum (Pennisetum americanum × P. purpureum, HP) CEL contained both Celluclast 1.5 L and Novozyme 188 preparations,
was supplied by the Beijing Research and Development Center for Grass which were dosed at 5 FPU/g DM and 500 nkat/g DM, respectively.
and Environment, Beijing Academy of Agriculture and Forestry Samples were withdrawn at 48 h and the glucose and xylose contents in
Sciences (Beijing, China). Before pretreatment, HP was milled and the supernatants were analyzed.
sieved through a 60 mesh (≦0.25 mm) screen scale. Then, the ground
HP samples were extracted with toluene-ethanol (2:1, v/v) in a Soxhlet 2.7. Carbohydrate analysis
to remove the wax and other extractives. The samples without ex-
tractives were then delignified with sodium chlorite (0.32 g/g HP) at pH The concentrations of glucose and xylose in the supernatants were
3.6–3.8, adjusted with acetic acid, at 70 °C for 10 (low delignification determined using Agilent 1260 infinity HPLC system (Agilent
severity, HP-L) and 90 min (high delignification severity, HP-H), re- Technologies, USA) as described previously (Yang et al., 2017). The
spectively. glucose and xylose yields from the hydrolysis of HP samples were cal-
culated by the following equations:
2.2. Alkaline-sulfite pretreatment Glucose released ×0.9
Glucose yield(%) = × 100
Theoretical amount of cellulose in substrates
The HP with/without delignification was pretreated with 5%
(4)
NaOH + 20% Na2SO3 (m/m) at 121 °C for 1 h at a solid to liquid ratio
of 1:10 (pH = 12.8). After pretreatment, solids were recovered by fil- Xylose released ×0.88
Xylose yield (%) = × 100
tration and washed repeatedly with distilled water until the washing Theoretical amount of xylose in substrates
fluid reached a neutral pH. Then, the solids were air-dried and stored at
(5)
−19 °C until chemical composition analysis and enzymatic hydrolysis.
The contents of cellulose, xylan and lignin were determined based upon
the National Renewable Energy Laboratory Analytical Procedure 3. Results and discussion
(Sluiter et al., 2012).
3.1. Chemical compositions
2.3. X-ray diffraction analysis
Contents of cellulose, xylan and lignin of native HP (HP) were
The crystallinity index (CrI) of HP samples was analyzed by X-ray 36.3%, 20.5% and 20.8%, respectively (Table 1). After low severity
diffraction (XRD) using a Rigaku D/max-3C generator (Rigaku delignification by sodium chlorite (HP-L), a reduction of lignin content
Corporation, Japan). The CrI was determined from XRD data according from 20.8% to 14.9% was observed with cellulose content change from
to the XRD spectral peak height (Segal et al., 1959). 36.3% to 38.1% and xylan content change from 20.5% to 21.6%. When
delignification severity increased (HP-H), the lignin content further
decreased to 11.2%, while little change in the relative cellulose and
2.4. X-ray photoelectron spectroscopy analysis xylan contents happened. The data indicated that partial delignification
showed little effect on the cell wall carbohydrate components, which
The X-ray photoelectron spectroscopy (XPS) analyses were per- were in good agreement with previous results (Sun et al., 2014).
formed as described previously (Yu et al., 2015). The surface coverages After ASP, the content of HP lignin decreased from 20.8% to 13.6%
of lignin (Slig) and carbohydrates (Scarb) were calculated based on the (HP-ASP), and the cellulose content was 48.7% with xylan content re-
average O/C values using the following equations (Yu et al., 2015): mained almost constant (Table 1). The increase of cellulose content
O/Cpretreated−O/Ccarbohydrate may be ascribed to the removal of lignin. The contents of cellulose in
Slig (%) = ∗100 the HP with partial delignification (HP-L and HP-H) increased after
O/Clignin−O/Ccarbohydrate (1)
ASP, which is more effective than that of HP without delignification.
Lower lignin contents were also noticed in all three samples after ASP,
Scarb = 1−Slig (2)
confirming the selective delignification capability of ASP (Mendes
et al., 2013; Tavares et al, 2018). Additionally, it was found that ASP
O Clignin = 0.33, O Ccarbohydrate = 0.83 (3)
exhibited higher delignifiability to the HP with higher lignin content.
CrI value of HP was 41.7%, compared with 46.4% and 47.9% of HP-
L and HP-H, respectively (Table 1). This increase in CrI, in part, resulted
2.5. Cellulases adsorption
from the removal of amorphous substance such as lignin. ASP showed
stronger ability to enhance the CrI of HP samples than that to enhance
Replicate adsorption experiments were performed in 10 mL test
tubes containing 0.05 M sodium citrate buffer (pH 5.0) at solids loading
Table 1
containing 2% (w/v) substrates at 4 °C to avoid hydrolysis. Cellulases Lignin, cellulose, and xylan contents and crystallinity index of alkaline sulfite
were dosed as 5 FPU Celluclast 1.5 L per gram DM (dry matter) and pretreated Hybrid Pennisetum (HP) with reduced lignin contents by sodium
500 nkat Novozyme 188 per gram DM. After 1 h, the samples were chlorite pretreatment.
centrifuged at 10, 000×g for 10 min. The FPU activity was measured
Sample Cellulose (%) Xylan (%) Lignin (%) CrI (%)
according to the IUPAC standard assay (Ghose, 1987), based on the
amount of reducing sugars released from Whatman number 1 filter HP 36.3 ± 0.2 20.5 ± 0.8 20.8 ± 0.0 41.7
paper using 2,4-dinitrosalicylic acid. After 1 h adsorption, the samples HP-ASP 48.7 ± 1.3 21.6 ± 1.1 13.6 ± 0.1 57.4
were centrifuged at 10,000g for 10 min. Cel3A and Cel5A activities in HP-L 38.1 ± 0.4 21.6 ± 0.2 14.9 ± 0.1 46.4
HP-L-ASP 56.5 ± 0.1 21.2 ± 0.6 10.0 ± 0.1 58.3
the hydrolysates were determined as the method of Bailey and
HP-H 40.0 ± 0.1 22.1 ± 0.2 11.2 ± 0.1 47.9
Nevalainen (1981), and Cel7A activity was determined as the method of HP-H-ASP 60.9 ± 0.5 22.2 ± 0.8 9.3 ± 0.1 61.5
Deshpande et al. (1984).

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J. Wang et al. Bioresource Technology 267 (2018) 793–796

Table 2 cellulose than on lignin (Ju et al., 2013). For HP-L and HP-H, ASP de-
Element composition and surface coverage by lignin (Slig) and carbohydrates creased the Cel5A adsorption on substrates (HP-L-ASP, HP-H-ASP),
(Scarb) on the surface of alkaline sulfite pretreated HP with reduced lignin which might be due to the reduction of unproductive binding of the
contents. enzymes on the residual lignin present in the pretreated materials
Pretreatment O/C Slig (%) Scarb (%) (Mendes et al., 2013) by the increase of the amount of sulfonic acid
groups and the enhancement of lignin hydrophilicity (Ju et al., 2013).
HP 0.55 55.2 44.8
Adsorbed Cel7A on HP samples (1.1%−14.8%) was less than Cel5A
HP-ASP 0.60 45.2 54.8
HP-L 0.54 58.7 41.3
(Table 3), which might be due to that the presence of Cel5A decreased
HP-L-ASP 0.61 43.6 56.4 the binding of Cel7A (Palonen et al., 2004). Cel3A (0.8%−9.2%) was
HP-H 0.54 58.1 41.9 hardly adsorbed on the HP samples compared to Cel5A and Cel7A.
HP-H-ASP 0.60 46.7 53.3 Generally, Cel3A have been thought to stay free in solution during
hydrolysis, which might be at least partially due to the absence of
cellulose-binding module of Cel3A (Várnai et al., 2011).
delignification.

3.2. XPS analysis 3.4. Enzymatic hydrolysis

The O/C ratio, the surface coverage by lignin, and the surface
coverage by carbohydrates of the HP samples were analyzed by XPS
(Table 2). The O/C ratio of HP was found to be 0.55. It has been re-
ported that the O/C ratios of un-extracted biomass were very close to
the theoretical value of O/C for lignin (0.33) (Nzokou and Kamdem,
2005). The higher O/C ratios obtained in this work might be due to the
removal of carbon-rich extractives such as fatty acids, terpenes, and
phenolics in the sample preparation procedures (Nzokou and Kamdem,
2005). The lignin surface coverage of the HP was 55.2%, showing that
more than half the surface of untreated sample is covered by lignin.
After delignification, however, slight increases in the lignin surface
coverage were observed, which might be due to a re-deposition of
dissolved lignin on its surface in cooling process during delignification.
After ASP, the surface lignin contents of HP, HP-L and HP-H were
decreased (Table 2). The reason might be that partial surface lignin was
dissolved in pretreatment reagent. Together with the results of the
chemical compositions, it can be concluded that delignification by so-
dium chlorite led to the reduction of total lignin content but showed
little effect on surface lignin content, while ASP resulted in the reduc-
tion of total lignin content and surface lignin content and exhibited
stronger capacity in the removal of surface lignin than delignification
by sodium chlorite.
3.3. Cellulases adsorption
Adsorbed Cel5A on untreated HP was 19.1%. After low and high
severity delignification by sodium chlorite (HP-L and HP-H), the ad-
sorbed Cel5A increased to 47.0% and 59.5%, respectively (Table 3),
which could be due to the increase of lignin removal and pore volume
(Yu et al., 2011) and thus increased adsorption of Cel5A. After ASP (HP-
ASP), adsorbed Cel5A increased from 19.1% to 44.0%. The reason
might be due to that ASP caused the removal of lignin content and the
reduction of surface lignin content (Table 1), exposed more cellulose for
Cel5A adsorption because that cellulases binds preferentially on
As expected, the hydrolysis yields of cellulose and xylan in un-
treated HP by CEL were low (less than 25%) due to plant cell wall
recalcitrance (Table 3). Delignification by sodium chlorite led to the
improvement of the hydrolysis yields, and glucose yields of HP-L and
HP-H were 31.4% and 53.1%, respectively. The glucose and xylose
yields of HP samples after ASP were 74.8%−90.8% and 65.9–79.5%,
respectively (Table 3), which were much higher than those of the
samples before ASP. The decrease of the bulk lignin (Table 1) and
surface lignin contents (Table 2), the enhanced adsorption of key cel-
lulases Cel7A onto samples (Table 3) together led to the improvement
of the hydrolysis yield of HP samples. Higher glucose (74.8%) and
xylose (65.9%) yields were observed from HP-ASP than those from HP-
L and HP-H (< 53.1%), suggesting that single delignification was not
enough to achieve high hydrolysis yield.
Besides, initial sodium chlorite delignification followed by a second
ASP step dramatically enhanced glucose and xylose yields of approxi-
mately 91% and 80% for HP-L-ASP, respectively, which was in good
line with previous results that lignocelluloses with lower lignin content
would be suitable for ASP pretreatment which caused only partial lignin
and hemicellulose removal (Mendes et al., 2011).
In this work, HP with reduced lignin content was subject to ASP,
which has been applied industrially. Delignification by sodium chlorite
did not reduce the surface lignin content but enhanced the adsorbed
amounts of cellulases and cellulose digestibility. HP with lower lignin
content, which could be achieved by delignification properly, would be
suitable for ASP, because a pretty perfect glucose yield (91%) of HP-L-
ASP was obtained using a low dosage of enzyme loadings
(5 FPU cellulases/g DM) together with the fact that ASP has been ap-
plied industrially. Therefore, the data provides key insight into the way
that HP with properly reduced lignin content obtained by several
breeding or transgenesis programs could be subject to ASP at a mild
condition for production of platform sugars at a very low cost.

Table 3
Percentage of theoretical activity of Cel5A, Cel7A and Cel3A in supernatants of Celluclast 1.5 L (5 FPU/g DM) and Novozyme 188 (500 nkat/g DM) after adsorption
on substrates (2%) at 4 °C for 1 h. Activity after adsorption without substrates were assumed to 100% of theoretical activity. Hydrolysis of HP substrates (2%) by
Celluclast 1.5 L (5 FPU/g DM) and Novozyme 188 (500 nkat/g DM) at 50 °C and pH 5.0 for 48 h. The error bars represent the standard errors of three independent
experiments.
Samples Activity in supernatant (% of theoretical activity) Hydrolysis yield (%)

Cel5A Cel7A Cel3A Glucose yield Xylose yield

HP 80.9 ± 0.0 98.9 ± 4.5 98 ± 0.0 24.6 ± 0.8 12.5 ± 0.3

HP-ASP 56.0 ± 3.8 93.7 ± 0.2 99.2 ± 3.0 74.8 ± 0.5 65.9 ± 1.0
HP-L 53.0 ± 5.1 92.2 ± 0.4 96.5 ± 0.0 31.4 ± 2.5 14.5 ± 1.1
HP-L-ASP 68.6 ± 4.6 95.8 ± 2.1 97.0 ± 3.6 90.8 ± 1.7 79.5 ± 2.5
HP-H 40.5 ± 3.3 89.3 ± 0.7 90.8 ± 0.2 53.1 ± 1.2 28.1 ± 0.5
HP-H-ASP 58.4 ± 1.0 85.2 ± 6.9 95.9 ± 2.2 78.3 ± 1.8 69.1 ± 1.3

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J. Wang et al.
4. Conclusions
ASP showed strong ability to remove total lignin and surface lignin
of HP substrates, exhibiting higher delignifiability to HP with higher
lignin content. Higher cellulose content and crystallinity index were
observed in alkaline sulfite-pretreated HP with lower lignin content.
The HP with moderate lignin after ASP (HP-L-ASP) achieved 91% cel-
lulose digestibility using a low loading of cellulases (5 FPU/g DM). The
results indicated that ASP was suitable for the HP with reduced lignin
content (properly) for production of platform sugars cost-effectively. In
addition, this work could also provide scientific guidance for ASP for
production of sugars from lignocelluloses.
Acknowledgements
This work was supported by the Natural Science Foundation of
China (No. 31670598), the Science and Technology Project of Yangling
Agricultural Hi-tech Industries Demonstration Zone, China (No.
2017NY-04) and the Science Foundation for Distinguished Young
Scholars of Northwest A&F University, China (No. 2452015098).
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