Bioresource Technology 183 (2015) 240–247

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

Ozonolysis pretreatment of maize stover: The interactive effect

h i g h l i g h t s

 Both particle size and moisture content had significant effects on ozonolysis.
 The ratio of free and bond water is a key factor for ozonolysis.
 Glucose yield after enzymatic hydrolysis increased as delignification rate increased.
 Washing ozonolyzed samples made no obvious difference to enzymatic digestibility.

a r t i c l e i n f o a b s t r a c t

Article history: Maize stover was ozonolyzed to improve the enzymatic digestibility. The interactive effect of sample
Received 14 November 2014 particle size and moisture content on ozonolysis was studied. After ozonolysis, both lignin and xylan
Received in revised form 6 January 2015 decreased while cellulose was only slightly affected in all experiments. It was also found that the smaller
Accepted 9 January 2015
particle size is better for ozonolysis. The similar water activity of the different optimum moisture
Available online 17 January 2015
contents for ozonolysis reveals that the free and bound water ratio is a key factor of ozonolysis. The best
result of ozonolysis was obtained at the mesh of 300 and the moisture of 60%, where up to 75% lignin
Keywords:
was removed. The glucose yield after enzymatic hydrolysis increased from 18.5% to 80%. Water washing
Lignocellulosic biomass
Maize stover
had low impact on glucose yield (less than 10% increases), but significantly reduced xylose yield (up to
Ozonolysis 42% decreases). The result indicates that ozonolysis leads to xylan solubilization.
Enzymatic hydrolysis Ó 2015 Elsevier Ltd. All rights reserved.
Particle size

1. Introduction Maize is one of the world’s widest planted crops. It has also
become the largest crop in China since 2012. Compared to other
In recent years, biorefinery technologies have attracted types of lignocellulosic material, maize stover has low lignification
significant attention due to the worldwide growing problems and high carbohydrate content making it an abundant and valuable
including oil shortage, pollution and excessive population. Unlike agricultural residue. However, maize stover is currently underex-
the first generation biofuel made of cereals, the second generation ploited. In China, only less than 10% of maize stover get in the
biofuel made from lignocellulosic materials has greater environ- pathway of high added-value processing while almost 40% is left
mental benefits and sustainability (Menon and Rao, 2012). Ligno- in the field or burned, causing severe waste and pollution. Other
cellulosic ethanol represents the most developed route of the crop straws are dealt with the same way. The Chinese government
second generation biofuel. Producing lignocellulosic ethanol had taken measures to change this situation. According to the
involves three steps: (a) pretreatment, which enhances the enzy- document (State Council, 2008), Opinions on Accelerating the
matic saccharification of biomass; (b) enzymatic hydrolysis, which Utilization of Crop Straw, great efforts will be taken to establish
converts structural polysaccharide into fermentable sugars; and (c) efficient straw collection and multi-grade utilization system. This
fermentation, which produces ethanol and other products using policy will propel the biorefinery industries based on maize stover
fermentable sugars (Sarkar et al., 2012). and other agricultural residues.
However, there are several limits that challenge biorefinery
⇑ Corresponding author. Tel./fax: +86 510 85197856. industrialization. The most crucial one is the low efficiency of
E-mail address: legend0318@hotmail.com (L. Wang).
enzymatic hydrolysis of structural polysaccharide. Several factors
1
These authors contributed equally to this work. have been proven to account for this, including particle size,

http://dx.doi.org/10.1016/j.biortech.2015.01.042
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
 C. Li et al. / Bioresource Technology 183 (2015) 240–247 241

cellulose crystallinity, degree of polymerization, and lignin content 150, 300 mesh. Thus five fractions obtained from the sieves were
(Hendriks and Zeeman, 2009). Among these factors, the lignin con- named as 20/40, 40/80, 80/150, 150/300 and 300, respectively.
tent shows the highest impact on biomass degradability. Ding et al. The 20/40 refers to the fraction from between 20 mesh and
(2012) found that lignin removal enhances binding of enzymes to 40 mesh sieves, and so on for the other fractions. Among the five
the cell walls and the extent of degradation. They concluded that fractions, 20/40, 80/150 and 300 was used as raw sample in this
the best pretreatments should maximize lignin removal. study. The major components of these three fractions were deter-
Various pretreatment technologies have been investigated, mined (Fig. 2), shown similar value to each other.
including physical, chemical and biological approaches (Agbor
et al., 2011; Alvira et al., 2010; Galbe and Zacchi, 2012; Hendriks 2.2. Complete delignified samples (CDL)
and Zeeman, 2009). Ozonolysis pretreatment is regarded as one
of the most promising chemical approaches. Due to its highly Complete delignified samples (CDL) were prepared using 20/40,
selective oxidation of compounds with high-electron-density 80/150 and 300 raw samples. Acid chlorite was used to remove
groups (mainly in lignin), ozonolysis shows high efficiency in lignin in the raw samples (Ding et al., 2012). The acid chlorite solu-
delignification and high recovery of carbohydrates. Furthermore, tion contains 0.1 N HCl and 10% NaClO2. Raw samples were added in
low inhibitor production was also reported (Alvira et al., 2010). the 1% (w/v) solution under magnetic stirred at room temperature
Ozonolysis has been tested on many types of biomass, such as for 12 h. The mixture was neutralized and vacuum filtrated. The
wheat straw (Binder et al., 1980; García-Cubero et al., 2009), sug- solid residue was washed and freeze dried to obtain the CDL.
arcane bagasse (Barros et al., 2013; Travaini et al., 2013), and ener-
gy grasses (Panneerselvam et al., 2013a,b). In these studies, lignin 2.3. Ozonolysis treatment
removal after ozonolysis improved the enzymatic hydrolysis yield.
García-Cubero et al. (2009) found that moisture content and bio- The ozonolysis reaction was performed in a glass column reac-
mass type are the two main factors of ozonolysis. They proposed tor (6 cm in diameter and 22.5 cm in height) at constant room tem-
a kinetic model for the ozonation reaction (García-Cubero et al., perature, 25 °C. The column was vertically placed. The gas flowed
2012). Travaini et al. (2013) found only xylitol, lactic, formic and from bottom to top. Ozone monitors were used to detect the ozone
acetic acid as oxidation products. Inhibitors like furfural and 5-hy- concentration in gas flow before and after the reactor. Ozone was
droxymethylfurfural were not detected. generated by an ozone generator (CF-G-3-20g, QingDao GuoLin
Comminution is an essential step before other processes. It aims industry Co., Ltd) with pure oxygen source. The flow rate and ozone
to increase the specific surface area and reduce the bulk volume. concentration maintained at 1.0 L/min and 60 mg/L during the
Studies have shown that individual application of comminution reaction, respectively.
in a specific range of meshes hardly improves hydrolysis (Chang The samples of 20/40, 80/150 and 300 was used in the treat-
et al., 1997; Chang and Holtzapple, 2000; Elshafei et al., 1991). ment. Each of the samples was adjusted to the moisture contents
Hence, focus has been shifted to the combination of comminution of 30%, 45%, 60% and 75% by adding distilled water before ozonoly-
with other pretreatment methods (Barros et al., 2013; Chundawat sis. The reactor was loaded with 10 g raw material (dry basis) and
et al., 2006; Miura et al., 2012; Souza-Correa et al., 2014). Barros the reaction time is 1 h for all tests.
et al. (2013) investigated the combination of wet disk milling
(WDM) and ozonolysis and found that ozonolysis before WDM 2.4. Compositional analysis
results in higher saccharification yield than the reverse and
reduces the WDM energy consumption. Souza-Correa et al. The moisture content was measured by a moisture analyzer
(2014) compared the ozonation process on substrates with a range (Ohaus MB23). The water extractives were determined by gravi-
of particle sizes. They found that the smaller particle size led to metric analysis before and after water wash. After water wash,
better ozonation result. insoluble components such as cellulose, xylan and lignin were
In this study, the interactive effect of sample moisture content determined following the NREL LAP ‘‘Determination of Structural
and particle size on ozonolysis of maize stover was investigated Carbohydrates and Lignin in Biomass’’ (Sluiter et al., 2012). The
from the perspectives of reaction dynamics, compositional varia- sugar analysis was conducted through high performance liquid
tion and hydrolysis yield. A series of samples with a wide range chromatography (Agilent 1260 infinity) equipped with differential
of particle sizes and moisture contents were ozonolyzed in a fixed refraction detector (RID). An Aminex HPX-87H (7.8  300 mm, Bio-
bed reactor under fixed conditions. The untreated sample (raw) Rad, USA) column was used at 60 °C with 5 mM H2SO4 as eluent,
and completely delignified sample (CDL) were set as blank control and the flow rate of eluent is 0.6 ml/min.
and positive control, respectively. To each experimental test, the
breakthrough ozone concentration versus reaction time was mea- 2.5. Enzymatic hydrolysis
sured, which revealed a dynamic profile of the ozonolysis reaction.
The major components, such as lignin and structural carbohy- Enzymatic hydrolysis was performed on both raw and pretreat-
drates, and enzymatic hydrolysis yield were analyzed to monitor ed maize stover, with or without washing, to verify the differences
the impact of ozone oxidation on samples. Water activity (Aw) of of enzymatic degradation. The hydrolysis was performed in 20 ml
each experimental test was determined to evaluate the free and 0.1 M pH 4.8 citrate buffer with 5% biomass (dry basis) suspended
bound water ratio. in it. The mixture was put in a 100 ml autoclavable glass bottle
with a sealing cap to ensure no evaporate losses during incubating.
Two enzymes were used in hydrolysis. One was Novozyme Cellu-
2. Methods clast 1.5 L with the activity of 33 FPU/ml. Another was cellobiase
from Jiangsu Ruiyang Biotech CO., LTD, which has an activity of
2.1. Raw material 60 IU/g. The dose of these two enzymes was 15 FPU and 5 IU per
gram of cellulose, respectively. The reaction was incubated at
Maize stover was collected in Liaoning province, China. It was 50 °C for 48 h with a shaking speed of 120 rpm. After incubation,
sundried and stored at 4 °C. The leaves, ears and rotten parts were the mixture was centrifugated and the supernate was collected
removed then grinded with a laboratory impact mill. The grinded for sugar analysis by HPLC with the same conditions as described
material was sieved with a set of standard sieves: 20, 40, 80, in Section 2.2.
242 C. Li et al. / Bioresource Technology 183 (2015) 240–247

2.6. Water activity from gas into water film, is k(c  cw) as above mentioned. The sur-
face area per unit volume of the bed is a, thus the ozone transfer
The water activity of all samples under adjusted moistures rate in control volume is aAdzNA. According to the mass equilibri-
before ozonolysis was measured by a water activity meter (GBX um analysis (Bird et al., 2007), the sum of the ozone outflow rate,
Fat-lab). the accumulation rate and the gas–water interface transfer rate is
equal to the inflow rate. And the expression can be written as
3. Results and discussion below with a boundary condition:
(
@c
@h
þ u @c
@z
þ kaðc  cw Þ ¼ 0
3.1. Model analysis of ozone concentration in reactor ð3Þ
8h > uz ; z ¼ 0; c ¼ ci
The sample bed for ozonolysis contains certain amount of
The expression is a first-order linear partial differential equa-
water, which forms water films on the surface of the sample parti-
tion. After solving it by the characteristics method, the results is:
cles. The gaseous ozone flows through the pore channels while part
   
of the gaseous ozone transfers into the water films through the exp kaz  1 cw þ ci
u
gas–water interface. Ozone decomposition may occurs in water c¼   ð4Þ
exp kaz
u
phase, however, the decomposition rate is very low compared to
the direct reaction of ozone with organic matters when pH < 12 Since exp(kaz/u) and ci are constant, the relationship between c
(Beltran, 2003). The ozone in the water films is consumed instan- and cw is positive linear. The evolution of c then depends on the
taneously due to the short diffusion path. Accordingly, the decom- evolution of cw in the bed. Eq. (4) describes the evolution of ozone
position of aqueous ozone can be neglected (Beltran, 2003). The concentration in the gas phase. When z represents the value of the
entire process is considered as two coupled steps: the ozone trans- thickness of the bed, c can be replaced by co, which was recorded
fer from gas to water films and the reaction of aqueous ozone with every 5 min during the process.
the samples. The transfer flux NA can be expressed as k(c  cw), Consumed ozone concentration (cc) was defined as the instanta-
where k is the overall mass transfer coefficient and cw is ozone con- neous decrement of ozone concentration for gas flow passing
centration in the water films. The reaction rate of ozone with the through the bed, mathematically expressed as cc = ci  co. As ci is
sample is ocr/oh, where cr is the reacted ozone concentration, h is a constant of 60 mg/L and co was recorded versus time, a set of cc
the reaction time. The variation of ozone concentration in the along with reaction time, was recorded for each ozonolysis group
water films is ocw/oh. At the beginning of the process, the reaction as a scatter plot (Fig. 1). Each set of scatter points was fitted with
rate is higher than the transfer rate. No ozone exists in the water Logistic model (R2 > 0.99 for all sets), the resulted fitting curves
films (cw = 0). The rate of the process is limited by the rate of ozone were used to integrate for total ozone consumption of the
transfer through gas–water interface, which is known as the mass ozonolysis, then calculating the ozone consumption per gram of
transfer control stage. As the process goes on, the reaction rate dry sample. The Logistic fitting expression f(h) is:
slows down and replaces the mass transfer rate to become the
A1  A2
limitation of the process rate. The process turns into the stage of cc ¼ f ðhÞ ¼  P þ A2 ð5Þ
reaction control stage. The mass equilibrium analysis gives the fol- 1 þ hh0
lowing equation (Bird et al., 2007):
where A1, A2, h0 and P were all determined through curve fitting. A1
dcw dcr
þ ¼ kaðc  cw Þ ð1Þ and A2 represent the initial value and the final value.
dh dh
From the above, cw was derived from by Eqs. (4) and (5) as:
For the reaction control stage, Eq. (1) becomes:  
(   exp kaz
1
cw ¼ c  ka expðkahÞ þ dcr cw ¼ c i   u f ðhÞ ð6Þ
dh
ð2Þ exp kaz
u
1
dcr
kaðc  cw Þ > dh
As previously discussed, exp(kaz/u) and ci are constants. Eq. (6)
Eq. (2) represents the ozone concentration in water film (cw) as shows cw as a function of f(h). And it is easily found that cw has a
a function of the reaction rate (dcr/dh). It is obvious that cw increas- negative linear relation with f(h). The analysis of Eq. (2) also reveals
es when reaction rate slows down. that cw negatively correlates with the reaction rate for the reaction
The ozonolysis process of the whole bed can be regarded as control stage. As a result, the reaction rate positively correlates
countless differential longitudinal sections that undergo the with f(h). The variation of the reaction rate can be estimated by
above-mentioned process and are stacked in series. -Based on this f(h). Fig. 1 shows the curves of consumed ozone concentration as
presumption, the model was theoretically deduced to describe the a function of ozonolysis time for samples with different moisture
distribution of ozone concentration and consumption along with contents and particle size. All the curves, except the ones with
the bed height. The bed is cylindrical. Its upward axial direction the moisture content of 75%, started at 60 mg/L and remained
was set as z axis, and the axis origin was set at the bottom of the the initial value for a while, which is called the initial stable stage.
bed. The gas flows into the bed from the bottom and out from Afterwards, decline stage started the initial stable stage here
the top. ci and co represents the ozone concentration in inlet flow reveals that the whole bed is under mass transfer control stage.
and outlet flow, respectively. A piece of bed with infinitesimal For samples of the same particle size, the curves at the moisture
thickness was selected as control volume. The thickness is dz. content of 30% had the longest initial stable stage and, then
The process is considered as plug-flow. The flow velocity u in the dropped sharply in decline stage. With the increase of moisture,
z direction is regarded as a constant. Because the flow rate is the length of the initial stable stage reduced, and the curves of
constant and the bed porosity does not change during the process. all the samples declined in a lower rate. Correspondingly, the lower
The cross sectional area of the bed is A. The ozone concentration of rate resulted in a longer reaction time. When moisture content
gas phase in the control volume is c. reached 75%, the curves dropped sharply at the very start without
The ozone mass inflow and outflow rates of the control volume an initial stable stage, then declined much slower than others. At
are Acu and A(cu+(@(cu)/oz)dz), respectively. The ozone accumulat- this moisture content, some of the pores are blocked with water
ed rate in control volume is (@c/@h)Adz. NA, the ozone transfer flux and part of the water films become thick enough. These changes
 C. Li et al. / Bioresource Technology 183 (2015) 240–247 243

Fig. 1. Consumed ozone concentration as a function of reaction time (h) for ozonolysis of samples with different moisture contents and particle sizes. Solid, dash, dot and
dash-dot lines represent the moisture content of 30%, 45%, 60% and 75%, respectively. Samples of different particle size are 20/40 (a), 80/150 (b) and 300 (c).

impede the aqueous ozone from transferring to and reacting with on the particle size. For the same moisture content, the lignin con-
the sample, leaving the reaction for a long time. Instead of reacting tent of the sample under the optimum moisture content declined
with the sample directly, the ozone in bulk water has enough time drastically as particle size went down. The lowest lignin content,
to decompose into hydroxyl radicals, which may lead to a reaction about 3.77%, was obtained under the condition of 300 mesh
pathway that differs from that of the lower moisture treatments and the moisture content of 60%. It is worth mentioning that the
(Hoigné et al., 1985). For samples with the same moisture content, AIL is mainly responsible for the variation of lignin, while ASL
the curves of the samples with smaller particle size show higher shows only slightly changes. Cellulose (as glucan) content does
values, which indicates that smaller particle size leads to higher not show a clear tendency among the samples. Some ozonolysis
reaction rate. Furthermore, these curves show a longer initial samples even have a slightly higher cellulose content than the cor-
stable stage and dropped slower when particle size was smaller. responding raw samples, which may be a result of low bias of
Therefore, size reduction improves not only the rate but also the water extractives due to volatile loss. Overall, it can be concluded
extent of the ozonolysis reaction. ozonolysis caused little loss of cellulose. The effect of ozonolysis
on xylan has the same trend as that of lignin in general, except
3.2. Effect of ozonolysis on sample composition for some small variations. For example, the lignin content of sam-
ples at the moisture content of 75% is relatively higher while the
Compositional analysis was performed on each ozonolysis sam- xylan content is relatively lower in comparison with those of sam-
ples to determine how ozonolysis impacted on the compositions ples at the moisture content of 30% in Fig. 2a and c, respectively. It
(Fig. 2). Main components such as lignin, glucan, xylan and water indicates the ozonolysis preferred to consume more xylan and less
extractives were compared as a function of different moisture con- lignin as the moisture content (above 60%) increased, But it is not
tents and particle sizes. Lignin was determined as two parts: acid proper to conclude that xylan is more vulnerable than lignin. As we
insoluble lignin (AIL) and acid soluble lignin (ASL). Glucan mainly know, Xylan has covalent linkage with lignin. When cleavage
forms as cellulose. Xylan is the major part of hemicellulose. Water occurs on double bonds and aromatic rings in lignin under the
extractives represent all water soluble components in samples. For attack of ozone, the cross-linked structure of xylan attached to
the same particle size, moisture has a remarkable effect on ozono- lignin is also damaged to some extent. Research has shown a
lysis in view of the variation of lignin content. The minimum lignin decreasing molar mass and an improving solubility of xylan after
content was got at the moisture content of 45% or 60% depending ozonation (å et al., 2003). This effect may cause a decrease in xylan
244 C. Li et al. / Bioresource Technology 183 (2015) 240–247

Fig. 2. Composition of the raw and ozonolysis samples. All contents express as dry basis. Lignin content (a) is the sum of AIL and ASL. Cellulose and hemicellulose here are
represented by glucan (b) and xylan (c), respectively. Water extractives (d) is the total amount of all water soluble fractions in samples.

content, since the xylan was determined after water wash. Water For samples with different particle size, the optimum moisture
extractives have an opposite trend with lignin and xylan. Com- contents of ozone consumption were not the same. Specifically, the
pared to raw samples, the sample after ozonolysis has increased optimum moisture content for delignification rate was 60% for
degradation products from lignin and xylan in the water extrac- 300 samples and 45% for others. This fact indicates that moisture
tives, probably including some solubilized xylan. and particle size have an interactive effect on ozonolysis. For sam-
To clearly reveal the relation between the extent of ozonolysis ples of different particle sizes but at the same moisture content,
and lignin/xylan variation, three parameters were defined. The first the state of water may be different. The water in samples can be
one is ozone consumption, which is calculated by dividing total divided into two groups: the bound water and free water (Pallatt
ozone consumption (obtained through integration of curves in Sec- and Thornley, 1990). The bound water refers to these absorbed
tion 3.1) by dry sample mass. The second one is delignification rate, to the solid surface tightly, forming water films with limited thick-
which is the percentage of removed lignin content in original lignin
content. The third one, xylan loss rate, is the percentage of removed
xylan content in original xylan content. Fig. 3 shows similar trend
among these three parameters. The data at the moisture content
of 30% and 75%, reveals the samples at 75% moisture content have
a higher ozone consumption and xylan loss rate, while their delig-
nification rate is the lowest. In other words, at the moisture content
of 75%, the ratio of delignification rate to ozone consumption is the
lowest. Therefore, ozonolysis of 75% samples shows low efficiency
in delignification and causes relatively more xylan loss, it is possible
that ozonolysis under this moisture content has a different
mechanism from others. It has been explained that the ozone
decomposition in water causes the high ozone consumption
(Oyama, 2000). The aqueous ozone decomposes into hydrophilic
hydroxyl radicals which may tend to attack xylan matrix and hardly
penetrates into the hydrophobic area of lignin (Grabber, 2005) Ozo-
nolysis at 75% moisture may have hydroxyl radicals reaction
involved. Therefore, a relative low delignification rate and high
xylan loss rate were observed. On the other hand, as the particle
size decreased, the ratio of delignification rate to ozone consump-
tion increased. It implies that the delignification efficiency gets
higher as particle size gets smaller. Besides, xylan loss rate Fig. 3. The ozone consumption (OC), delignification rate (DLR) and xylan loss rate
(XLR) of ozonolysis samples. Ozone consumption is defined as the micrograms of
increased slower than delignification rate as particle size reduced.
ozone consumed by 1 g dry sample after ozonolysis. Delignification rate is defined
At the optimum condition (300, 60%), the delignification reached as the percentage of reduced lignin content in the original lignin content. Xylan loss
to about 78% and ozone consumption is about 256 mg/g. rate is a similar concept to delignification rate.
 C. Li et al. / Bioresource Technology 183 (2015) 240–247 245

ness. The water which cannot form strong bonds to the solid is It was found that samples with their Aw below the optimum
regarded as free water. The amount of bound water depends on point (about 0.775) had a good linearity between their OC and
the specific surface area of the particles if the water is sufficient. LRA or XRA. However, when Aw was above the optimum point,
The larger the specific surface area is, the more water can be tightly there was no clear correlation (data was not given). Fig. 4c shows
adsorbed on the surface to form water films, resulting in more the linear fitting for the correlation of OC with LRA and XRA
bound water and less free water. Water activity (Aw) is used to when Aw is under 0.775. The R2 of OC-LRA and OC-XRA linear fit-
measure the ratio of free and bound water in samples, reflecting ting are 0.9503 and 0.9478, respectively. High linearity indicates
the water state quantitatively. Fig. 4 shows the relation of ozone that most ozone was consumed in the degradation of lignin and
consumption with moisture content (a) and Aw (b). The points of xylan.
each particle size were connected with smooth curves. And the
peaks of these curves were marked out with vertical dash lines. 3.3. The effect of ozonolysis on enzymatic hydrolysis
Fig. 4a shows the peak of 20/40 is at about 48% moisture content,
different from that of the 80/150 and 300. In Fig. 5b, all Aw peaks Enzymatic hydrolysis was performed on all ozonolysis samples,
lie in the small range of 0.77–0.78. In other words, despite the dif- before and after water washing, raw samples and complete delig-
ferent moisture contents and particle sizes, the optimum level of nified samples (CDL) of the three particle sizes (20/40, 80/150
the free and bound water ratio in the sample is very close. This and 300). The hydrolysates were analyzed by HPLC to determine
result also indicates that the effect of water on ozonolysis is highly the amount of glucose and xylose. The glucose or xylose yield was
associated with water state in the sample. calculated as a ratio of the amount of glucan or xylan that hydro-
With the composition results of all the tests, the relation lyzed into monomers to the original amount of glucan or xylan pre-
between ozone consumption (OC) and lignin/xylan variation sented in the samples. CDL was obtained by treating the raw
was investigated. Here, the amount (grams) of lignin and xylan samples with sodium chlorite solution overnight. The treatment
decreased during ozonolysis was used, as lignin removal amount caused complete delignification while the structural carbohydrates
(LRA) and xylan removal amount (XRA), to evaluate their change. were slightly affected. Since these effects improve the enzymatic

Fig. 4. (a) The ozone consumption as a function of moisture content, (b) water activity for different particle sizes and (c) lignin removal amount (LRA) and xylan removal
amount (XRA) as a function of ozone consumption (OC). All data of (c) came from the ozonolysis of samples of different particle size and the moisture content of 30% and 45%.
The R2 of OC-LRA fitting and OC-XRA fitting are 0.9503 and 0.9478, respectively.
246 C. Li et al. / Bioresource Technology 183 (2015) 240–247

Fig. 5. The glucose (a) and xylose (b) yield of the enzymatic hydrolysis on raw, CDL and ozonolysis samples. For ozonolysis samples, hydrolysis was performed before and
after water washing. (c) Glucose yield as a function of delignification rate (DLR).

hydrolysis greatly, CDL is the goal to be achieved through ozonoly- shown in Fig. 5c. The glucose yield increased as DLR increasing
sis in this research. The sugar yields of raw and CDL after hydroly- in a linear manner. All of the three trends have a similar slope,
sis were used as controls. Fig. 5a and b shows the glucose yield and revealing that the relation between glucose yield and DLR is
xylose yield of ozonolysis samples under different moisture con- independent of particle size. To put it in another way, under dif-
tents and particle sizes compared with the raw samples and CDL ferent DLR levels, the particle size reduction improves glucose
in corresponding particle size. Particle size reduction causes slight yield to the same extent. Namely, particle size reduction
increases in both glucose and xylose yield for raw and CDL. Size improves hydrolysis in a small extent, no matter how much
reduction has only a small effect on enzymatic hydrolysis, which the DLR is. But on the other hand, particle size reduction greatly
has been reported several times (Chang et al., 1997; Chang and enhances the extent of ozonolysis reaction by increasing DLR and
Holtzapple, 2000; Chundawat et al., 2006). For samples of 20/40 promoting enzymatic hydrolysis.
and 80/150, the glucose yield reached the maximum at the mois-
ture content of 45%, as high as 40% and 60%, respectively. For
300 samples, the maximum glucose yield, about 80%, was got 4. Conclusions
at the moisture content of 60%. This is similar to the trend of the
delignification rate in Fig. 3. Water washing was used to remove This study shows that both particle size and moisture have sig-
any potential enzyme inhibitor, expecting to improve the sugar nificant effects on the ozonolysis process, specifically on the ozone
yield. Only a few samples showed slight glucose and xylose yield transfer rate and reaction extent. The interactive effect of particle
increase after washing. In most samples, water washing caused size and moisture reveals that the free and bound water ratio is a
obvious decreases in xylose yield. As previously discussed, ozono- key factor of ozonolysis. The optimum water activity and particle
lysis partly improved the solubility of xylan. And the xylan analysis size is about 0.775 and 300, respectively. The removed lignin
procedure was performed on extractive-free samples, the solubi- and xylan both linearly correlate with ozone consumption. The
lized part of xylan was removed through water washing thus not compositional analysis and enzymatic hydrolysis shows that glu-
counted in. Therefore, the decrease of xylose yield after washing cose yield positively correlates with delignification rate in an
may be a result of removal of solubilized xylan. Since there are only almost linear manner.
slightly changes in glucose yield and the xylose yield after wash-
ing, it seems unnecessary to apply water washing step before
Acknowledgements
hydrolysis.
The ozonolysis samples were divided into three groups by
This work was funded by the Fundamental Research Funds for
their particle size. In each groups, the samples got different
the Central Universities (JUSRP51302A) and the Jiangsu Provincial
DLR through ozonolysis under different moisture contents. The
Science and Technology Support Program (BE2012428). We also
relationship between glucose yield and DLR in each group was
thank Dr. Jun Wang for his assistance in the experiments.
 C. Li et al. / Bioresource Technology 183 (2015) 240–247 247

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