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Ozonolysis pretreatment of maize stover: The interactive effect of sample particle size and moisture on ozonolysis process
Accepted Manuscript Ozonolysis Pretreatment of Maize Stover: the Interactive Effect of Sample Particle Size and Moisture on Ozonolysis Process Cheng Li, Li Wang, Zhengxing Chen, Yongfu Li, Ren Wang, Xiaohu luo, Guolin Cai, Yanan Li, Qiusheng Yu, Jian Lu PII: DOI: Reference:
S0960-8524(15)00062-0 http://dx.doi.org/10.1016/j.biortech.2015.01.042 BITE 14474
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
14 November 2014 6 January 2015 9 January 2015
Please cite this article as: Li, C., Wang, L., Chen, Z., Li, Y., Wang, R., luo, X., Cai, G., Li, Y., Yu, Q., Lu, J., Ozonolysis Pretreatment of Maize Stover: the Interactive Effect of Sample Particle Size and Moisture on Ozonolysis Process, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.01.042
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Ozonolysis Pretreatment of Maize Stover: the Interactive Effect of Sample Particle Size and Moisture on Ozonolysis Process Cheng Li†, Li Wang†*, Zhengxing Chen, Yongfu Li, Ren Wang, Xiaohu luo, Guolin Cai, Yanan Li, Qiusheng Yu, Jian Lu State Key Laboratory of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, and Key Laboratory of Carbohydrate Chemistry & Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, People's Republic of China †These authors contributed equally to this work *Corresponding author. Fax: +86-510-85197856; Tel: +86-510-85197856; E-mail address: [email protected]
Abstract Maize stover was ozonolyzed to improve the enzymatic digestibility. The interactive effect of sample particle size and moisture content on ozonolysis was studied. After ozonolysis, both lignin and xylan decreased while cellulose was only slightly affected in all experiments. It was also found that the smaller particle size is better for ozonolysis. The similar water activity of the different optimum moisture contents for ozonolysis
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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 was removed. The glucose yield after enzymatic hydrolysis increased from 18.5% to 80%. Water washing had low impact on glucose yield (less than 10% increases), but significantly reduced xylose yield (up to 42% decreases). The result indicates that ozonolysis leads to xylan solubilization. Key words: Lignocellulosic biomass; Maize stover; Ozonolysis; Enzymatic hydrolysis; Particle size
1. Introduction In recent years, biorefinary technologies have attracted significant attention due to the worldwide growing problems including oil shortage, pollution and excessive population. Unlike the first generation biofuel made of cereals, the second generation biofuel made from lignocellulosic materials has greater environmental benefits and sustainability (Menon & Rao, 2012). Lignocellulosic ethanol represents the most developed route of the second generation biofuel. Producing lignocellulosic ethanol involves three steps: (a) pretreatment, which enhances the enzymatic saccharification of biomass; (b) enzymatic hydrolysis, which converts structural polysccharide into fermentable sugars; and (c) fermentation, which produces ethanol and other products using fermentable sugars (Sarkar et al., 2012).
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Maize is one of the world’s widest planted crops. It has also become the largest crop in China since 2012. Compared to other types of lignocellulosic material, maize stover has low lignification and high carbohydrate content making it an abundant and valuable agricultural residue. However, maize stover is currently underexploited. . In China, only less than 10% of maize stover get in the pathway of high added-value processing while almost 40% is left in the field or burned, causing severe waste and pollution. Other crop straws are dealt with the same way. The Chinese government had taken measures to change this situation. According to the document (State Council, 2008), Opinions on Accelerating the Utilization of Crop Straw, great efforts will be taken to establish efficient straw collection and multi-grade utilization system. This policy will propel the biorefinary industries based on maize stover and other agricultural residues. However, there are several limits that challenge biorefinary industrialization. The most crucial one is the low efficiency of enzymatic hydrolysis of structural polysaccharide. Several factors have been proven to account for this, including particle size, cellulose crystallinity, degree of polymerization, and lignin content (Hendriks & Zeeman, 2009). Among these factors, the lignin content shows the highest impact on biomass degradability. Ding et al. (2012) found that lignin removal enhances binding of enzymes to the cell walls and the extent of degradation. They concluded that the best pretreatments should maximize lignin removal. Various pretreatment technologies have been investigated, including physical, chemical
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and biological approaches (Agbor et al., 2011; Alvira et al., 2010; Galbe & Zacchi, 2012; Hendriks & Zeeman, 2009). Ozonolysis pretreatment is regarded as one of the most promising chemical approaches. Due to its highly selective oxidation of compounds with high-electron-density groups (mainly in lignin), ozonolysis shows high efficiency in delignification and high recovery of carbohydrates. Furthermore, low inhibitor production was also reported (Alvira et al., 2010). Ozonolysis has been tested on many types of biomass, such as wheat straw (Binder et al., 1980; García-Cubero et al., 2009), sugarcane bagasse (Barros et al., 2013; Travaini et al., 2013), and energy grasses (Panneerselvam et al., 2013a; Panneerselvam et al., 2013b). In these studies, lignin removal after ozonolysis improved the enzymatic hydrolysis yield. GarcíaCubero et al. (2009) found that moisture content and biomass type are the two main factors of ozonolysis. They proposed a kinetic model for the ozonation reaction (GarcíaCubero et al., 2012). Travaini et al. (2013) found only xylitol, lactic, formic and acetic acid as oxidation products. Inhibitors like furfural and 5-hydroxymethylfurfural were not detected. Comminution is an essential step before other processes. It aims to increase the specific surface area and reduce the bulk volume. Studies have shown that individual application of comminution in a specific range of meshes hardly improves hydrolysis (Chang et al., 1997; Chang & Holtzapple, 2000; Elshafei et al., 1991). Hence, focus has been shifted to the combination of comminution with other pretreatment methods (Barros et al.,
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2013; Chundawat et al., 2006; Miura et al., 2012; Souza-Correa et al., 2014). Barros et al. (2013) investigated the combination of wet disk milling (WDM) and ozonolysis and found that ozonolysis before WDM results in higher saccharification yield than the reverse and reduces the WDM energy consumption. Souza-Correa et al. (2014) compared the ozonation process on substrates with a range of particle sizes. They found that the smaller particle size led to better ozonation result. In this study, the interactive effect of sample moisture content and particle size on ozonolysis of maize stover was investigated from the perspectives of reaction dynamics, compositional variation and hydrolysis yield. A series of samples with a wide range of particle sizes and moisture contents were ozonolyzed in a fixed bed reactor under fixed conditions. The untreated sample (raw) and completely delignified sample (CDL) were set as blank control and positive control, respectively. To each experimental test, the breakthrough ozone concentration versus reaction time was measured, which revealed a dynamic profile of the ozonolysis reaction. The major components, such as lignin and structural carbohydrates, and enzymatic hydrolysis yield were analyzed to monitor the impact of ozone oxidation on samples. Water activity (Aw) of each experimental test was determined to evaluate the free and bound water ratio. 2. Materials and methods 2.1 Raw material Maize stover was collected in Liaoning province, China. It was sundried and stored at 4
5
o
C. The leaves, ears and rotten parts were removed then grinded with a laboratory
impact mill. The grinded material was sieved with a set of standard sieves: 20, 40, 80, 150, 300 mesh. Thus five fractions obtained from the sieves were named as 20/40, 40/80, 80/150, 150/300 and -300, respectively. The 20/40 refers to the fraction from between 20 mesh and 40 mesh sieves, and so on for the other fractions. Among the five fractions, 20/40, 80/150 and -300 was used as raw sample in this study. The major components of these three fractions were determined (Fig. 2), shown similar value to each other. 2.2 Complete delignified samples (CDL) Complete delignified samples (CDL) were prepared using 20/40, 80/150 and -300 raw samples. Acid chlorite was used to remove lignin in the raw samples (Ding et al., 2012). The acid chlorite solution contains 0.1 N HCl and 10% NaClO2. Raw samples were added in the 1% (w/v) solution under magnetic stirred at room temperature for 12 h. The mixture was neutralized and vacuum filtrated. The solid residue was washed and freeze dried to obtain the CDL. 2.3 Ozonolysis treatment The ozonolysis reaction was performed in a glass column reactor (6 cm in diameter and 22.5 cm in height) at constant room temperature, 25 oC. The column was vertically placed. The gas flowed from bottom to top. Ozone monitors were used to detect the ozone concentration in gas flow before and after the reactor. Ozone was generated by an
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ozone generator (CF-G-3-20g, QingDao GuoLin industry Co., Ltd) with pure oxygen source. The flow rate and ozone concentration maintained at 1.0 L/min and 60 mg/L during the reaction, respectively. The samples of 20/40, 80/150 and -300 was used in the treatment. Each of the samples was adjusted to the moisture contents of 30%, 45%, 60% and 75% by adding distilled water before ozonolysis. The reactor was loaded with 10 g raw material (dry basis) and the reaction time is 1 h for all tests. 2.4 Compositional analysis The moisture content was measured by a moisture analyzer (Ohaus MB23). The water extractives were determined by gravimetric analysis before and after water wash. After water wash, insoluble components such as cellulose, xylan and lignin were determined following the NREL LAP “Determination of Structural Carbohydrates and Lignin in Biomass” (Sluiter et al., 2012). The sugar analysis was conducted through high performance liquid chromatography (Agilent 1260 infinity) equipped with differential refraction detector (RID). An Aminex HPX-87H (7.8×300 mm, Bio-Rad, USA) column was used at 60 oC with 5 mM H2SO4 as eluent, and the flow rate of eluent is 0.6 ml/min. 2.5 Enzymatic hydrolysis Enzymatic hydrolysis was performed on both raw and pretreated maize stover, with or without washing, to verify the differences of enzymatic degradation. The hydrolysis was
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performed in 20 ml 0.1 M pH 4.8 citrate buffer with 5% biomass (dry basis) suspended 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 Celluclast 1.5 L with the activity of 33 FPU/ml. Another was cellobiase from Jiangsu Ruiyang Biotech CO., LTD, which has an activity of 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 50 oC for 48h with a shaking speed of 120 rpm. After incubation, the mixture was centrifugated and the supernate was collected for sugar analysis by HPLC with the same conditions as described in section 2.2. 2.6 Water activity The water activity of all samples under adjusted moistures before ozonolysis was measured by a water activity meter (GBX Fat-lab). 3. Results and discussion 3.1 Model analysis of ozone concentration in reactor The sample bed for ozonolysis contains certain amount of water, which forms water films on the surface of the sample particles. The gaseous ozone flows through the pore channels while part of the gaseous ozone transfers into the water films through the gaswater interface. Ozone decomposition may occurs in water phase, however, the decomposition rate is very low compared to the direct reaction of ozone with organic matters when pH < 12 (Beltran, 2003). The ozone in the water films is consumed
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instantaneously due to the short diffusion path. Accordingly, the decomposition of aqueous ozone can be neglected (Beltran, 2003). The entire process is considered as two coupled steps: the ozone transfer from gas to water films and the reaction of aqueous ozone with the samples. The transfer flux NA can be expressed as k(c-cw)， where k is the overall mass transfer coefficient and cw is ozone concentration in the water films. The reaction rate of ozone with the sample is ∂cr/∂θ, where cr is the reacted ozone concentration, θ is the reaction time. The variation of ozone concentration in the water films is ∂cw/∂θ. At the beginning of the process, the reaction rate is higher than the transfer rate. No ozone exists in the water films (cw=0). The rate of the process is limited by the rate of ozone transfer through gas-water interface, which is known as the mass transfer control stage. As the process goes on, the reaction rate slows down and replaces the mass transfer rate to become the limitation of the process rate. The process turns into the stage of reaction control stage. The mass equilibrium analysis gives the following equation (Bird et al., 2007):
dcw dcr + = ka (c − cw ) (1) dθ dθ For the reaction control stage, Eqs. (1) becomes: 1 dcr cw = c - ka exp(-kaθ ) + dθ (2) ka (c - c ) > dcr w dθ
Eqs. (2) represents the ozone concentration in water film (cw) as a function of the reaction rate (dcr/dθ). It’s obvious that cw increases when reaction rate slows down. 9
The ozonolysis process of the whole bed can be regarded as countless differential longitudinal sections that undergo the above-mentioned process and are stacked in series. -Based on this presumption, the model was theoretically deduced to describe the distribution of ozone concentration and consumption along with the bed height. The bed is cylindrical. Its upward axial direction was set as z axis, and the axis origin was set at the bottom of the bed. The gas flows into the bed from the bottom and out from the top. ci and co represents the ozone concentration in inlet flow and outlet flow, respectively. A piece of bed with infinitesimal thickness was selected as control volume. The thickness is dz. The process is considered as plug-flow. The flow velocity u in the z direction is regarded as a constant. Because the flow rate is constant and the bed porosity doesn’t change during the process. The cross sectional area of the bed is A. The ozone concentration of gas phase in the control volume is c. The ozone mass inflow and outflow rates of the control volume are Acu and A(cu+(∂(cu)/∂z)dz), respectively. The ozone accumulated rate in control volume is (∂c/∂θ)Adz. NA, the ozone transfer flux from gas into water film, is k(c-cw) as above mentioned. The surface area per unit volume of the bed is a, thus the ozone transfer rate in control volume is aAdzNA. According to the mass equilibrium analysis (Bird et al., 2007), the sum of the ozone outflow rate, the accumulation rate and the gas-water interface transfer rate is equal to the inflow rate. And the expression can be written as below with a boundary condition:
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∂c ∂c ∂θ + u ∂z + ka (c − cw ) = 0 (3) ∀θ > z , z = 0, c = c i u The expression is a first-order linear partial differential equation. After solving it by the characteristics method, the results is: [exp( c=
kaz ) − 1]cw + ci u (4) kaz exp( ) u
Since exp(kaz/u) and ci are constant, the relationship between c and cw is positive linear. The evolution of c then depends on the evolution of cw in the bed. Eqs. (4) describes the evolution of ozone concentration in the gas phase. When z represents the value of the thickness of the bed, c can be replaced by co, which was recorded every 5 min during the process. Consumed ozone concentration (cc) was defined as the instantaneous decrement of ozone concentration for gas flow passing through the bed, mathematically expressed as cc=ci-co. As ci is a constant of 60 mg/L and co was recorded versus time, a set of cc along with reaction time, was recorded for each ozonolysis group as a scatter plot (Fig. 1). Each set of scatter points was fitted with Logistic model (R2 >0.99 for all sets), the resulted fitting curves were used to integrate for total ozone consumption of the ozonolysis, then calculating the ozone consumption per gram of dry sample. The Logistic fitting expression f(θ) is:
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cc = f (θ ) =
A1 − A2 1+ (
θ P ) θ0
+ A2 (5)
Where A1, A2, θ0 and P were all determined through curve fitting. A1 and A2 represent the initial value and the final value. From the above, cw was derived from by Eqs. (4) and (5) as: kaz ) u cw = ci − f (θ ) (6) kaz exp( ) −1 u exp(
As previously discussed, exp(kaz/u) and ci are constants. Eqs. (6) shows cw as a function of f(θ). And it’s easily found that cw has a negative linear relation with f(θ). The analysis of Eqs. (2) also reveals that cw negatively correlates with the reaction rate for the reaction control stage. As a result, the reaction rate positively correlates with f(θ). The variation of the reaction rate can be estimated by f(θ). Fig. 1 shows the curves of consumed ozone concentration as a function of ozonolysis time for samples with different moisture contents and particle size. All the curves, except the ones with the moisture content of 75%, started at 60 mg/L and remained the initial value for a while, which is called the initial stable stage. Afterwards, decline stage started The initial stable stage here reveals that the whole bed is under mass transfer control stage. For samples of the same particle size, the curves at the moisture content of 30% had the longest initial stable stage and, then dropped sharply in decline stage. With the increase of moisture, the length of the initial stable stage reduced, and the curves of all the
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samples declined in a lower rate. Correspondingly, the lower rate resulted in a longer reaction time. When moisture content reached 75%, the curves dropped sharply at the very start without an initial stable stage, then declined much slower than others. At this moisture content, some of the pores are blocked with water and part of the water films become thick enough. These changes impede the aqueous ozone from transferring to and reacting with the sample, leaving the reaction for a long time. Instead of reacting with the sample directly, the ozone in bulk water has enough time to decompose into hydroxyl radicals, which may lead to a reaction pathway that differs from that of the lower moisture treatments (Hoigné et al., 1985). For samples with the same moisture content, the curves of the samples with smaller particle size show higher values, which indicates that smaller particle size leads to higher reaction rate. Furthermore, these curves show a longer initial stable stage and dropped slower when particle size was smaller. Therefore, size reduction improves not only the rate but also the extent of the ozonolysis reaction. 3.2 Effect of ozonolysis on sample composition Compositional analysis was performed on each ozonolysis samples to determine how ozonolysis impacted on the compositions (Fig. 2). Main components such as lignin, glucan, xylan and water extractives were compared as a function of different moisture contents and particle sizes. Lignin was determined as two parts: acid insoluble lignin (AIL) and acid soluble lignin (ASL). Glucan mainly forms as cellulose. Xylan is the
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major part of hemicellulose. Water extractives represent all water soluble components in samples. For the same particle size, moisture has a remarkable effect on ozonolysis in view of the variation of lignin content. The minimum lignin content was got at the moisture content of 45% or 60% depending on the particle size. For the same moisture content, the lignin content of the sample under the optimum moisture content declined drastically as particle size went down. The lowest lignin content, about 3.77%, was obtained under the condition of -300 mesh and the moisture content of 60%. It is worth mentioning that the AIL is mainly responsible for the variation of lignin, while ASL shows only slightly changes. Cellulose (as glucan) content doesn’t show a clear tendency among the samples. Some ozonolysis samples even have a slightly higher cellulose content than the corresponding raw samples, which may be a result of low bias of water extractives due to volatile loss. Overall, it can be concluded ozonolysis caused little loss of cellulose. The effect of ozonolysis on xylan has the same trend as that of lignin in general, except for some small variations. For example, the lignin content of samples at the moisture content of 75% is relatively higher while the xylan content is relatively lower in comparison with those of samples at the moisture content of 30% in Fig. 2a and Fig. 2c, respectively. It indicates the ozonolysis preferred to consume more xylan and less lignin as the moisture content (above 60%) increased, But it’s not proper to conclude that xylan is more vulnerable than lignin. As we know, Xylan has covalent linkage with lignin. When cleavage occurs on double bonds and aromatic rings in lignin
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under the attack of ozone, the cross-linked structure of xylan attached to lignin is also damaged to some extent. Research has shown a decreasing molar mass and an improving solubility of xylan after ozonation (å et al., 2003). This effect may cause a decrease in xylan content, since the xylan was determined after water wash. Water extractives have an opposite trend with lignin and xylan. Compared to raw samples, the sample after ozonolysis has increased degradation products from lignin and xylan in the water extractives, probably including some solubilized xylan. To clearly reveal the relation between the extent of ozonolysis and lignin/xylan variation, three parameters were defined. The first one is ozone consumption, which is calculated by dividing total ozone consumption (obtained through integration of curves in section 3.1) by dry sample mass. The second one is delignification rate, 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 delignification 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
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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) Ozonolysis 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 consumption increased. It implies that the delignification efficiency gets higher as particle size gets smaller. Besides, xylan loss rate increased slower than delignification rate as particle size reduced. At the optimum condition (300, 60%), the delignification reached to about 78% and ozone consumption is about 256 mg/g. For samples with different particle size, the optimum moisture contents of ozone consumption were not the same. Specifically, the optimum moisture content for delignification rate was 60% for -300 samples and 45% for others. This fact indicates that moisture and particle size have an interactive effect on ozonolysis. For samples of different particle sizes but at the same moisture content, the state of water may be different. The water in samples can be divided into two groups: the bound water and free water (Pallatt & Thornley, 1990). The bound water refers to these absorbed to the solid surface tightly, forming water films with limited thickness. The water which can’t form strong bonds to the solid is regarded as free water. The amount of bound water
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depends on the specific surface area of the particles if the water is sufficient. The larger the specific surface area is, the more water can be tightly adsorbed on the surface to form water films, resulting in more bound water and less free water. Water activity (Aw) is used to measure the ratio of free and bound water in samples, reflecting the water state quantitively. Fig. 4 shows the relation of ozone consumption with moisture content (a) and Aw (b). The points of each particle size were connected with smooth curves. And the peaks of these curves were marked out with vertical dash lines. 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 lie in the small range of 0.77 to 0.78. In other words, despite the different moisture contents and particle sizes, the optimum level of the free and bound water ratio in the sample is very close. This result also indicates that the effect of water on ozonolysis is highly associated with water state in the sample. With the composition results of all the tests, the relation between ozone consumption (OC) and lignin/xylan variation was investigated. Here, the amount (grams) of lignin and xylan decreased during ozonolysis was used, as lignin removal amount (LRA) and xylan removal amount (XRA), to evaluate their change. It was found that samples with their Aw below the optimum point (about 0.775) had a good linearity between their OC and LRA or XRA. However, when Aw was above the optimum point, there was no clear correlation (data was not given). Fig. 4c shows the linear fitting for the correlation of OC with LRA and XRA when Aw is under 0.775. The R2 of OC-LRA and OC-XRA
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linear fitting are 0.9503 and 0.9478, respectively. High linearity indicates that most ozone was consumed in the degradation of lignin and xylan. 3.3 The effect of ozonolysis on enzymatic hydrolysis Enzymatic hydrolysis was performed on all ozonolysis samples, before and after water washing, raw samples and complete delignified samples (CDL) of the three particle sizes (20/40, 80/150 and -300). The hydrolysates were analysed by HPLC to determine the amount of glucose and xylose. The glucose or xylose yield was calculated as a ratio of the amount of glucan or xylan that hydrolyzed into monomers to the original amount of glucan or xylan presented in the samples. CDL was obtained by treating the raw samples with sodium chlorite solution overnight. The treatment caused complete delignification while the structural carbohydrates were slightly affected. Since these effects improve the enzymatic hydrolysis greatly, CDL is the goal to be achieved through ozonolysis in this research. The sugar yields of raw and CDL after hydrolysis were used as controls. Fig. 5a and Fig. 5b shows the glucose yield and xylose yield of ozonolysis samples under different moisture contents and particle sizes compared with the raw samples and CDL in corresponding particle size. Particle size reduction causes slight increases in both glucose and xylose yield for raw and CDL. Size reduction has only a small effect on enzymatic hydrolysis, which has been reported several times (Chang et al., 1997; Chang & Holtzapple, 2000; Chundawat et al., 2006). For samples of 20/40 and 80/150, the glucose yield reached the maximum at the moisture content of
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45%, as high as 40% and 60%, respectively. For -300 samples, the maximum glucose yield, about 80%, was got 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 any potential enzyme inhibitor, expecting to improve the sugar yield. Only a few samples showed slight glucose and xylose yield increase after washing. In most samples, water washing caused obvious decreases in xylose yield. As previously discussed, ozonolysis partly improved the solubility of xylan. And the xylan analysis procedure was performed on extractive-free samples, the solubilized part of xylan was removed through water washing thus not counted in. Therefore, the decrease of xylose yield after washing may be a result of removal of solubilized xylan. Since there are only slightly changes in glucose yield and the xylose yield after washing, it seems unnecessary to apply water washing step before hydrolysis. The ozonolysis samples were divided into three groups by their particle size. In each groups, the samples got different DLR through ozonolysis under different moisture contents. The relationship between glucose yield and DLR in each group was shown in Fig. 5c. The glucose yield increased as DLR increasing in a linear manner. All of the three trends have a similar slope, revealing that the relation between glucose yield and DLR is independent of particle size. To put it in another way, under different DLR levels, the particle size reduction improves glucose yield to the same extent. Namely, particle size reduction improves hydrolysis in a small extent, no matter how much the
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DLR is. But on the other hand, particle size reduction greatly enhances the extent of ozonolysis reaction by increasing DLR and promoting enzymatic hydrolysis. 4. Conclusions
This study shows that both particle size and moisture have significant effects on the ozonolysis process, specifically on the ozone transfer rate and reaction extent. The interactive effect of particle size and moisture reveals that the free and bound water ratio is a key factor of ozonolysis. The optimum water activity and particle size is about 0.775 and -300, respectively. The removed lignin and xylan both linearly correlate with ozone consumption. The compositional analysis and enzymatic hydrolysis shows that glucose yield positively correlates with delignification rate in an almost linear manner. Acknowledgements
This work was funded by the Fundamental Research Funds for the Central Universities (JUSRP51302A) and the Jiangsu provincial science and technology support program (BE2012428). We also thank Dr. Jun Wang for his assistance in the experiments.
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18. Hoigné, J., Bader, H., Haag, W.R., Staehelin, J. 1985. Rate constants of reactions of ozone with organic and inorganic compounds in water—III. Inorganic compounds and radicals. Water Research, 19(8), 993-1004. 19. Menon, V., Rao, M. 2012. Trends in bioconversion of lignocellulose: Biofuels, platform chemicals & biorefinery concept. Progress in Energy and Combustion Science, 38(4), 522-550. 20. Miura, T., Lee, S.-H., Inoue, S., Endo, T. 2012. Combined pretreatment using ozonolysis and wet-disk milling to improve enzymatic saccharification of Japanese cedar. Bioresource Technology, 126, 182-186. 21. Oyama, S.T. 2000. Chemical and catalytic properties of ozone. Catalysis ReviewsScience and Engineering, 42(3), 279-322. 22. Pallatt, N., Thornley, D. 1990. The role of bound water and capillary water in the evaluation of porosity in reservoir rocks. Advances in Core Evaluation: Accuracy and Precision in Reserves Estimation, 21-23. 23. Panneerselvam, A., Sharma-Shivappa, R.R., Kolar, P., Clare, D.A., Ranney, T. 2013a. Hydrolysis of ozone pretreated energy grasses for optimal fermentable sugar production. Bioresource Technology, 148, 97-104. 24. Panneerselvam, A., Sharma-Shivappa, R.R., Kolar, P., Ranney, T., Peretti, S. 2013b. Potential of ozonolysis as a pretreatment for energy grasses. Bioresource Technology, 148, 242-248.
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25. Sarkar, N., Ghosh, S.K., Bannerjee, S., Aikat, K. 2012. Bioethanol production from agricultural wastes: An overview. Renewable Energy, 37(1), 19-27. 26. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D. 2012. Determination of Structural Carbohydrates and Lignin in Biomass. Laboratory analytical procedure. 27. Souza-Correa, J.A., Oliveira, C., Nascimento, V.M., Wolf, L.D., Gomez, E.O., Rocha, G.J.M., Amorim, J. 2014. Atmospheric Pressure Plasma Pretreatment of Sugarcane Bagasse: the Influence of Biomass Particle Size in the Ozonation Process. Applied Biochemistry and Biotechnology, 172(3), 1663-1672. 28. State Council. 2008. Opinions on Accelerating the Utilization of Crop Straw. Guobanfa No. 105 [2008] 29. Travaini, R., Otero, M.D.M., Coca, M., Da-Silva, R., Bolado, S. 2013. Sugarcane bagasse ozonolysis pretreatment: Effect on enzymatic digestibility and inhibitory compound formation. Bioresource Technology, 133, 332-339.
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Figure captions Fig. 1 Consumed ozone concentration as a function of reaction time (θ) for ozonolysis
of samples with different moisture contents and particle sizes. Solid, dash, dot and dashdot 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). 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. 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 ozone consumed by 1 g dry sample after ozonolysis. Delignification rate is defined as the percentage of reduced lignin content in the original lignin content. Xylan loss rate is a similar concept to delignification rate. 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.
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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).
26
Figures
Fig. 1 Consumed ozone concentration as a function of reaction time ( ) for or oozonolysis of samples with different moisture contents and and particle sizes. Solid, dash, dot and dashdot 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).
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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.
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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 ozone consumed by 1 g dry sample after ozonolysis. Delignification rate is defined as the percentage of reduced lignin content in the original lignin content. Xylan loss rate is a similar concept to delignification rate.
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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.
30
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).
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Highlights 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.
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S0960-8524(15)00062-0 http://dx.doi.org/10.1016/j.biortech.2015.01.042 BITE 14474
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
14 November 2014 6 January 2015 9 January 2015
Please cite this article as: Li, C., Wang, L., Chen, Z., Li, Y., Wang, R., luo, X., Cai, G., Li, Y., Yu, Q., Lu, J., Ozonolysis Pretreatment of Maize Stover: the Interactive Effect of Sample Particle Size and Moisture on Ozonolysis Process, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.01.042
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Ozonolysis Pretreatment of Maize Stover: the Interactive Effect of Sample Particle Size and Moisture on Ozonolysis Process Cheng Li†, Li Wang†*, Zhengxing Chen, Yongfu Li, Ren Wang, Xiaohu luo, Guolin Cai, Yanan Li, Qiusheng Yu, Jian Lu State Key Laboratory of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, and Key Laboratory of Carbohydrate Chemistry & Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, People's Republic of China †These authors contributed equally to this work *Corresponding author. Fax: +86-510-85197856; Tel: +86-510-85197856; E-mail address: [email protected]
Abstract Maize stover was ozonolyzed to improve the enzymatic digestibility. The interactive effect of sample particle size and moisture content on ozonolysis was studied. After ozonolysis, both lignin and xylan decreased while cellulose was only slightly affected in all experiments. It was also found that the smaller particle size is better for ozonolysis. The similar water activity of the different optimum moisture contents for ozonolysis
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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 was removed. The glucose yield after enzymatic hydrolysis increased from 18.5% to 80%. Water washing had low impact on glucose yield (less than 10% increases), but significantly reduced xylose yield (up to 42% decreases). The result indicates that ozonolysis leads to xylan solubilization. Key words: Lignocellulosic biomass; Maize stover; Ozonolysis; Enzymatic hydrolysis; Particle size
1. Introduction In recent years, biorefinary technologies have attracted significant attention due to the worldwide growing problems including oil shortage, pollution and excessive population. Unlike the first generation biofuel made of cereals, the second generation biofuel made from lignocellulosic materials has greater environmental benefits and sustainability (Menon & Rao, 2012). Lignocellulosic ethanol represents the most developed route of the second generation biofuel. Producing lignocellulosic ethanol involves three steps: (a) pretreatment, which enhances the enzymatic saccharification of biomass; (b) enzymatic hydrolysis, which converts structural polysccharide into fermentable sugars; and (c) fermentation, which produces ethanol and other products using fermentable sugars (Sarkar et al., 2012).
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Maize is one of the world’s widest planted crops. It has also become the largest crop in China since 2012. Compared to other types of lignocellulosic material, maize stover has low lignification and high carbohydrate content making it an abundant and valuable agricultural residue. However, maize stover is currently underexploited. . In China, only less than 10% of maize stover get in the pathway of high added-value processing while almost 40% is left in the field or burned, causing severe waste and pollution. Other crop straws are dealt with the same way. The Chinese government had taken measures to change this situation. According to the document (State Council, 2008), Opinions on Accelerating the Utilization of Crop Straw, great efforts will be taken to establish efficient straw collection and multi-grade utilization system. This policy will propel the biorefinary industries based on maize stover and other agricultural residues. However, there are several limits that challenge biorefinary industrialization. The most crucial one is the low efficiency of enzymatic hydrolysis of structural polysaccharide. Several factors have been proven to account for this, including particle size, cellulose crystallinity, degree of polymerization, and lignin content (Hendriks & Zeeman, 2009). Among these factors, the lignin content shows the highest impact on biomass degradability. Ding et al. (2012) found that lignin removal enhances binding of enzymes to the cell walls and the extent of degradation. They concluded that the best pretreatments should maximize lignin removal. Various pretreatment technologies have been investigated, including physical, chemical
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and biological approaches (Agbor et al., 2011; Alvira et al., 2010; Galbe & Zacchi, 2012; Hendriks & Zeeman, 2009). Ozonolysis pretreatment is regarded as one of the most promising chemical approaches. Due to its highly selective oxidation of compounds with high-electron-density groups (mainly in lignin), ozonolysis shows high efficiency in delignification and high recovery of carbohydrates. Furthermore, low inhibitor production was also reported (Alvira et al., 2010). Ozonolysis has been tested on many types of biomass, such as wheat straw (Binder et al., 1980; García-Cubero et al., 2009), sugarcane bagasse (Barros et al., 2013; Travaini et al., 2013), and energy grasses (Panneerselvam et al., 2013a; Panneerselvam et al., 2013b). In these studies, lignin removal after ozonolysis improved the enzymatic hydrolysis yield. GarcíaCubero et al. (2009) found that moisture content and biomass type are the two main factors of ozonolysis. They proposed a kinetic model for the ozonation reaction (GarcíaCubero et al., 2012). Travaini et al. (2013) found only xylitol, lactic, formic and acetic acid as oxidation products. Inhibitors like furfural and 5-hydroxymethylfurfural were not detected. Comminution is an essential step before other processes. It aims to increase the specific surface area and reduce the bulk volume. Studies have shown that individual application of comminution in a specific range of meshes hardly improves hydrolysis (Chang et al., 1997; Chang & Holtzapple, 2000; Elshafei et al., 1991). Hence, focus has been shifted to the combination of comminution with other pretreatment methods (Barros et al.,
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2013; Chundawat et al., 2006; Miura et al., 2012; Souza-Correa et al., 2014). Barros et al. (2013) investigated the combination of wet disk milling (WDM) and ozonolysis and found that ozonolysis before WDM results in higher saccharification yield than the reverse and reduces the WDM energy consumption. Souza-Correa et al. (2014) compared the ozonation process on substrates with a range of particle sizes. They found that the smaller particle size led to better ozonation result. In this study, the interactive effect of sample moisture content and particle size on ozonolysis of maize stover was investigated from the perspectives of reaction dynamics, compositional variation and hydrolysis yield. A series of samples with a wide range of particle sizes and moisture contents were ozonolyzed in a fixed bed reactor under fixed conditions. The untreated sample (raw) and completely delignified sample (CDL) were set as blank control and positive control, respectively. To each experimental test, the breakthrough ozone concentration versus reaction time was measured, which revealed a dynamic profile of the ozonolysis reaction. The major components, such as lignin and structural carbohydrates, and enzymatic hydrolysis yield were analyzed to monitor the impact of ozone oxidation on samples. Water activity (Aw) of each experimental test was determined to evaluate the free and bound water ratio. 2. Materials and methods 2.1 Raw material Maize stover was collected in Liaoning province, China. It was sundried and stored at 4
5
o
C. The leaves, ears and rotten parts were removed then grinded with a laboratory
impact mill. The grinded material was sieved with a set of standard sieves: 20, 40, 80, 150, 300 mesh. Thus five fractions obtained from the sieves were named as 20/40, 40/80, 80/150, 150/300 and -300, respectively. The 20/40 refers to the fraction from between 20 mesh and 40 mesh sieves, and so on for the other fractions. Among the five fractions, 20/40, 80/150 and -300 was used as raw sample in this study. The major components of these three fractions were determined (Fig. 2), shown similar value to each other. 2.2 Complete delignified samples (CDL) Complete delignified samples (CDL) were prepared using 20/40, 80/150 and -300 raw samples. Acid chlorite was used to remove lignin in the raw samples (Ding et al., 2012). The acid chlorite solution contains 0.1 N HCl and 10% NaClO2. Raw samples were added in the 1% (w/v) solution under magnetic stirred at room temperature for 12 h. The mixture was neutralized and vacuum filtrated. The solid residue was washed and freeze dried to obtain the CDL. 2.3 Ozonolysis treatment The ozonolysis reaction was performed in a glass column reactor (6 cm in diameter and 22.5 cm in height) at constant room temperature, 25 oC. The column was vertically placed. The gas flowed from bottom to top. Ozone monitors were used to detect the ozone concentration in gas flow before and after the reactor. Ozone was generated by an
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ozone generator (CF-G-3-20g, QingDao GuoLin industry Co., Ltd) with pure oxygen source. The flow rate and ozone concentration maintained at 1.0 L/min and 60 mg/L during the reaction, respectively. The samples of 20/40, 80/150 and -300 was used in the treatment. Each of the samples was adjusted to the moisture contents of 30%, 45%, 60% and 75% by adding distilled water before ozonolysis. The reactor was loaded with 10 g raw material (dry basis) and the reaction time is 1 h for all tests. 2.4 Compositional analysis The moisture content was measured by a moisture analyzer (Ohaus MB23). The water extractives were determined by gravimetric analysis before and after water wash. After water wash, insoluble components such as cellulose, xylan and lignin were determined following the NREL LAP “Determination of Structural Carbohydrates and Lignin in Biomass” (Sluiter et al., 2012). The sugar analysis was conducted through high performance liquid chromatography (Agilent 1260 infinity) equipped with differential refraction detector (RID). An Aminex HPX-87H (7.8×300 mm, Bio-Rad, USA) column was used at 60 oC with 5 mM H2SO4 as eluent, and the flow rate of eluent is 0.6 ml/min. 2.5 Enzymatic hydrolysis Enzymatic hydrolysis was performed on both raw and pretreated maize stover, with or without washing, to verify the differences of enzymatic degradation. The hydrolysis was
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performed in 20 ml 0.1 M pH 4.8 citrate buffer with 5% biomass (dry basis) suspended 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 Celluclast 1.5 L with the activity of 33 FPU/ml. Another was cellobiase from Jiangsu Ruiyang Biotech CO., LTD, which has an activity of 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 50 oC for 48h with a shaking speed of 120 rpm. After incubation, the mixture was centrifugated and the supernate was collected for sugar analysis by HPLC with the same conditions as described in section 2.2. 2.6 Water activity The water activity of all samples under adjusted moistures before ozonolysis was measured by a water activity meter (GBX Fat-lab). 3. Results and discussion 3.1 Model analysis of ozone concentration in reactor The sample bed for ozonolysis contains certain amount of water, which forms water films on the surface of the sample particles. The gaseous ozone flows through the pore channels while part of the gaseous ozone transfers into the water films through the gaswater interface. Ozone decomposition may occurs in water phase, however, the decomposition rate is very low compared to the direct reaction of ozone with organic matters when pH < 12 (Beltran, 2003). The ozone in the water films is consumed
8
instantaneously due to the short diffusion path. Accordingly, the decomposition of aqueous ozone can be neglected (Beltran, 2003). The entire process is considered as two coupled steps: the ozone transfer from gas to water films and the reaction of aqueous ozone with the samples. The transfer flux NA can be expressed as k(c-cw)， where k is the overall mass transfer coefficient and cw is ozone concentration in the water films. The reaction rate of ozone with the sample is ∂cr/∂θ, where cr is the reacted ozone concentration, θ is the reaction time. The variation of ozone concentration in the water films is ∂cw/∂θ. At the beginning of the process, the reaction rate is higher than the transfer rate. No ozone exists in the water films (cw=0). The rate of the process is limited by the rate of ozone transfer through gas-water interface, which is known as the mass transfer control stage. As the process goes on, the reaction rate slows down and replaces the mass transfer rate to become the limitation of the process rate. The process turns into the stage of reaction control stage. The mass equilibrium analysis gives the following equation (Bird et al., 2007):
dcw dcr + = ka (c − cw ) (1) dθ dθ For the reaction control stage, Eqs. (1) becomes: 1 dcr cw = c - ka exp(-kaθ ) + dθ (2) ka (c - c ) > dcr w dθ
Eqs. (2) represents the ozone concentration in water film (cw) as a function of the reaction rate (dcr/dθ). It’s obvious that cw increases when reaction rate slows down. 9
The ozonolysis process of the whole bed can be regarded as countless differential longitudinal sections that undergo the above-mentioned process and are stacked in series. -Based on this presumption, the model was theoretically deduced to describe the distribution of ozone concentration and consumption along with the bed height. The bed is cylindrical. Its upward axial direction was set as z axis, and the axis origin was set at the bottom of the bed. The gas flows into the bed from the bottom and out from the top. ci and co represents the ozone concentration in inlet flow and outlet flow, respectively. A piece of bed with infinitesimal thickness was selected as control volume. The thickness is dz. The process is considered as plug-flow. The flow velocity u in the z direction is regarded as a constant. Because the flow rate is constant and the bed porosity doesn’t change during the process. The cross sectional area of the bed is A. The ozone concentration of gas phase in the control volume is c. The ozone mass inflow and outflow rates of the control volume are Acu and A(cu+(∂(cu)/∂z)dz), respectively. The ozone accumulated rate in control volume is (∂c/∂θ)Adz. NA, the ozone transfer flux from gas into water film, is k(c-cw) as above mentioned. The surface area per unit volume of the bed is a, thus the ozone transfer rate in control volume is aAdzNA. According to the mass equilibrium analysis (Bird et al., 2007), the sum of the ozone outflow rate, the accumulation rate and the gas-water interface transfer rate is equal to the inflow rate. And the expression can be written as below with a boundary condition:
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∂c ∂c ∂θ + u ∂z + ka (c − cw ) = 0 (3) ∀θ > z , z = 0, c = c i u The expression is a first-order linear partial differential equation. After solving it by the characteristics method, the results is: [exp( c=
kaz ) − 1]cw + ci u (4) kaz exp( ) u
Since exp(kaz/u) and ci are constant, the relationship between c and cw is positive linear. The evolution of c then depends on the evolution of cw in the bed. Eqs. (4) describes the evolution of ozone concentration in the gas phase. When z represents the value of the thickness of the bed, c can be replaced by co, which was recorded every 5 min during the process. Consumed ozone concentration (cc) was defined as the instantaneous decrement of ozone concentration for gas flow passing through the bed, mathematically expressed as cc=ci-co. As ci is a constant of 60 mg/L and co was recorded versus time, a set of cc along with reaction time, was recorded for each ozonolysis group as a scatter plot (Fig. 1). Each set of scatter points was fitted with Logistic model (R2 >0.99 for all sets), the resulted fitting curves were used to integrate for total ozone consumption of the ozonolysis, then calculating the ozone consumption per gram of dry sample. The Logistic fitting expression f(θ) is:
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cc = f (θ ) =
A1 − A2 1+ (
θ P ) θ0
+ A2 (5)
Where A1, A2, θ0 and P were all determined through curve fitting. A1 and A2 represent the initial value and the final value. From the above, cw was derived from by Eqs. (4) and (5) as: kaz ) u cw = ci − f (θ ) (6) kaz exp( ) −1 u exp(
As previously discussed, exp(kaz/u) and ci are constants. Eqs. (6) shows cw as a function of f(θ). And it’s easily found that cw has a negative linear relation with f(θ). The analysis of Eqs. (2) also reveals that cw negatively correlates with the reaction rate for the reaction control stage. As a result, the reaction rate positively correlates with f(θ). The variation of the reaction rate can be estimated by f(θ). Fig. 1 shows the curves of consumed ozone concentration as a function of ozonolysis time for samples with different moisture contents and particle size. All the curves, except the ones with the moisture content of 75%, started at 60 mg/L and remained the initial value for a while, which is called the initial stable stage. Afterwards, decline stage started The initial stable stage here reveals that the whole bed is under mass transfer control stage. For samples of the same particle size, the curves at the moisture content of 30% had the longest initial stable stage and, then dropped sharply in decline stage. With the increase of moisture, the length of the initial stable stage reduced, and the curves of all the
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samples declined in a lower rate. Correspondingly, the lower rate resulted in a longer reaction time. When moisture content reached 75%, the curves dropped sharply at the very start without an initial stable stage, then declined much slower than others. At this moisture content, some of the pores are blocked with water and part of the water films become thick enough. These changes impede the aqueous ozone from transferring to and reacting with the sample, leaving the reaction for a long time. Instead of reacting with the sample directly, the ozone in bulk water has enough time to decompose into hydroxyl radicals, which may lead to a reaction pathway that differs from that of the lower moisture treatments (Hoigné et al., 1985). For samples with the same moisture content, the curves of the samples with smaller particle size show higher values, which indicates that smaller particle size leads to higher reaction rate. Furthermore, these curves show a longer initial stable stage and dropped slower when particle size was smaller. Therefore, size reduction improves not only the rate but also the extent of the ozonolysis reaction. 3.2 Effect of ozonolysis on sample composition Compositional analysis was performed on each ozonolysis samples to determine how ozonolysis impacted on the compositions (Fig. 2). Main components such as lignin, glucan, xylan and water extractives were compared as a function of different moisture contents and particle sizes. Lignin was determined as two parts: acid insoluble lignin (AIL) and acid soluble lignin (ASL). Glucan mainly forms as cellulose. Xylan is the
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major part of hemicellulose. Water extractives represent all water soluble components in samples. For the same particle size, moisture has a remarkable effect on ozonolysis in view of the variation of lignin content. The minimum lignin content was got at the moisture content of 45% or 60% depending on the particle size. For the same moisture content, the lignin content of the sample under the optimum moisture content declined drastically as particle size went down. The lowest lignin content, about 3.77%, was obtained under the condition of -300 mesh and the moisture content of 60%. It is worth mentioning that the AIL is mainly responsible for the variation of lignin, while ASL shows only slightly changes. Cellulose (as glucan) content doesn’t show a clear tendency among the samples. Some ozonolysis samples even have a slightly higher cellulose content than the corresponding raw samples, which may be a result of low bias of water extractives due to volatile loss. Overall, it can be concluded ozonolysis caused little loss of cellulose. The effect of ozonolysis on xylan has the same trend as that of lignin in general, except for some small variations. For example, the lignin content of samples at the moisture content of 75% is relatively higher while the xylan content is relatively lower in comparison with those of samples at the moisture content of 30% in Fig. 2a and Fig. 2c, respectively. It indicates the ozonolysis preferred to consume more xylan and less lignin as the moisture content (above 60%) increased, But it’s not proper to conclude that xylan is more vulnerable than lignin. As we know, Xylan has covalent linkage with lignin. When cleavage occurs on double bonds and aromatic rings in lignin
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under the attack of ozone, the cross-linked structure of xylan attached to lignin is also damaged to some extent. Research has shown a decreasing molar mass and an improving solubility of xylan after ozonation (å et al., 2003). This effect may cause a decrease in xylan content, since the xylan was determined after water wash. Water extractives have an opposite trend with lignin and xylan. Compared to raw samples, the sample after ozonolysis has increased degradation products from lignin and xylan in the water extractives, probably including some solubilized xylan. To clearly reveal the relation between the extent of ozonolysis and lignin/xylan variation, three parameters were defined. The first one is ozone consumption, which is calculated by dividing total ozone consumption (obtained through integration of curves in section 3.1) by dry sample mass. The second one is delignification rate, 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 delignification 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
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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) Ozonolysis 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 consumption increased. It implies that the delignification efficiency gets higher as particle size gets smaller. Besides, xylan loss rate increased slower than delignification rate as particle size reduced. At the optimum condition (300, 60%), the delignification reached to about 78% and ozone consumption is about 256 mg/g. For samples with different particle size, the optimum moisture contents of ozone consumption were not the same. Specifically, the optimum moisture content for delignification rate was 60% for -300 samples and 45% for others. This fact indicates that moisture and particle size have an interactive effect on ozonolysis. For samples of different particle sizes but at the same moisture content, the state of water may be different. The water in samples can be divided into two groups: the bound water and free water (Pallatt & Thornley, 1990). The bound water refers to these absorbed to the solid surface tightly, forming water films with limited thickness. The water which can’t form strong bonds to the solid is regarded as free water. The amount of bound water
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depends on the specific surface area of the particles if the water is sufficient. The larger the specific surface area is, the more water can be tightly adsorbed on the surface to form water films, resulting in more bound water and less free water. Water activity (Aw) is used to measure the ratio of free and bound water in samples, reflecting the water state quantitively. Fig. 4 shows the relation of ozone consumption with moisture content (a) and Aw (b). The points of each particle size were connected with smooth curves. And the peaks of these curves were marked out with vertical dash lines. 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 lie in the small range of 0.77 to 0.78. In other words, despite the different moisture contents and particle sizes, the optimum level of the free and bound water ratio in the sample is very close. This result also indicates that the effect of water on ozonolysis is highly associated with water state in the sample. With the composition results of all the tests, the relation between ozone consumption (OC) and lignin/xylan variation was investigated. Here, the amount (grams) of lignin and xylan decreased during ozonolysis was used, as lignin removal amount (LRA) and xylan removal amount (XRA), to evaluate their change. It was found that samples with their Aw below the optimum point (about 0.775) had a good linearity between their OC and LRA or XRA. However, when Aw was above the optimum point, there was no clear correlation (data was not given). Fig. 4c shows the linear fitting for the correlation of OC with LRA and XRA when Aw is under 0.775. The R2 of OC-LRA and OC-XRA
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linear fitting are 0.9503 and 0.9478, respectively. High linearity indicates that most ozone was consumed in the degradation of lignin and xylan. 3.3 The effect of ozonolysis on enzymatic hydrolysis Enzymatic hydrolysis was performed on all ozonolysis samples, before and after water washing, raw samples and complete delignified samples (CDL) of the three particle sizes (20/40, 80/150 and -300). The hydrolysates were analysed by HPLC to determine the amount of glucose and xylose. The glucose or xylose yield was calculated as a ratio of the amount of glucan or xylan that hydrolyzed into monomers to the original amount of glucan or xylan presented in the samples. CDL was obtained by treating the raw samples with sodium chlorite solution overnight. The treatment caused complete delignification while the structural carbohydrates were slightly affected. Since these effects improve the enzymatic hydrolysis greatly, CDL is the goal to be achieved through ozonolysis in this research. The sugar yields of raw and CDL after hydrolysis were used as controls. Fig. 5a and Fig. 5b shows the glucose yield and xylose yield of ozonolysis samples under different moisture contents and particle sizes compared with the raw samples and CDL in corresponding particle size. Particle size reduction causes slight increases in both glucose and xylose yield for raw and CDL. Size reduction has only a small effect on enzymatic hydrolysis, which has been reported several times (Chang et al., 1997; Chang & Holtzapple, 2000; Chundawat et al., 2006). For samples of 20/40 and 80/150, the glucose yield reached the maximum at the moisture content of
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45%, as high as 40% and 60%, respectively. For -300 samples, the maximum glucose yield, about 80%, was got 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 any potential enzyme inhibitor, expecting to improve the sugar yield. Only a few samples showed slight glucose and xylose yield increase after washing. In most samples, water washing caused obvious decreases in xylose yield. As previously discussed, ozonolysis partly improved the solubility of xylan. And the xylan analysis procedure was performed on extractive-free samples, the solubilized part of xylan was removed through water washing thus not counted in. Therefore, the decrease of xylose yield after washing may be a result of removal of solubilized xylan. Since there are only slightly changes in glucose yield and the xylose yield after washing, it seems unnecessary to apply water washing step before hydrolysis. The ozonolysis samples were divided into three groups by their particle size. In each groups, the samples got different DLR through ozonolysis under different moisture contents. The relationship between glucose yield and DLR in each group was shown in Fig. 5c. The glucose yield increased as DLR increasing in a linear manner. All of the three trends have a similar slope, revealing that the relation between glucose yield and DLR is independent of particle size. To put it in another way, under different DLR levels, the particle size reduction improves glucose yield to the same extent. Namely, particle size reduction improves hydrolysis in a small extent, no matter how much the
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DLR is. But on the other hand, particle size reduction greatly enhances the extent of ozonolysis reaction by increasing DLR and promoting enzymatic hydrolysis. 4. Conclusions
This study shows that both particle size and moisture have significant effects on the ozonolysis process, specifically on the ozone transfer rate and reaction extent. The interactive effect of particle size and moisture reveals that the free and bound water ratio is a key factor of ozonolysis. The optimum water activity and particle size is about 0.775 and -300, respectively. The removed lignin and xylan both linearly correlate with ozone consumption. The compositional analysis and enzymatic hydrolysis shows that glucose yield positively correlates with delignification rate in an almost linear manner. Acknowledgements
This work was funded by the Fundamental Research Funds for the Central Universities (JUSRP51302A) and the Jiangsu provincial science and technology support program (BE2012428). We also thank Dr. Jun Wang for his assistance in the experiments.
References
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Figure captions Fig. 1 Consumed ozone concentration as a function of reaction time (θ) for ozonolysis
of samples with different moisture contents and particle sizes. Solid, dash, dot and dashdot 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). 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. 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 ozone consumed by 1 g dry sample after ozonolysis. Delignification rate is defined as the percentage of reduced lignin content in the original lignin content. Xylan loss rate is a similar concept to delignification rate. 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.
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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).
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Figures
Fig. 1 Consumed ozone concentration as a function of reaction time ( ) for or oozonolysis of samples with different moisture contents and and particle sizes. Solid, dash, dot and dashdot 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).
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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.
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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 ozone consumed by 1 g dry sample after ozonolysis. Delignification rate is defined as the percentage of reduced lignin content in the original lignin content. Xylan loss rate is a similar concept to delignification rate.
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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.
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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).
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Highlights 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.
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