Yuanyuan Li,Yelong Zhang ,Hongbo Zheng ,Jian Du ,Hongman Zhang ,*,Juanzi Wu ,He Huang

1 College of Science,Nanjing Tech University,Nanjing 210009,China

2 China State Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 210009,China

3 Institute of Animal Science,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China

Keywords:Energy crop Elephant grass Dilute sulfuric acid pretreatment Enzymatic hydrolysis Glucose

ABSTRACT Five elephant grass cultivars,Pennisetum purpureum.cv.Huanan(Huanan),P.purpureum.cv.N51(N51),P.purpureum.cv.Sumu No.2(Sumu-2),(Pennisetum americanum×P.purpureum)×P.purpureum cv.Guimu No.1(Guimu-1)and P.americanum cv.Tift23A×P.purpureum cv.Tift N51(Hybrid Pennisetum),at three harvest stages were studied.With dilute sulfuric acid pretreatment followed by enzymatic hydrolysis,it is found that cellulose conversion of the five elephant grass cultivars harvested in August and September is higher than that harvested in October.The cellulose conversion for elephant grass cultivars harvested in August and September follows an order of Hybrid Pennisetum＞Sumu-2＞Huanan＞Guimu-1＞N51.This may be explained by the fact that lignification is gradually strengthened with time,inhibiting degradation of cellulose and hemicellulose.Moreover,cellulose conversions of Hybrid Pennisetum,Sumu-2 and Huanan harvested in August and September are higher based on hierarchical clustering results.

1.Introduction

The research on biomass conversion is receiving much attention all over the world as energy shortage and environmental pollution problems become more and more serious[1–3].Bioconversion refers to the release and fermentation of sugars from biomass,in which carbohydrates are converted into sugars and the mixed sugars are further fermented microbially into ethanol or other chemicals.At present,there are two main procedures in sugar conversion process[4],concentrated acid or multi-step dilute acid pretreatment followed by enzymatic hydrolysis.Selig et al.have concluded that the overall effect of biochemical transformation is the best after dilute acid pretreatment for the present[5].

Since the 1980s,perennial grasses have been studied as energy crops.Nowadays,extensive research on perennial grasses is focused on switchgrass,miscanthus,Yi grass,bamboo reed,etc.[6,7].Elephant grass belongs to high-yield perennial grasses of poaceae and pennisetum.Its dry matter biomass production is more than twice that of sugarcane and switchgrass per unit area.Thus,elephant grass is well-recognized as an energy crop.The root of elephant grass is transplanted from greenhouse to outside in April every year when the ambient temperature is above 4°C.After three months of growth,the plant can reach 2 m in height,and cellulose and hemicellulose contents accumulate gradually.In October,when the temperature is below 4°C,the root should be moved into the greenhouse again for survival.Therefore,the optimal harvest time for elephant grass is between August and October,and the annual dry biomass can reach 40–48 t·hm-2[8–12].

Various factors affect the yield and composition of perennial grass biomass,such as cultivar,harvest time,and environmental conditions.It is found that cultivar and harvest time affect biomass yield and composition of switchgrass significantly.With the delay of harvest time,the mass fraction of cellulose,hemicelluloses and lignin increase gradually while sugar yield decreases[13–15].In this paper, five elephant grass cultivars,including Pennisetum purpureum.cv.Huanan(Huanan)[16],P.purpureum.cv.N51(N51)[17],Sumu No.2(Sumu-2)[18,19],(Pennisetum americanum×P.purpureum)×P.purpureum cv.Guimu No.1(Guimu-1)[20],and P.americanum cv.Tift23A×P.purpureum cv.Tift N51(Hybrid Pennisetum)[21],are studied and the contents of cellulose,hemicellulose and Klason lignin are determined by National Renewable Energy Laboratory(NREL)method.In addition,sugar yield is investigated under the same pretreatment and enzymatic hydrolysis conditions to screen out the cultivar(s)with higher cellulose conversion to provide theoretical foundation for further study.

2.Methods

2.1.Feedstock materials

Samples of five elephantgrass cultivars,Huanan(HN),N51,Sumu-2(SM),Guimu-1(GM)and Hybrid Pennisetum(HP),were obtained from Jiangsu Academy of Agricultural Sciences,Nanjing,China.They were planted in May 2011 and harvested in August,September or October in the same year.These elephant grass cultivars were dried at 105°C for 12 h and milled until the samples passed a standard 2 mm sieve[24].The samples were kept in the dryer at room temperature before composition analysis and pretreatment.

Elephant grass cultivars evaluated in this study are given in Table 1 with a description of their origins.

2.2.Pretreatment of elephant grass with dilute H2SO4

Milled elephant grass was soaked in 1%(by mass)dilute sulfuric acid solution at room temperature with a solid loading of 10%(by mass).The presoaked biomass slurry was transferred to four Hastelloy C-276 batch tube reactors(5.1 cm×11 cm,total volume 50 ml each,sealed with Swagelok fittings and Teflon gaskets at both ends)and pretreated in an oilbath(HH-S,Jiangsu,China)at140°C for20 min.After the reaction was completed,the reactors were quenched in ice bath until the temperature dropped below 100°C.The slurry was vacuum filtered immediately through a glass fiber filter with the temperature kept above 60°C.The filtrate was collected with a 40 ml centrifuge tube and stored at-80°C for the analysis of monosaccharides.The solid residue was washed with deionized water until the filtrate pH was above 6,and stored at 4°C for composition analysis and enzymatic hydrolysis[22].

2.3.Enzymatic hydrolysis

An enzyme system,Cellic CTec2,purchased from Novozymes(Beijing,China),was used for enzymatic hydrolysis experiments.By the filter paper assay[23],the activity of this enzyme was measured and the value was 90 FPU·ml-1.The enzyme loading was 7 FPU·(g dry matter)-1in the hydrolytic reaction and the pH value of the substrate was adjusted to 4.8 before enzyme addition with 50 mmol·L-1acetate buffer.Then the reaction mixture(70 ml)of enzymatic hydrolysis was taken into flasks and incubated at50 °C on a rotary shaker at100 r·min-1.Aliquots of 0.5 ml were sampled at 12,24,36,48,60 and 72 h and centrifuged at 12,000 r·min-1for 5 min.Finally,the supernatant was filtered through a 0.22 μm membrane and stored at-4 °C for further analysis.

2.4.Analytical procedure

The composition of elephant grass was determined by NREL standard laboratory analytical procedures[24],and components including cellulose,hemicellulose,Klason lignin,other extractives and ash were analyzed.Concentrations of monosaccharides were measured by HPLC(Shimazu,LC-20A)equipped with a refractive index detector.A Bio-Rad Aminex HPX-87H column was used for quantification of concentrations of sugars.The mobile phase of the column was 0.05 mol·L-1H2SO4at a flow rate of 0.6 ml·min-1and the column temperature was 65°C[25].

2.5.Statistical analysis

All treatments in this study were conducted in triplicate under the same conditions,with their average values reported.The squared Euclidean distance was used for distance measure and transform measure was rescaled to 0–1 range in cluster analysis.Based on the treatments,the similarity matrix was obtained and hierarchical clustering was performed with SPSS 18.0 software.

3.Results and Discussion

3.1.Composition of elephant grass cultivars and harvests

Five elephant grass cultivars harvested at three stages of growth were studied.Their total contents of cellulose,hemicellulose and Klason lignin were above 60%(dry mass)(Tables 2–4).No significant difference is found for chemical compositions among the five cultivars harvested at the same stage,while the contents of cellulose and hemicellulose increase gradually and the Klason lignin content increases dramatically with the delay of harvest time.It may be explained by the fact that the lignification is enhanced with time.Similar results have been observed by Xie et al.[26–28].

Table 2 Composition of elephant grass cultivars harvested in August

Table 3 Composition of elephant grass cultivars harvested in September

Table 4 Composition of elephant grass cultivars harvested in October

3.2.Dilute acid pretreatment and enzymatic hydrolysis of elephant grass cultivars

3.2.1.Sugar concentrations of prehydrolyzate

The hemicellulose in the elephant grasses is mostly degraded and mainly turns to xylose after dilute sulfuric acid pretreatment.Fig.1 shows that the prehydrolyzate contains glucose and xylose with xylose yield much higher than that of glucose,indicating slight cellulose degradation in the pretreatment process.With the delay of harvest time,the yields of xylose and glucose in the prehydrolyzate decrease.

Fig.1.Yields of glucose and xylose in the prehydrolyzate of elephant grass cultivars at three harvest stages.A—August;S—September;O—October.

3.2.2.Chemical compositions of pretreated solid

Tables 2–4 and Fig.2 show that hemicellulose content in the elephant grasses decreases from about 18%to less than 7%after pretreatment while the contents of cellulose and lignin increase,with a high cellulose content of more than 50%.Fig.3 shows that there is no difference in crystalline and non-crystalline regions after dilute sulfuric acid pretreatment but the shape changes from a long strip crystal to flocculent structure.A lot of irregular holes are on the surface of pretreated elephant grasses,showing loose texture.For pretreated HP,irregular holes are more for the sample harvested in August than that harvested in September and October,indicating that HP harvested in August has better enzyme accessibility.Similar results were obtained for samples of other four elephant grass cultivars(data not shown).Harvest time has more impact on the performance than elephant grass variety.

The above results could be explained by the fact that the elephant grasses harvested earlier are less lignified,leading to better hemicellulose degradation.With the delay of harvest time,the lignification may increase lignin–carbohydrate complex gradually,preventing hemicellulose degradation in the pretreatment process[29].

3.2.3.Enzymatic hydrolysis of pretreated solid

Fig.4 shows that pretreated elephant grasses are enzymatically hydrolyzed.For the same elephant grass cultivar,cellulose conversion for samples harvested in August and September is much higher than that harvested in October.According to literature[30,31],syringyl(S)unit and guaiacyl(G)unit are the two important monolignols incorporated into the lignin polymer,and the ratio S/G has a different influence on sugar yield for different samples.For elephant grass cultivars harvested at the same time,the order of cellulose conversion is HP＞SM＞HN＞GM＞N51.Tables2–4 and Fig.4 also show that lignin content of the elephant grasses increases with harvest time,reducing the accessibility of cellulase enzyme and cellulose conversion.Weng et al.[32]and Xu et al.[33]had similar conclusions when using other biomass to prepare cellulosic ethanol.

3.3.Cluster analysis

Tables 5 and 6 and Figs.5 and 6 are similarity matrix based on squared Euclidean distance and hierarchical clustering dendrogram based on cellulose conversion for different elephant grass cultivars harvested at different time.HP,SM and HN have a close squared Euclidean distance to each other.The squared Euclidean distances between HN and SM,HN and HP are 0.022 and 0.053,respectively.The squared Euclidean distance between HP and SM is 0.00.According to hierarchical clustering(Fig.5),the five cultivars are divided into two groups.HP and SM are categorized into one group and connected with HN,while GM and N51 are grouped together.In addition,the squared Euclidean distance between harvest stages August and September is 0.00,so there is no significant difference in the cellulose conversion of these samples.Therefore,the harvest time is also divided into two groups,August and September,and October only(Fig.6).The cellulose conversions of HP,SM and HN harvested in August and September are higher,consistent with the results with pretreatment.

Fig.2.Compositions of elephant grass cultivars at three harvest stages after pretreatment with 1%H2SO4 at 150°C for 20 min.A—August;S—September;O—October.

Fig.3.Scanning electron microscope images of the pretreated HP.(a)August;(b)September;(c)October.

Fig.4.Enzymatic hydrolysis ofHP(a),GM(b),N51(c),SM(d),and HN(e)after pretreatment.Enzymatic hydrolysis:50 °C,100 r·min-1,50 mmol·L-1 acetate buffer(pH 4.8),20 FPU·g-1 cellulose.

Table 5 Similarity matrix based on squared Euclidean distance for the five elephant grass cultivars with transform measure rescaled to 0–1 range

Table 6 Similarity matrix based on squared Euclidean distance for the three harvest stages with transform measure rescaled to 0–1 range

4.Conclusions

Five elephant grass cultivars harvested at different times were investigated for their ferment able sugar production potential.After dilute sulfuric acid pretreatment and enzymatic hydrolysis,the cellulose conversion for the elephant grass cultivars harvested at the same time followed an order of HP＞SM＞HN＞GM＞N51.Cellulose conversion for the five elephant grass cultivars harvested in August and September was higher than that harvested in October.

According to the linkage method between groups with hierarchical clustering with SPSS 18.0 software,cellulose conversion of HP,SM and HN harvested in August and September was higher,which is consistent with the pretreatment results.

With the delay of harvest time,the lignification would gradually increase lignin–carbohydrate complex,preventing the degradation of hemicellulose in the pretreatment.Lignification also reduced the accessibility of cellulase enzyme and the cellulose conversion of elephant grasses.

Fig.5.Linkage method between groups with hierarchical clustering dendrogram for the five elephant grass cultivars,using the squared Euclidean distance,with the similarity between cultivars reflected by the branch length to the node as referenced by the base scale.

Fig.6.Linkage method between groups with hierarchical clustering dendrogram for the three harvest stages,using the squared Euclidean distance,with the similarity between harvest stages reflected by the branch length to the node as referenced by the base scale.