Huisheng Lü,Jinyi Zhou ,Jiatao Liu ,Chunliu Lü,Feng Lian ,Yonghui Li,*

1 Key Laboratory for Green Chemical Technology of Ministry of Education,R&DCenter for Petrochemical Technology,Tianjin University,Tianjin 300072,China

2 Collaborative Innovation Center of Chemical Science and Engineering,Tianjin 300072,China

Keywords:Waste treatment Biofuel Hydrothermal Cassava anaerobic residue Co-utilization Response surface methodology

ABSTRACT The development of a process that could recover biofuel from industrial cellulose waste can not only reduce the negative environmental impactsby using fossil fuels,but also bring a green idea for the waste's disposing.In this study,hydrothermal pretreatment w as optimized for cassava anaerobic residue,a cellulosic w aste from cassava ethanol industry,to co-utilize xylose and glucose for producing bioethanol.The effect of the main pretreatment conditions,namely,temperature,solid content and time,w as explored for the highest recovery of xylose in prehydrolysate and glucose in enzymatic hydrolysate.The single factor experiment results showed that the conditions for maximum xylose recovery in prehydrolysate and glucose recovery in enzymatic hydrolysate were 60 °C,75 min,10%solids and 160 °C,75 min,10%solids,respectively.Whereafter,response surface methodology(RSM)was applied to further optimize the pretreatment conditions for the maximum theoretical ethanol production through utilizing both xylose and glucose.A treatment at 163°C,for 59 min and w ith 9.5%solids was found optimal,with the highest ethanol production of 20.2 mg·g-1 raw material.Furthermore,in order to assess the impacts of the pretreatment on cassava anaerobic residue,the changes in crystallinity and morphology for untreated and pretreated solids were investigated.

1.Introduction

Bioethanol,an important renewable clean energy source,contributes to reducing the negative environmental impacts which w ere generated by the worldw ide utilization of fossil fuels[1].To date,sugar and starch based materials such as sugarcane and grains are tw o main resources for industrial bioethanol production[2].With the gradual maturity of cellulosic technology[3]and the breakthrough of pentose and hexose co-fermentation strains[4],it has become a reality to use cellulose and hemicellulose in lignocellulosic materials for replacing food crops to produce bioethanol.Lignocellulosic w astes are one of the best choices in the future,w hich possess the virtues of low cost and no pollution[5].Furthermore,the development of a process that could recover chemical resources from biomass w aste is desirable for environmental protection and energy saving.

Cassava serves as one of the three major tuber crops in the w orld,which acts asan important food and biomassenergy crops[6].In industrial processing of cassava,processing 250-300 tons of cassava tubers will generate about 1.6 tons of solid peelsand about 280 tonsof residue with a high moisture content of 85%[7].Currently,the most economical treatment of cassava residue is anaerobic fermentation for biogas;however,there are still plenty of solid anaerobic waste residues after anaerobic fermentation[8].Cassava anaerobic residue lacking of starch and other nutrientsis generally identi fi ed asinferior material[9],w hich is usually discarded as environmentally hazardous land fi ll or burned after pow er-w asting drying.The discarded residue is also a serious concern to the environment.How ever,it contains abundant cellulose and hemicellulose,w hich is primarily hydrolyzed into fermentable sugars,namely xylose and glucose.If the cellulose and hemicellulose can be recovered and used,it w ill not only make a signi fi cant contribution to the protection of the environment but also bring very considerable economic bene fi ts.

Theuse of agricultural organic residuesasraw materialsfor producing ethanol and other chemicals has been widely reported,such as wheat straw[10],corn straw[11],rapeseed straw[12],and olive tree biomass[13].How ever,up to date,there are very few published w orks for coutilization of celluloseand hemicellulosein fermented anaerobic residues.Pretreatment is the key process for maximum use of agricultural organic residuesduringthewholeprocessing.Thisisdueto thecompact structure of cellulose as well as the physical-chemical barrier formed by lignin and hemicelluloses[12,14],which are recalcitrant to chemical and biological decomposition[15].In the past decades,various pretreatment methods have been investigated for lignocellulosematerials,which can besummarized in physical,physico-chemical,chemical and biological methods[11-13,16-19].Among these,hydrothermal reaction hasbeen considered as a cost-effective pretreatment method,because this approach does not require the addition and recovery of any other chemicals besides water,w hich is low-cost,non-toxic and environment-friendly.Speci fi cally,the structureof cassavaanaerobic residuehasbeen partially destroyed during the anaerobic fermentation process,and it makes them more easy to decompose than other agricultural organic residues.Therefore,a relatively mild pretreatment is required to realize a high sugar recovery.Hydrothermal pretreatment can be a promising method for ef fi cient utilization of cassava anaerobic residues.

This work aimed to explore an optimal hydrothermal pretreatment condition for reaching maximum theoretical ethanol production through utilizing both xylose and glucose in cassava anaerobic residue.The main operation variables,that is,temperature,solid content and time,w ere investigated in hydrothermal pretreatment for the maximum xylose yield in prehydrolysate and the highest glucose yield in enzymatic hydrolysis,respectively.In addition,the RSM w as applied to optimize hydrothermal pretreatment for reaching the maximum ethanol production through assuming fermentation of both xylose and glucose.Furthermore,the effect of hydrothermal pretreatment on cassava anaerobic residue w as assessed by SEM and XRD.The cassava anaerobic residue can be used as a probe on behalf of other anaerobic residues for recovering cellulose and hemicellulose.

2.Materials and Methods

2.1.Feedstock

Cassava anaerobic residue w as obtained from the Tianjin Guayue Group Co.,China.On average,the cassava anaerobic residue contained 50.1 mg·g-1cellulose,30.4 mg·g-1hemicellulose,and 180 mg·g-1lignin(dry mass basis).It w as dried at 150°C for 5 h under air atmosphere.Particles in the size ranged from to 250-420μm w ere used in the experiments.The particles w ere dried again in a vacuum drying oven at 105°Cto a constant mass and stored in a desiccator for further use.

2.2.Hydrothermal pretreatment

The hydrothermal pretreatment was carried out in a high pressure reactor(Parr 4843,Parr Instrument Co.).20-60 g cassava anaerobic residue w asmixed w ith 400 ml deionized w ater to maintain a solid-liquid ratio(w/v)of 0.05-0.15,and then packed in to a 1000 ml reaction vessel.The magnetic agitator w as operated at 300 r·min-1,and w hen the mixturewasheated to thedesign temperature(120-240°C),the timers w ere started.Reaction time w as set to 45-60 min.After completion of the reaction,the reactor was rapidly cooled to about 30°C.The schematic diagram of experimental facilities applied in this study w as show n in Fig.1.The liquid fraction w as separated by fi ltration for analyzingcompositions,and the unhydrolyzed solidsweredried and stored in a desiccator for further enzymatic hydrolysis.

2.3.Enzymatic hydrolysis

After hydrothermal pretreatment,the unhydrolyzed solidswere enzymatically hydrolyzed by using NS50013(12.08 mg·(g glucan)-1;Novozymes;Beijing,China)and NS50010(31 mg·(g glucan)-1;Novozymes;Beijing,China)enzyme cocktail.The enzymatic hydrolysis mixture consisted of 1 g unhydrolyzed solids,30 g buffer solution(0.1 mol·L-1acetic acid-sodium acetate,p H=4),and 1 g enzyme cocktail(0.95 g of NS50013 and 0.05 g of NS50010).This mixture w as conducted in a 100 ml Erlenmeyer fl ask,and then all fl asks were incubated in a shaker(ZHWY-2102C)at 50°C w ith a speed of 120 r·min-1for 72 h.The digestibility of enzymatic hydrolysiswascalculated as a reliable indicator for assessing glucose recovery.The percentage of enzymatic digestibility w as calculated according to Eq.(1).

2.4.Analytical methods

The celluloseand hemicellulosein solid compositionsw ereanalyzed according to the“Determination of Structural Carbohydratesand Lignin in Biomass”provided by the National Renew able Energy Laboratory(NREL)[20].The monosaccharide components and small molecule byproducts in liquid compositions w ere analyzed by HPLCequipped with an RIDdetector(AGILENT-1100,Agilent Technologies Inc.,USA).The speci fi c parameters w ere:Aminex HPX-87H column at 40°C,RID detector at 35 °C,and mobile phase(4 m M H2SO4)at 0.6 ml·min-1.The yields of xylose and glucose in prehydrolysate w ere calculated according to Eqs.(2)and(3),respectively.

Fig.1.Schematic diagram of experimental facilities.

2.5.Crystallinity and morphology analysis

Crystallinity w asakey index to re fl ect the degree of removing hemicellulose or lignin as w ell as crystal structure changes of cellulose[20,21].The crystallinity of cassava anaerobic residue w as conducted by XRD.The w orking conditions w ere as follow s:patterns with Cu radiation,angular range of 10°-40°,and the scanning speed at 5(°)·min-1.The crystallinity index(Cr I)of biomass w as calculated according to the following formula[22].

where I002is the intensity of the peak at 2θ =22°,Iamis the intensity of the background at 2θ=18°.

The microstructures of cassava anaerobic residue before and after pretreatment w ere observed by SEM.The w orking conditions w ere as follow s:the acceleration voltage of 0.1-30 k V,the step of 1 k V,the magni fi cation of 120 thousand times,the resolution of 1 nm,the gold fi lm thickness of 5-10 nm,and the sample stage movement of 50 mm for X and Y and 30 mm for Z.

3.Results and Discussion

3.1.Effect of single factorsin hydrothermal pretreatment

In order to explore the most suitablepretreatment conditionsfor xylose and glucose recovery,three single factors,namely temperature(120 °C-240 °C),solid-liquid ratio(5%-15%)and time(15 min-120 min)w ere researched.Taking yields of xylose in prehydrolysate and the enzymatic digestibility in enzymatic hydrolysis as evaluation parameters,the effect of the three single factors w as investigated.The enzymatic digestibility,namely,the glucose recovery during enzymatic hydrolysis,w as an important index for evaluating the effect of the hydrothermal pretreatment on enzymatic hydrolysis process and the most direct indicator for evaluating the digestibility of cellulose[23].

3.1.1.Effect of temperature

Fig.2.The yields of xylose and glucose in prehydrolysate at different temperatures.

Pretreatment temperature played a key role in sugar recovery in the prehydrolysate.Hydrothermal reaction w as performed w ith 10%solidliquid ratio for 90 min and the reaction temperature of 120 °C-160 °C,the result is show n in Fig.2.Increasing temperature from 120°Cto 160°Cresulted in a signi fi cant increase in xylose yield w ith peaking(50.76%)at 160°C.How ever,the xylose yield started to decreased w hen the temperature exceeds 160°C.This drop could be explained by the degradation of the xylose to furfural at high temperature[23].In contrast,the glucose yield sustained a low level(around 2%)over the w hole temperature range.This indicated that cellulose w as dif fi cult to bedecomposed by thehydrothermal pretreatment,and most of them retained in unhydrolyzed solids for enzymatic hydrolysis.The suitable temperature for xylose recovery was160°Cwith the maximum xylose yield of 50.76%.

Fig.3 show ed the enzymatic digestibility under different pretreatment temperatures(120 °C-240 °C)w ith 10%solid-liquid ratio for 90 min.With the temperature rising from 120 °Cto 160 °C,the enzymatic digestibility went up from 52.67%to 78.12%.While,the enzymatic digestibility decreased lightly after temperature reached above 160°C.Because higher temperature w ould promote cellulose hydrolysis in the pretreatment processand caused the cellulosic loss.The highest enzymatic digestibility of 78.12%w as obtained at 160°C.

Fig.3.Enzymatic digestibility at different temperatures.

3.1.2.Effect of solid-liquid ratio

Fig.4.The yields of xylose and glucose in prehydrolysate at different solid-liquid ratios.

Theeffect of solid-liquid ratio[5%(w/v)to 15%(w/v)]on xylose and glucose recovery in prehydrolysate at 160°Cand for 90 min wasshow n in Fig.4.Xylose yield in prehydrolysate reduced from 53.42%to 36.12%accompanied w ith the increase of solid-liquid ratio from 5%to 15%.The decreasewasparticularly pronounced when solid-liquid ratio exceeded 10%.The main reason w as that the sugar formed in the surface of cassava anaerobic residue needs to diffuse into bulk solution in favor of further reaction,and low solid content contributed to this diffusion.How ever,note that low solid content w ould cause low concentration of sugar which was uneconomical for quantity production.Improving solid-liquid ratio could solve this problem but there w as another problem following.Wall sticking occurred when solid content wastoo high,w hich w ould cause the incomplete reaction and reduce the ef fi ciency of hydrothermal reaction.Compared w ith xylose,the yield of glucose w as lower than 1.5%as well as had little change in the all range.Taking these factors into consideration,the solid-liquid ratio should be properly controlled at 10%to balance a relatively high sugar yield and economic ef fi ciency.

Enzymatic digestibility under different pretreatment solid-liquid ratios[5%(w/v)to 15%(w/v)]w ith 160°Cand for 90 min w as illustrated in Fig.5.The enzymatic digestibility declined from 82.52%to 72.54%w ith the solid-liquid ratio increase from 5%to 15%.A signi fi cant drop occurs w hen solid-liquid ratio exceeded 0.1.Low solid content seemed to result in high enzymatic digestibility;however,it meant low concentration of sugars w hich led to a reduction in the economy.For this reason,the 10%solids w as a nice compromise betw een the tw o aspects,accompanying the enzymatic digestibility of 78.12%.

Fig.5.Enzymatic digestibility at different solid-liquid ratios.

3.1.3.Effect of time

Thepretreatment time wasan essential factor that affected thetreatment process.Fig.6 show ed the effect of time(15 min-120 min)on xylose yield in hydrothermal reaction at 160°Cand w ith 10%solids.It can be seen that the xylose yields increased at fi rst and then decreased as time goes on.The xylose yield rose signi fi cantly from 30.20%to 55.13%w ith the time increasing from 15 min to 45 min.Whereas,as the time continued to increase to 120 min,the xylose yields reduced to 42.70%.At the w hole time range,glucose yield continued to creep up from 1.081%to 2.325%,w hich w as still far below than xylose yield.The optimal pretreatment time w as 45 min,w ith the highest xylose yield of 55.13%.

At 160°C and w ith 10%solids,the effect of pretreatment time(15 min-120 min)on enzymatic digestibility was show ed in Fig.7.As thereaction timeincreased,the effect of hydrothermal pretreatment increased.The hemicellulose could be further removed as the reaction time increased.Due to the further removal of hemicellulose,more cellulose could be exposed,which could enhance access to enzyme.Hence,the enzymatic digestibility of cellulose improved.The enzymatic digestibility increased signi fi cantly from 46.73%to 79.23%w ith the grow th of time from 15 min to 75 min.While a dow ntrend w as show n w hen the time exceeded 75 min.To get the highest enzymatic digestibility of 79.23%,the time should be fi xed at 75 min.

Fig.6.The yields of xylose and glucose in prehydrolysate at different reaction times.

Fig.7.Enzymatic digestibility at different reaction times.

Based on the above investigations,the temperature,solid-liquid ratio and time w ere analyzed by using the yields of xylose in prehydrolysate and the enzymatic digestibility in enzymatic hydrolysis as the index,respectively.The maximum xylose yield of 55.13%w as achieved at 160°Cfor 45 min and with 10%solids.Moreover the enzymatic digestibility got the highest value of 79.23%at 160°Cfor 75 min and w ith 10%solids.Conclusions show ed that the temperature for hydrothermal pretreatment of cassava anaerobic residue was below the typical temperatures(about 200°C)for other agricultural residues[12].The optimal conditions for the highest xylose yield in prehydrolysate and theenzymatic digestibility in enzymatic hydrolysiswereinconsistent.And in this range,the highest overall yield of the sum of xylose and glucose could reach the maximum.Therefore,RSM w as applied to optimize hydrothermal pretreatment conditions exactly for realizing the maximum utilization of xylose and glucose in cassava anaerobic residue.

3.2.Multifactor optimization

3.2.1.Statistical design of experiments

The multifactor interaction experiments w ere designed to investigate the combine action among temperature,solid-liquid ratio and time to obtain the optimal pretreatment conditions for the maximum theoretical ethanol production.The theoretical ethanol production from assuming fermentation of both xylose in prehydrolysate and glucose in enzymatic hydrolysate w as selected for target value,and was calculated according to Eq.(5)[24,25].

According to the optimal results in hydrolysis experiment,the Box-Behnken design of RSM in three factors and three levels w as selected to investigate the signi fi cance of temperature,solidliquid ratio and time.The Design-Expert 8.0.6(State-Ease Inc.,Minneapolis,USA)w as applied to build the experimental designs,analyze experimental data,imitate regression equation,and plot response surface[26].

The independent variables of temperature,solid-liquid ratio and time w ere transformed to range betw een-1 and+1 for the assessments of factors.The three variables w ere coded by the follow ing Eq.(6).The theoretical ethanol production w as chosen for response target Y.Multifactor experimental design w as presented in Table 1.The Box-Behnken experimental design scheme and results w ere shown in Table 2.

where xiand Xiw ere the coded valuesand actual valuesof the independent variable i;X0w as the actual value of the independent variable at the center point and X was a dimensionless value standing for the step change of Xicorresponding to a unit variation.

Table 1 Multifactor experimental design

Table 2 CCD design and corresponding theoretical ethanol production

3.2.2.Model fi tting

According to the design experimental data shown in Table 2,a second-order polynomial model w as applied to express the theoretical ethanol production[26,27].The model w hich consisted of main effects,two factor interactions,and curvature effects were shown as the following equation:

w here Y was the predicted theoretical ethanol production(mg·g-1raw material),X1w as the temperature,X2w as the time and X3w as the solid-liquid ratio.

The results of the analysis of variance(ANOVA)for the response surface model w ere show n in Table 3.A second-order polynomial response surface model w ith a higher F-value and R2-value as w ell as low er Lack of Fit(LOF)and P-value could re fl ect the signi fi cances of themodel.Themodel's F-value of 11.07 implied that themodel w assigni fi cant.The LOF F-value of 6.85 in the model implied that the LOFwas signi fi cant to the pure error,and there w as only a 4.70%opportunity that a LOF F-value this large could happen due to noise.The coef fi cient of determination(R2)value could examine the goodness of fi t of the model.Many literatures suggested that the R2value should be at least 0.80 for a good fi t of a model[15].The R2of 94.43%indicated that only 5.57%of the total variation could not be explained by this model.In addition,the model's P-value of 0.0022 more less than 0.05 indicated statistically signi fi cance and expresses the goodness of the fi t[26,28].

Table 3 ANOVA for second-order polynomial response surface model

3.2.3.Effect of parameterson responses

The Design-Expert 8.0.6 w as applied to draw the response surface plots[26].The surface plots and the corresponding contour plots were show n in Fig.8(a)and(b).Thesegraphsevaluated theeffect of pairw ise interaction betw een tw o variables and their optimum levels.Thus,the optimal level range could be found on the vertex area of the surface.A gentle slope of the response surface meant that the theoretical ethanol production could tolerate the variation of the conditions without big change.Conversely,if a slope of the response surface w as quite steep,it indicated that the theoretical ethanol production w ould be fairly sensitive to the change of the conditions.In addition,the contour line shape could re fl ect the intensity of the pairwise interaction.In general,an oval contour lineindicated strong pairw ise interaction,and for circle,represented w eak pairw ise interaction,on the contrary[29].

Fig.8.Response surface plot and contour plot:(a)under the combined effects of temperature and solid-liquid ratio;(b)under the combined effects of temperature and time.

Fig.8 showed that the response surface plots graphically represent the regression equation.The pairw ise interaction betw een temperature and solid-liquid ratio(Fig.8(a))w as investigated w hen the time w as kept at 60 min.With increase of temperature and solid-liquid ratio,ethanol production gradually increased at fi rst and then it reduced.This trend w as reasonable because xylose w as a sensitive sugar in high temperatures,and rapidly converted to furfural.Meanw hile,high solid-liquid ratio prevented the diffusion of degradation products,thus it inhibited the hydrolysis process.At medium temperature and solid-liquid ratio,the ethanol production reached the maximum.As can be seen in the contour plot,the effect of solid-liquid ratio on the production of ethanol w as more conspicuous than temperature.The contour line w as nearly elliptical shaped,w hich meant a strong interaction between temperature and solid-liquid ratio.The pairw ise interaction of temperature and time w ith 10%solid-liquid ratio w as plotted in Fig.8(b).The production of ethanol increased gradually w ith temperatureand timeuntil middlepoint.Themore harsh pretreatment conditions(higher temperature and more reaction time)resulted in low er ethanol production.Furthermore,the maximum production of ethanol w as located in the central regions of the response surface and the contour lines w ere close to round,which means the two factors had similar in fl uences on the production of ethanol,and relatively w eak interaction.By contrasting Fig.8(a)and(b),theeffect of solid-liquid ratio on ethanol production w as relatively more conspicuous than temperature and time.A maximum ethanol production of 19.5 mg·g-1(raw material)w as obtained at w here the temperature,solid-liquid ratio and time w ere 163°C,9.6%and 59 min,respectively.3.2.4.Validation of predictive model

Validation experiments w ere adopted to verify the fi tnessand effectiveness of the model.Under the optimal conditions above,the yield of xylose in prehydrolysate w as 53.9%and the enzymatic digestibility in enzymatic hydrolysiswas75.7%.Additionally,theproduction of ethanol obtained from co-fermentation of glucose and xylose w as 20.2 mg·g-1(raw material)w hich w as close to the predicted value of 19.5 mg·g-1(raw material),meanw hile,the predictive accuracy w as approximately 97%.Therefore the optimization model was reliable and feasible to predictive the experimental results.

3.3.Comparative analysis of the different conditions

Fig.9.Theoretical ethanol production under different pretreatment conditions.

Three pretreatment conditions w ere found through the single factor and multifactor optimization experiments,and they w ere 160°C-45 min-10%solids for maximum xylose yield,160°C-75 min-10%solids for maximum glucose yield and 163°C-59 min-9.6%solids optimized by multifactor optimization experiments.Theoretical ethanol production under the three pretreatment conditions has been compared.Asshown in Fig.9,thetheoreticalethanol production ranged from 17.9 mg·g-1to 20.2 mg·g-1(raw material).The highest value w as obtained under the condition of 163°C-59 min-9.6%solids,which illustrated the validity of multifactor optimization experiments.

3.4.Crystallinity and morphology

3.4.1.Crystallinity analysis

The crystallinestructure of cellulosew asgenerally seen asoneof the important factors w hich hindered the cellulose hydrolysis process.Cellulose had a regular crystal structure,w hile hemicellulose and lignin have an amorphous structure in cassava anaerobic residue,so that the XRDcould re fl ect the crystal structure changes of cellulose.The crystallinity index w as calculated as Eq.(4).As can be seen from Table 4,the crystallinity index of cassava anaerobic residue samples drops to 11.32%from 23.92%after the hydrothermal treatment,and the crystallinity index reduces to about 12.5%,which indicated the destruction of the crystalline structure of cellulose by hydrothermal treatment.

Table 4 The crystallinity index of cassava anaerobic residue before and after pretreatment

3.4.2.Morphology analysis

To investigate the microstructure changes,the cassava anaerobic residue before and after the pretreatment under optimal condition w ere analyzed by SEM,and the results w ere presented in Fig.10.As show n in Fig.10(a),the raw cassava anaerobic residue exhibited a smooth and ridge surface.However,the cassava anaerobic residue after pretreatment show ed cracks and even signs of structural breakdow n,as illustrated in Fig.10(b).This phenomenon indicated that hydrothermal pretreatment hydrolyzed the hemicellulose as w ell as destroyed the dense structure formed by hemicellulose,cellulose and lignin.

4.Conclusions

Hydrothermal pretreatment w as an effective and environmentalfriendly method for releasing sugars from cassava anaerobic residue.Based on the single factor experiments,the highest xylose yield of 55.13%w as obtained under the conditions of 160°C,45 min and 10%solids,w hile the conditions for maximum glucose yield of 79.23%were 160°C,75 min and 10%solids.In multifactor optimization experiments,the maximum theoretical ethanol production of 19.5 mg·g-1raw material by co-utilization of xylose and glucose w as obtained at 163°C,for 59 min and with 9.5%solids.Additionally,the actual production of ethanol w as 20.2 mg·g-1raw material in experiment.The optimal conditions not only made the most of xylose and glucose in cassava anaerobic residue but also provided an important reference for the hydrothermal process of other anaerobic residue.

In thefuture prospects,thesugar-rich prehydrolysateand enzymatic hydrolysate of cellulose w aste can serve as mixing w ater for feedstock in starch ethanol industry.This mode not only improves the sugar content of the mash for ethanol fermentation,thusreducing the wastew ater for producing a certain amount of ethanol,but also realizes the full utilization of starch and cellulose composition in feedstock w ithout additional fermentation equipment.

Fig.10.SEM images of cassava anaerobic residue:(a)raw cassava anaerobic residue;(b)cassava anaerobic residue after the pretreatment.

Acknowledgment

Theauthorsaregrateful to the fi nancial support from Key Laboratory for Green Chemical Technology of Ministry of Education,R&DCenter for Petrochemical Technology,Tianjin University.