Bioresource Technology 169 (2014) 9–18

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Simultaneous saccharification and co-fermentation of aqueous ammoniapretreated corn stover with an engineered Saccharomyces cerevisiaeSyBE005

http://dx.doi.org/10.1016/j.biortech.2014.06.0850960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Key Laboratory of Systems Bioengineering, Ministry ofEducation, Tianjin University, Tianjin 300072, PR China. Tel.: +86 22 27406491; fax:+86 22 27403888.

E-mail address: bzli@tju.edu.cn (B.-Z. Li).

Jia-Qing Zhu, Lei Qin, Bing-Zhi Li ⇑, Ying-Jin YuanKey Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, PR ChinaCenter of Synthetic Biology, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University,Tianjin 300072, PR China

h i g h l i g h t s

� Strain SyBE005 exhibited excellent performance on xylose utilization in SSCF.� Cellulase loading was significantly reduced with the addition of Tween 20.� Yeast feeding was efficient to improve SSCF with high solid loading.� The ethanol concentration reached to 70.1 g/L at 12% glucan loading.

a r t i c l e i n f o

Article history:Received 7 May 2014Received in revised form 23 June 2014Accepted 24 June 2014Available online 1 July 2014

Keywords:Simultaneous saccharification and co-fermentationPre-hydrolysisSurfactantsFermentation with high solid contentFed-batch SSCF

a b s t r a c t

Co-fermentation of glucose and xylose from lignocelluloses is an efficient approach to increasing ethanolproduction. Simultaneous saccharification and co-fermentation (SSCF) of corn stover pretreated withaqueous ammonia was performed using engineered yeast with xylose utilization pathway. Thus far,the effect of the several key factors on SSCF was investigated, including temperature, inoculation size,pre-hydrolysis and pH. Ethanol concentration was achieved to 36.5 g/L during SSCF process with 6%glucan loading. The addition of Tween 20 reduced enzyme loading, i.e., from 15 to 7.5 FPU/g glucan withthe same final ethanol concentration. The ethanol concentration was achieved to 70.1 g/L at 12% glucanloading. Yeast feeding, combined with substrate and enzyme feeding, was proved to be an efficientapproach for SSCF with high solid loading.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Lignocellulosic biomass contains 15–35% of hemicellulose typi-cally, which is primarily composed of xylose (Girio et al., 2010).Efficient conversion of xylose, together with cellulose-derivedglucose, into ethanol generates higher fuel yields and greaterreturn on investment (Sassner et al., 2007). Therefore, engineeringSaccharomyces cerevisiae for efficient xylose utilization by intro-ducing a xylose metabolic pathway has attracted great interest(Zha et al., 2013). Even though some genetic engineered cells arecapable of utilizing xylose, xylose consumption is still poor in

co-fermentation of xylose and glucose (Li et al., 2010; Jin et al.,2012). Pre-fermenting free glucose in the liquid (Bertilsson et al.,2009), enzyme feeding (Olofsson et al., 2010a) or both enzymeand substrate feeding (Olofsson et al., 2010b) and glucose feeding(Erdei et al., 2012) were applied to keep the ratio of glucose toxylose low, which is very beneficial to promote utilization of xylose(Subtil and Boles, 2012). However, the residual xylose in the finalbroths is still an issue to be solved. Simultaneous saccharificationand co-fermentation (SSCF) process is another efficient approachto keeping the glucose concentration low and enhancing xyloseutilization (Jin et al., 2012).

Compared with separate hydrolysis and co-fermentation(SHCF), SSCF is an alternative process that encompasses two stepswithin the same vessel simultaneously. SSCF is more favorable forlignocellulosic ethanol production than SHCF due to its lower cost,shorter processing time, higher sugar conversions, higher ethanol

Table 1The compositions of untreated corn stover and high temperature aqueous ammoniapretreated corn stover.

UT-CSa AAP-CSb

Glucan 31.70 ± 2.3% 68.34 ± 0.4%Xylan 17.10 ± 1.2% 18.24 ± 0.9%Arabina 2.60 ± 0.5% ndc

AILd 12.60 ± 1.6% 7.99 ± 1.1%Extractives 23.50 ± 1.1% ndc

Ash 4.30 ± 0.8% ndc

a UT-CS stands for untreated corn stover.b AAP-CS stands for aqueous ammonia pretreated corn stover.c nd stands for not determined.d AIL stands for acid insoluble lignin.

10 J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18

yields and lower contamination risk (Taherzadeh and Karimi,2007). Ethanol yield in SSCF process is affected by many factors,such as temperature, inoculum size, pre-hydrolysis time, pH,enzyme loading, solid content and so on (Laluce et al., 2009), whichmainly affect the efficiency of hydrolysis and fermentation. Notonly the yield but also the ethanol concentration is important forthe production of lignocellulosic ethanol, because the distillationcosts decrease with the final ethanol concentration increasing(Sassner et al., 2007). Lignocellulosic ethanol would become eco-nomically feasible only when the concentration exceeds 40 g/L(Zacchi and Axelsson, 1989). To increase the ethanol concentration,high loading of dry matter (DM) is necessary in the fermentation.However, high DM loading results in high viscosity of the mediumand leads to severe mixing problems (Bertilsson et al., 2009). Inorder to overcome this problem, effective strategies, such as pre-hydrolysis and fed-batch operation, were applied (Hoyer et al.,2013; Lan et al., 2013; Pessani et al., 2011). The main advantageof the pre-hydrolysis is to reduce viscosity of the medium (Hoyeret al., 2013). Fed-batch process could decrease the viscosity, toxic-

Table 2Fed-batch SSCF with six different feed profiles at 9% and 12% glucan loading.

Feed profile �12 h �6 h

A Substrate 3% 3%Enzyme All –Inoculation size (g/L) – –

B Substrate 3% 3%Enzyme 1/3a 1/6Inoculation size (g/L) – –

C Substrate 3% 3%Enzyme 1/3 –Inoculation size (g/L) – –

D Substrate 3% 1.5%Enzyme 1/3 –Inoculation size (g/L) – –

E Substrate 3% –Enzyme 1/3 –Inoculation size (g/L) – –

F Substrate 3% –Enzyme 1/3 –Inoculation size (g/L) – –

A0 Substrate 4% 4%Enzyme All –Inoculation size (g/L) – –

E0 Substrate 4% –Enzyme 1/3 –Inoculation size (g/L)

F0 Substrate 4%Enzyme 1/3Inoculation size (g/L) – –

a Based on the total enzyme loading (15 FPU/g glucan).

ity and increase xylose uptake (Lan et al., 2013; Pessani et al.,2011).

In this present study, engineered xylose-fermenting yeastSyBE005 (Zha et al., 2013) was used, and the effect on ethanol yieldof temperature, inoculation size, pre-hydrolysis time, and pH wasinvestigated in SSCF. The effect of three additives on SSCF wasstudied to reduce the enzyme loading. Different feeding strategieswere investigated in the fermentation with high solid loading.

2. Methods

2.1. Materials

Corn stover used in this study was harvested from the suburb ofTianjin, China. The corn stover was milled and screened and frac-tions between 20 and 80 meshes were collected. Following theLaboratory Analytical Procedure (LAP) of the National RenewableEnergy Laboratory (NREL) the compositions of corn stover weredetermined.

Enzymes used in this experiment are as follows: Accellerase1500 (Genencor, 89 mg/mL, 77 FPU/mL), Novozyme 188(Sigma–Aldrich, 67 mg/mL, 850 CBU/mL) and Multifect xylanase(Genencor, 42 mg/mL). The activity of enzymes is determinedaccording to LAP method. Additives used in this study are asfollows: bovine serum albumin (GenView, USA), Tween 20 andTween 80 (Ding Guo Biotech, China).

2.2. Pretreatment of corn stover

Milled corn stover was pretreated using aqueous ammonia athigh temperature according to previous report (Qin et al.,2013).The biomass slurry after pretreatment was then pressedthrough a filtration cloth in order to separate free liquid fromsolids. Then the solid fraction derived from biomass with 16 g

0 h 12 h 24 h 36 h 48 h

3% – – – –– – – – –2.5 – – – –

3% – – – –1/6 – 1/6 – 1/62.5 – – – –

3% – – – –– 1/6 1/6 1/6 1/62.5 – – – –

1.5% 1.5% 1.5% – –– 1/6 1/6 1/6 1/62.5 – – – –

– 1.5% 1.5% 1.5% 1.5%– 1/6 1/6 1/6 1/62.5 – – – –

– 1.5% 1.5% 1.5% 1.5%– 1/6 1/6 1/6 1/61.0 – 1.0 – 0.5

4% – – – –– – – – –2.5 – – – –

– 2% 2% 2% 2%– 1/6 1/6 1/6 1/62.5 – – – –

2% 2% 2% 2%1/6 1/6 1/6 1/6

1.0 – 1.0 – 0.5

Fig. 1. SSCF performance at different temperature. The experiments were con-ducted at the following conditions: inoculation size 2.5 g/L, 24 h pre-hydrolysis, pH5.2 and 6% glucan loading (equivalent to 8.8% DM loading).

J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18 11

dry matter was washed three times using 300 mL water. The solidfraction was dried at room temperature until the moisture was lessthan 10% and composition of the solid fraction, named pretreatedcorn stover, was analyzed. The compositions of untreated and pre-treated corn stover were listed in Table 1.

2.3. Microorganism and seed culture preparation

An engineered S. cerevisiae SyBE005 with genetically con-structed xylose utilizing pathway (Zha et al., 2013) was used in thisstudy. Seed culture was prepared by inoculating a single colony of

S. cerevisiae SyBE005 from a YPX-Agar plate (per liter: 10 g yeastextract, 20 g peptone, 20 g xylose, and 20 g agar) into a tubecontaining 5 mL YPX medium (per liter: 10 g yeast extract, 20 gpeptone and 20 g xylose) at 30 �C, 250 rpm for 24 h; and then500 lL culture was inoculated into 100 mL YPX medium in a250 mL conical flask and incubated for approximately 18 h at30 �C and 250 rpm. The final OD600 was around 5. The cells wereharvested for fermentation inoculation by centrifugation at 4000rpm for 5 min.

2.4. Simultaneous saccharification and co-fermentation

All the SSCF experiments were carried out in duplicates underanaerobic conditions using shaking flasks in an orbital incubatorat 150 rpm. Unless otherwise specified, the enzyme loading in SSCFexperiment was: 15 FPU/g glucan Accellerase 1500, 30 CBU/g glu-can Novozyme 188 and 0.15 mL/g glucan Multifect xylanase. Torelease the carbon dioxide produced during fermentation, all theflasks were sealed by a rubber stopper with a syringe needle inside.All of the pre-hydrolysis experiments were conducted at 50 �C and200 rpm. Ampicillin with finial concentration of 50 mg/L was usedto prevent bacterial contamination. No any other extra organicnutrients and inorganic salt were added to the SSCF medium.

In the additive assays, two enzyme loadings for cellulases wereused: 15 and 7.5 FPU/g glucan. Twelve hour pre-hydrolysis wascarried out to avoid the inhibition of surfactant additives to yeaststrain. The additives of 0.05 g/g DM was added at the beginningof pre-hydrolysis.

For fed-batch fermentation, a standard fed-batch SSCF with allenzymes initially added to the broth was carried out as a reference(Feed profile A). Five different feed profiles (Table 2) were investi-gated in SSCF. Substrate were all fed during the pre-hydrolysis pro-cess in feed profile A, B and C while part of the substrate were fedduring the fermentation process and the enzymes were fed alongwith the substrate in feed profile D, E and F.

2.5. Chemical analysis and measurement of viable cell density

Samples from SSCF were centrifuged at 12,000 rpm for 5 minand the supernatant was filtered with a 0.22 lm filter. Ethanol,glucose, xylose and glycerol in the sample were analyzed usingHPLC with a Aminex HP-87H column (Bio-Rad, Hercules, CA,USA) at 65 �C, using 5 mM H2SO4 as eluent, at a flow rate of0.6 mL/min (Qin et al., 2012).

Fermentation samples were collected and diluted using sterilewater. One hundred microliter of each diluted sample was platedon a YPD agar medium (per liter: 10 g yeast extract, 20 g peptone,20 g glucose, and 20 g agar). The dilution rate for each sample wasvaried in order to obtain 20–200 colonies on a single plate. Theplates were then cultured at 30 �C for 36 h. Single colonies formedon the plates were counted and viable cell density was calculatedaccordingly.

2.6. Mass balance

Mass balance calculation was based on the analysis of ethanol,monomeric, oligomeric, and polymeric sugars during pretreatment(Li et al., 2010; Liu et al., 2013) and SSCF process (Jin et al., 2012). Inorder to facilitate the calculation, content of glucan and xylan inthe pretreated corn stover and solid residue were converted intothe content of glucose and xylose. The theoretical amount of glu-cose released during SSCF is 1.11 times the amount of glucan and1.13 times the amount of xylan in the solid material, respectively.Glucan conversion, xylan conversion, ethanol metabolic yield andethanol yield were calculated as follows:

Fig. 2. SSCF performance at different inoculation size (IS). The experiments were conducted at the following conditions: 38 �C, 12 h pre-hydrolysis, pH 5.2, and 6% glucanloading.

12 J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18

Glucan conversion ð%Þ ¼ ðglucose AAP-CS � glucose solid residueÞ=� glucose AAP-CS � 100%

Xylan conversion ð%Þ ¼ ðxylose AAP-CS � xylose solid residueÞ=� xylose AAP-CS � 100%

Ethanol metabolic yield ð%Þ ¼ ethanol SSCF=0:51� ðglucose AAP-CS

� glucose solid residue � glucose SSCF

� GO SSCF þ xylose AAP-CS

� xylose solid residue � xylose SSCF

� XO SSCFÞ � 100%

Ethanol yield ð%Þ ¼ ethanolSSCF=0:51� ðglucose AAP-CS

þ xylose AAP-CSÞ � 100%

where GO stands for glucose oligomers, XO stands for xyloseoligomers.

3. Results and discussion

3.1. The effect of temperature on SSCF

The trade-off phenomena of temperature have been investi-gated extensively in SSF or SSCF (Öhgren et al., 2007; Park et al.,2010). The optimal temperature for SSCF is determined by several

factors, such as the tolerance of cellulases to low temperature andthe tolerance of the fermentation strains to high temperature. Theeffect of temperature on SSCF was investigated at different temper-atures (30, 35, 38, 42 and 45 �C) using an engineered yeast withxylose utilizing pathway (Fig. 1). At lower temperatures (30, 35,38 �C), glucose and xylose was consumed rapidly, glucose andxylose was almost completely consumed (concentrations below1.5 g/L) in 12 h and 24 h, respectively. However, at higher temper-atures (42 and 45 �C), glucose and xylose gradually accumulatedduring the late fermentation phase, which resulted in low ethanolfinal concentrations. Two reasons might lead to the result. Highertemperature inhibited strain growth and then sugar consumptionrate significantly declined. On the other hand, the rate of hydroly-sis, obviously affected by temperature, is increased and a higherrate of hydrolysis resulted in a faster release of glucose and xylose.

The fastest ethanol productivity (1.02 g/L/h in the first 24 h) andthe highest ethanol concentration (30.4 g/L at 72 h) were achievedat 38 �C. The fastest xylose consumption rate (0.53 g/L/h in the first12 h, Fig. 1) was also observed at 38 �C. Rudolf et al. (2008) con-cluded that more xylose was consumed by TMB3400 at 32 �C incomparison with 37 �C during SSCF of sugar cane bagasse. S. cere-visiae 424A was used in SSCF at 30 �C (Jin et al., 2012). However,35 �C was more favorable for SSCF with KE6-12 (Erdei et al.,2013). Based on the above, the effect of temperature on SSCF ismainly depends on the strain background of thermal tolerance.The optimal temperature using S. cerevisiae SyBE005 is 38 �C forSSFC of aqueous ammonia pretreated corn stover.

Fig. 3. SSCF performance at different pre-hydrolysis time (PT). The experiments were conducted at the following conditions: 38 �C, inoculation size 2.5 g/L, pH 5.2 and 6%glucan loading.

J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18 13

3.2. The effect of inoculation size on SSCF

The effect of the inoculation size was investigated with fourinitial inoculation sizes (0.5, 2.5, 5.0 and 7.5 g/L) on SSCF frompretreated corn stover at 38 �C (Fig. 2). It took 24 h to completelyconsume glucose under the condition of inoculation size 0.5 g/L,and 8 h for the condition of inoculation size 2.5 g/L. When the inoc-ulation size is 5.0 and 7.5 g/L, it only took 6 h to consume glucose.The fast xylose consumption was also observed when the inocula-tion size is 5.0 and 7.5 g/L (Fig. 2). The fastest rate of xylose con-sumption reached to 0.87 g/L/h. However, rapid consumption ofglucose and xylose did not result in high final ethanol concentra-tion. Despite the higher ethanol productivity during the beginning12 h at bigger inoculation sizes, the highest ethanol concentration(36.1 g/L) was achieved at the inoculation size 0.5 g/L.

Jin et al. (2010) found that higher inoculation size resulted inhigher xylose consumption rates and higher ethanol yield. Jinet al. (2010) achieved the highest ethanol concentration (36.1 g/L) at the initial OD600 = 16 using xylose-fermenting S. cerevisiae424A(LNH-ST). The final xylose concentration at the initialOD600 = 1 is much higher than that at the initial OD600 = 16.However, much lower final xylose concentration (0.86 g/L) wasachieved at the inoculation size 0.5 g/L (Fig. 2) in this study.SyBE005 showed an outstanding ability to metabolize xylose evenat lower strain loading. The low final ethanol concentration at biginoculation size may be due to the more sugar consumption forstrain maintenance for survival. The viable cell density in SSCF

with different inoculation size was determined (Fig. 2), and theresults indicated that higher viable cell density was kept duringSSCF with the big inoculation size. More sugars used for straingrowth resulted in the loss of carbon source and energy, and thenethanol yield would decrease with high strain density (Öhgrenet al., 2007). In addition, low strain loading would decrease the costof strain seed, but long fermentation time is needed at low stainloading, which would increase the cost of fermentation process.How to balance the initial strain loading and fermentation timeneeds further investigation. In this study, 2.5 g/L was consideredas the optimal inoculation size for SSCF.

3.3. The effect of the pre-hydrolysis time on SSCF

Pre-hydrolysis process is generally considered to reduce the vis-cosity of fermentation broths and the glucose generated duringpre-hydrolysis process is favorable for strain growth (Jin et al.,2010). The effect of pre-hydrolysis was investigated in SSCF at38 �C and inoculation size 2.5 g/L. Different pre-hydrolysis time(0, 12, 24 and 48 h) was used in this study (Fig. 3).

Ethanol productivity in the first 12 h was higher with longerpre-hydrolysis time. However, the higher ethanol concentrations(36.5 and 35.8 g/L) were achieved without pre-hydrolysis and with12 h pre-hydrolysis. These results indicated that pre-hydrolysishad positive impact on the ethanol productivity during the first12 h but negative impact on the overall ethanol yield. The lowerethanol yield with long pre-hydrolysis may be caused by several

Table 3A brief summary of SSCF results with different engineered xylose-fermenting yeasts.

Strain Raw biomass Type ofpretreatment

Amount ofsolids

Mode ofoperation

Prehydrolysiscondition

Finalethanolconc. (g/L)

Ethanolyield(%)

Xyloseconsumption(%)

References

S. cerevisiaeTMB3400

Spruce Steam-pretreated

7%WISa Batch NO 33.9 91.0 84 Bertilsson et al.(2009)

S. cerevisiaeTMB3400

Spruce Steam-pretreated

10%WIS Fed-batch NO 45.2 85.0 77 Bertilsson et al.(2009)

S. cerevisiaeTMB3400

Wheat straw Steam-pretreated

11%WIS Fed-batch NO 38.0 69.0 49 Olofsson et al.(2010b)

S. cerevisiaeTMB3400

Spruce Steam-pretreated

8%WIS Fed-batch NO 35.2 78.0 85 Olofsson et al.(2010a)

S. cerevisiae 424A Switchgrass AFEXb

pretreatment6% glucanloading

Batch 8 h 35.1 55.5 79 Jin et al. (2010)

S. cerevisiae 424A Corn stover AFEXpretreatment

6% glucanloading

Batch 6 h 39.9 61.2 88 Jin et al. (2012)

S. cerevisiae 424A Corn stover AFEXpretreatment

6% glucanloading

Batch 24 h 37.9 59.8 83 Jin et al. (2012)

S. cerevisiae KE6–12

Wheat mealandwheat straw

Steam-pretreated

7.5%WIS Fed-batch(SWM 48–96 h)c

NO 60.0 76.0 31 Erdei et al. (2013)

S. cerevisiae KE6–12

Wheat mealandwheat straw

Steam-pretreated

7.5%WIS Fed-batch(SWM 24–96 h)

NO 58.8 74.0 21 Erdei et al. (2013)

S. cerevisiae Y35 Corn stover AFEXpretreatment

6% Glucanloading

Batch 6 h 33.3 ± 0.5 NAd 51 Jin et al. (2013)

S. cerevisiae Y35 Corn stover AFEXpretreatment

9% Glucanloading

Batch 6 h 45.5 ± 0.1 NA 21 Jin et al. (2013)

S. cerevisiaeSyBE005

Corn stover AAP 6% Glucanloading

Batch NO 36.1 83.8 93 This study

S. cerevisiaeSyBE005

Corn stover AAP 9% Glucanloading

Fed-batch 12 h 52.9 81.9 88 This study

S. cerevisiaeSyBE005

Corn stover AAP 12% Glucanloading

Fed-batch 12 h 68.8 79.8 89 This study

a WIS stands for water insoluble solid.b AFEX stands for ammonia fiber expansion.c SWM 48–96 h means saccharified wheat meal addition during fermentation 48–96 h.d NA stands for not available.

14 J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18

reasons. During pre-hydrolysis, monosaccharides were released aswell as other degradation products, such as xylooligomers andglucoligomers, which had adverse effect on the enzymatic hydroly-sis (Qing et al., 2010). Enzyme deactivation is temperature andtime dependant. In the SSCF with pre-hydrolysis the enzymesunderwent higher temperature at pre-hydrolysis stage, whichcould lead to higher degree of deactivation of enzymes and thuslower hydrolysis and ethanol production at the end of the SSCF(Öhgren et al., 2007).

The glucose concentration was 46.5, 53.2 g/L respectively after24, 48 h of pre-hydrolysis (Fig. 3), which led to high osmotic stressfor yeast cells at the beginning of SSCF. Osmostress response wouldbe induced by changes in the osmotic pressure, and production ofglycerol is one way for the yeast cells to adapt to the change inosmotic pressure (Albertyn et al., 1994). As Fig. 3 shows, glycerolconcentration was 22.4% higher with 24 h pre-hydrolysis com-pared with that without pre-hydrolysis. Ethanol production wouldbe lowered by the accumulation of glycerol due to the loss of car-bon source and energy (Öhgren et al., 2007). Therefore, suddenchanges in the osmotic pressure in the surroundings of fermenta-tion workhorses could also be the reason for the lower ethanolyield in the SSCF with longer time pre-hydrolysis.

3.4. The effect of pH on SSCF

In addition to the factors discussed above, pH was found to beanother key factor for SSCF using S cerevisiae BY4742 with NaOHpretreated wheat straw (Zhang et al., 2013). It was commonlybelieved and widely practiced that pH 4.8 is optimal for enzymatic

hydrolysis (Wang et al., 2013b), while pH 5.5 was recommendedfor the yeast fermentation (Jin et al., 2010). In order to balancethe fermentation and enzymatic hydrolysis, the effect of pH wasinvestigated in SSCF using the engineered S. cerevisiae SyBE005.Three initial pH values (4.8, 5.2 and 5.6) were used according theoptimal pH of cellulases and S. cerevisiae.

Glucose, xylose consumption and final ethanol concentration inSSCF were not significantly changes at different initial pH values(Supplementary Material Fig. 1). S. cerevisiae SyBE005 showed goodfermentative ability at a pH range of 4.8–5.6. Xylose was almostcompletely consumed (concentration below 1 g/L) in the beginning24 h in all the SSCF. A relatively high xylose consumption rate(0.88 g/L/h) in the beginning 12 h was also achieved. The finalxylose concentration in this study was very low using S. cerevisiaeSyBE005 compared to other studies. A brief summary of SSCFresults with different engineered xylose-fermenting yeasts wasshowed in Table 3. These results indicate that S. cerevisiae SyBE005is an excellent strain to ferment xylose.

3.5. The effect of additives on the SSCF

Surfactant additives were effective to improve enzymatichydrolysis (Li et al., 2011). The effect of surfactants on SSCF wasinvestigated using the optimal conditions established above.Non-ionic surfactants have been proven to be effective in increas-ing cellulose hydrolysis, whereas anionic and cationic surfactantsdecrease cellulose hydrolysis (Park et al., 1992). Two kinds ofnon-ionic surfactants (Tween 20 and Tween 80) and one kind ofprotein (BSA) were used in this study.

Fig. 4. The effects of three additives on SSCF. A1, B1, C1 were conducted at enzyme loading of 15 FPU/g glucan. A2, B2, C2 were conducted at enzyme loading of 7.5 FPU/g glucan.

J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18 15

With the addition of additives, glucose and xylose concentra-tion at the beginning of the fermentation was much higher thanwithout additives (Fig. 4). That indicated that the additivesimproved enzymatic hydrolysis as reported in other study (Liet al., 2011). Among three additives, Tween 80 showed the bestimprovement on enzymatic hydrolysis.

At high enzyme loading (15 FPU/g glucan) the final ethanol yieldincreased by 9% with the addition of Tween 20. The maximum eth-anol concentration was reached at 48 h in SSCF with additives, while

it took 72 h without additives (Fig. 4). At low enzyme loading of7.5 FPU/g glucan, Tween 20 increased the final ethanol concentra-tion by 47.5% compared to that without additives. Tween 80 andBSA also increased final ethanol concentration by 38% and 42.8%,respectively. The fermentation time to reach the maximum ethanolconcentration was shortened from 96 h to 72 h with additives addi-tion (Fig. 4). It is interesting that more sugars are released in pre-hydrolysis with Tween 80 than that with Tween 20, but SSCF withTween 20 resulted in highest final ethanol concentration.

Fig. 5. Performance of fed-batch SSCF with different feed profiles at 9% (A) and 12%(B) glucan loading (equivalent to 13.2% and 17.6% DM loading, respectively).

16 J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18

Techno-economical calculations have indicated that a 50%reduction of enzyme loading is beneficial if the yield decreases lessthan 6–7% and required residence time is not increased by morethan 30% (Sassner et al., 2007). SSCF was considered to releasethe inhibition of monosugars to enzyme, and surfactant additiveswas proved to enhance the enzymatic hydrolysis. Therefore, wetested to reduce the enzyme loadings in SSCF with surfactant addi-tives. Almost the same final ethanol concentration was achieved inSSCF with additive using 7.5 FPU/g glucan enzyme loading com-pared with that in SSCF without additive using 15 FPU/g glucanenzyme loading.

3.6. Fed-batch SSCF

To increase the final ethanol concentration, high loading of drymatter (DM) is necessary for SSCF process. However, a high DMloading results in a high viscosity of the medium and leads tosevere mixing problems (Bertilsson et al., 2009). In order to over-come this problem, pre-hydrolysis and fed-batch operations wereapplied in the SSCF with high glucan loadings (9% and 12%) in thisstudy. Five different feed profiles were used, including strain feed-ing, biomass feeding, enzyme feeding. Biomass feedings were usedto reduce the real-time solid loading and reduce the mixing prob-

lems. Enzyme feedings were used to keep the enzymatic activitiesin a long time during SSCF. The strain feedings were used to keepviable cell density at a high level.

Feed profile A, B and C gave low final ethanol concentration butmuch higher ethanol productivity. The ethanol productivityreached 1.73 g/L/h and 2.15 g/L/h during the first 24 h at 9% and12% glucan loading, respectively (Feed profile A). Obviously, sub-strate all fed during the pre-hydrolysis course had a positive effecton ethanol productivity at the first time in SSCF (Feed profile A, Band C).

Fig. 5 shows that feed profile B and C did not increase final eth-anol concentration compared with the reference fed-batch case(Feed profile A) while a little higher ethanol concentration wasachieved in feed profile D, E and F, which was in agreement withprevious study (Olofsson et al., 2010b). These results indicated thatfeedings of biomass and enzymes in the SSCF process couldincrease ethanol yield. These feeding profiles (D, E and F) provideda relative constant release of glucose, which is very beneficial tokeep strain viability to increase xylose uptake and ethanol produc-tion (Subtil and Boles, 2012). The maximum ethanol concentration(53.8 g/L and 70.1 g/L at 9% and 12% glucan loading, respectively)was achieved with feed profile F, which included strain feedings,could further enhance cell viability during SSCF (Koppram andOlsson, 2014). In addition, some inhibitors existed in hydrolysisof pretreated biomass, and the inhibitors could reduce strainviability and be degraded by yeast (Wang et al., 2013a). The firstfeeding strains may be inhibited by the inhibitors, while thesestrains could degrade the inhibitors. Therefore, the feeding strainsafter the first feeding could keep high viability due to the removalof inhibitors.

3.7. Mass balance of different SSCF processes

At 6% glucan loading, 374.32 g ethanol per kg aqueous ammoniapretreated corn stover was achieved in the SSCF processes withTween 20 addition at 7.5 FPU/g glucan and the ethanol yield was82.55% (Fig. 6A). Only a little higher ethanol production(379.98 g) and ethanol yield (83.79%) were achieved during SSCFprocesses at 15 FPU/g glucan without Tween 20 (Fig. 6B). Theseresults indicate that the addition of 5% Tween 20 successfullyreduced enzyme dosages by 50%.

Mass balance studies of SSCF at high DM content were con-ducted using feed profile F (Table 2). Ethanol yield at 9 and 12%glucan loading were achieved to 81.92 and 79.83% (Fig. 6C andD), which was only a little lower than that at 6% glucan loading(83.79%, Fig. 6B). These results indicated that yeast feeding, com-bined with substrate feeding and enzyme feeding was an efficientapproach for SSCF with high DM loading. According to the massbalance analysis, two aspects could be further increase ethanolproduction in SSCF. For the first, some oligomeric sugar (sugarsunutilized in the form of oligosaccharides), especially oligomericxylose at higher DM loading was observed, which not only led tosugar loss (Jin et al., 2010, 2012), but also inhibited enzymatichydrolysis (Qing et al., 2010). For the second, some amount of solidresidues was observed and further releasing of glucan and xylanwere needed.

4. Conclusion

SSCF process was performed using S. cerevisiae SyBE005. Thisstrain showed an excellent ability to utilize xylose at a relativelyhigh temperature 38 �C and a pH range of 4.8–5.6. Applying addi-tives and fed-batch model improve the SSCF process economically.With the addition of Tween 20, the enzyme dosage was success-fully reduced by 50%. A relatively high final ethanol concentration

Fig. 6. Mass balance for SSCF. For A, SSCF were conducted at 6% glucan loading with the addition of Tween 20 and the enzyme loading was 7.5 FPU/g glucan. For B, SSCF wereconducted at 6% glucan loading with the enzyme loading 15 FPU/g glucan. For C and D, SSCF were conducted at 9% and 12% glucan loading, respectively, with the addition ofTween 20 and 12 h pre-hydrolysis and the enzyme loading was 15 FPU/g glucan.

J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18 17

(70.1 g/L) was achieved at 12% glucan loading when fed-batch SSCFwas conducted. Yeast feeding, combined with substrate feedingand enzyme feeding, was demonstrated to be an efficient approachfor SSCF with t high DM loading.

Acknowledgements

This study was financially supported by the National BasicResearch Program of China (‘‘973’’ Program: 2013CB733600), theNational Natural Science Foundation of China (Major InternationalJoint Research Project: 21020102040) and the International JointResearch Project of Tianjin (11ZCGHHZ00500). The authors aregrateful for Genencor International Corporation (Suzhou, China)kindly providing the enzymes.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.06.085.

References

Albertyn, J., Hohmann, S., Thevelein, J.M., Prior, B.A., 1994. GPD1, which encodesglucerol-3-phosphate dehydrogenase, is essential for growth under osmoticstress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol. Cell. Biol. 14, 4135–4144.

Bertilsson, M., Olofsson, K., Lidén, G., 2009. Prefermentation improves xyloseutilization in simultaneous saccharification and co-fermentation of pretreatedspruce. Biotechnol. Biofuels 2, 8.

Erdei, B., Frankó, B., Galbe, M., Zacchi, G., 2012. Separate hydrolysis and co-fermentation for improved xylose utilization in integrated ethanol productionfrom wheat meal and wheat straw. Biotechnol. Biofuels 5, 12.

18 J.-Q. Zhu et al. / Bioresource Technology 169 (2014) 9–18

Erdei, B., Hancz, D., Galbe, M., Zacchi, G., 2013. SSF of steam-pretreated wheat strawwith the addition of saccharified or fermented wheat meal in integratedbioethanol production. Biotechnol. Biofuels 6, 169.

Girio, F.M., Fonseca, C., Carvalheiro, F., Duarte, L.C., Marques, S., Bogel-Lukasik, R.,2010. Hemicelluloses for fuel ethanol: a review. Bioresour. Technol. 101, 4775–4800.

Hoyer, K., Galbe, M., Zacchi, G., 2013. The effect of prehydrolysis and improvedmixing on high-solids batch simultaneous saccharification and fermentation ofspruce to ethanol. Process Biochem. 48, 289–293.

Jin, M.J., Gunawan, C., Balan, V., Lau, M.W., Dale, B.E., 2012. Simultaneoussaccharification and co-fermentation (SSCF) of AFEX™ pretreated corn stoverfor ethanol production using commercial enzymes and Saccharomyces cerevisiae424A(LNH-ST). Bioresour. Technol. 110, 587–594.

Jin, M.J., Lau, M.W., Balan, V., Dale, B.E., 2010. Two-step SSCF to convert AFEX-treated switchgrass to ethanol using commercial enzymes and Saccharomycescerevisiae 424A(LNH-ST). Bioresour. Technol. 101, 8171–8178.

Jin, M.J., Sarks, C., Gunawan, C., Bice, B.D., Simonett, S.P., Narasimhan, R.A., Willis,L.B., Dale, B.E., Balan, V., Sato, T.K., 2013. Phenotypic selection of a wildSaccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX™ pretreated corn stover. Biotechnol. Biofuels 6, 108.

Koppram, R., Olsson, L., 2014. Combined substrate, enzyme and yeast feed insimultaneous saccharification and fermentation allow bioethanol productionfrom pretreated spruce biomass at high solids loadings. Biotechnol. Biofuels 7,54.

Laluce, C., Tognolli, J.O., De Oliveira, K.F., Souza, C.S., Morais, M.R., 2009.Optimization of temperature, sugar concentration, and inoculum size tomaximize ethanol production without significant decrease in yeast cellviability. Appl. Microbiol. Biotechnol. 83, 627–637.

Lan, T.Q., Gleisner, R., Zhu, J.Y., Dien, B.S., Hector, R.E., 2013. High titer ethanolproduction from SPORL-pretreated lodgepole pine by simultaneous enzymaticsaccharification and combined fermentation. Bioresour. Technol. 127, 291–297.

Li, B.Z., Balan, V., Yuan, Y.J., Dale, B.E., 2010. Process optimization to convert forageand sweet sorghum bagasse to ethanol based on ammonia fiber expansion(AFEX) pretreatment. Bioresour. Technol. 101, 1285–1292.

Li, J., Li, S.Z., Fan, C.Y., Yan, Z.P., 2011. The mechanism of polyethylene glycol (PEG)4000 effect on enzymatic hydrolysis of lignocellulose. Colloids Surf. B 89, 203–210.

Liu, Z.H., Qin, L., Jin, M.J., Pang, F., Li, B.Z., Kang, Y., Dale, B.E., Yuan, Y.J., 2013.Evaluation of storage methods for the conversion of corn stover biomass tosugars based on steam explosion pretreatment. Bioresour. Technol. 132, 5–15.

Olofsson, K., Wiman, M., Lidén, G., 2010a. Controlled feeding of cellulases improvesconversion of xylose in simultaneous saccharification and co-fermentation forbioethanol production. J. Biotechnol. 145, 168–175.

Olofsson, K., Palmqvist, B., Lidén, G., 2010b. Improving simultaneoussaccharification and co-fermentation of pretreated wheat straw using bothenzyme and substrate feeding. Biotechnol. Biofuels 3, 17.

Öhgren, K., Vehmaanperä, J., Siika-Aho, M., Galbe, M., Viikari, L., Zacchi, G., 2007.High temperature enzymatic prehydrolysis prior to simultaneous

saccharification and fermentation of steam pretreated corn stover for ethanolproduction. Enzyme Microb. Technol. 40, 607–613.

Park, J.W., Takahata, Y., Kajiuchi, T., Akehata, T., 1992. Effects of nonionic surfactanton enzymatic-hydrolysis of used newspaper. Biotechnol. Bioeng. 39, 117–120.

Park, I., Kim, I., Kang, K., Sohn, H., Rhee, I., Jin, I., 2010. Cellulose ethanol productionfrom waste newsprint by simultaneous saccharification and fermentation usingSaccharomyces cerevisiae KNU5377. Process Biochem. 45, 487–492.

Pessani, N.K., Atiyeh, H.K., Wilkins, M.R., Bellmer, D.D., Banat, I.M., 2011.Simultaneous saccharification and fermentation of Kanlow switchgrass bythermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading,temperature and higher solid loadings. Bioresour. Technol. 102, 10618–10624.

Qin, L., Liu, Z.H., Li, B.Z., Dale, B.E., Yuan, Y.J., 2012. Mass balance and transformationof corn stover by pretreatment with different dilute organic acids. Bioresour.Technol. 112, 319–326.

Qin, L., Liu, Z.H., Jin, M.J., Li, B.Z., Yuan, Y.J., 2013. High temperature aqueousammonia pretreatment and post-washing enhance the high solids enzymatichydrolysis of corn stover. Bioresour. Technol. 146, 504–511.

Qing, Q., Yang, B., Wyman, C.E., 2010. Xylooligomers are strong inhibitors ofcellulose hydrolysis by enzymes. Bioresour. Technol. 101, 9624–9630.

Rudolf, A., Baudel, H., Zacchi, G., Hahn-Hägerdal, B., Lidén, G., 2008. Simultaneoussaccharification and fermentation of steam pretreated bagasse usingSaccharomyces cerevisiae TMB3400 and Pichia stipites CBS6054. Biotechnol.Bioeng. 99, 783–790.

Sassner, P., Galbe, M., Zacchi, G., 2007. Techno-economic evaluation of bioethanolproduction from three different lignocellulosic materials. Biomass Bioenergy10, 014.

Subtil, T., Boles, E., 2012. Competition between pentoses and glucose during uptakeand catabolism in recombinant Saccharomyces cerevisiae. Biotechnol. Biofuels 5,14.

Taherzadeh, M.J., Karimi, K., 2007. Enzyme-based hydrolysis processes for ethanolfrom lignocellulosic materials: a review. Bioresources 2, 707–738.

Wang, X., Li, B.Z., Ding, M.Z., Zhang, W.W., Yuan, Y.J., 2013a. Metabolomic analysisreveals key metabolites related to the rapid adaptation of Saccharomycescerevisiae to multiple Inhibitors of furfural, acetic acid, and phenol. OMICS 17,150–159.

Wang, Z.J., Lan, T.Q., Zhu, J.Y., 2013b. Lignosulfonate and elevated pH can enhanceenzymatic saccharification of lignocelluloses. Biotechnol. Biofuels 6, 9.

Zacchi, G., Axelsson, A., 1989. Economic evaluation of preconcentration inproduction of ethanol from dilute sugar solutions. Biotechnol. Bioeng. 34,223–233.

Zha, J., Shen, M.H., Hu, M.L., Song, H., Yuan, Y.J., 2013. Enhanced expression of genesinvolved in initial xylose metabolism and the oxidative pentose phosphatepathway in the improved xylose-utilizing Saccharomyces cerevisiae throughevolutionary engineering. J. Ind. Microbiol. Biotechnol. 41, 27–39.

Zhang, W., Lin, Y., Zhang, Q., Wang, X.Z., Wu, D.Y., Kong, H.N., 2013. Optimisation ofsimultaneous saccharification and fermentation of wheat straw for ethanolproduction. Fuel 112, 331–337.