Bioresource Technology 100 (2009) 3245–3251

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /b ior tech

Simultaneous saccharification and fermentation of lignocellulosic residuespretreated with phosphoric acid–acetone for bioethanol production

Hui Li a,b,1, Nag-Jong Kim a,1, Min Jiang b, Jong Won Kang a, Ho Nam Chang a,*

a Biochemical Engineering Lab, Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Koreab College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 210009, PR China

a r t i c l e i n f o

Article history:Received 16 October 2008Received in revised form 13 January 2009Accepted 14 January 2009Available online 16 March 2009

Keywords:Simultaneous saccharification andfermentationLignocellulosePretreatmentPhosphoric acid–acetoneBioethanol

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.01.021

* Corresponding author. Tel.: +82 42 350 3952; faxE-mail address: hnchang@kaist.edu (H.N. Chang).

1 Authors with equal contributions.

a b s t r a c t

Bermudagrass, reed and rapeseed were pretreated with phosphoric acid–acetone and used for ethanolproduction by means of simultaneous saccharification and fermentation (SSF) with a batch and fed-batchmode. When the batch SSF experiments were conducted in a 3% low effective cellulose, about 16 g/L ofethanol were obtained after 96 h of fermentation. When batch SSF experiments were conducted with ahigher cellulose content (10% effective cellulose for reed and bermudagrass and 5% for rapeseed), higherethanol concentrations and yields (of more than 93%) were obtained. The fed-batch SSF strategy wasadopted to increase the ethanol concentration further. When a higher water-insoluble solid (up to36%) was applied, the ethanol concentration reached 56 g/L of an inhibitory concentration of the yeaststrain used in this study at 38 �C. The results show that the pretreated materials can be used as good feed-stocks for bioethanol production, and that the phosphoric acid–acetone pretreatment can effectivelyyield a higher ethanol concentration.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The unavoidable depletion of the world petroleum supply andthe increasing problem of greenhouse gas effects have strength-ened the worldwide interest in alternative, nonpetroleum-basedsources of energy. The use of fuel ethanol will significantly reducethe net carbon dioxide emission once it replaces fossil fuels be-cause fermentation-derived ethanol is already a part of the globalcarbon cycle. However, the production cost should be reduced inorder to increase the market position of the biofuel. Nowadays,the raw materials and the production of enzymes are the two maincontributors to the overall costs; thus, the use of lignocellulosicresidues as a feedstock could reduce costs. Lignocellulosic ethanolproduction is attractive because the nonfood portion of the plantcan be used to produce ethanol; hence, there is no competitionfor feedstock with the food industry. Indeed a key trend in the mar-ket today is a move away from food crops to nonfood oilseed crops.

Lignocellulose is the most plentiful renewable biomass pro-duced from photosynthesis, and its annual production was esti-mated in 1 � 1010 MT worldwide (Sánchez and Cardona, 2008).The potential for using lignocellulosic materials in bioethanol pro-duction is well recognized. However, the task of hydrolyzing ligno-cellulose to fermentable monosugars is still technically

ll rights reserved.

: +82 42 350 3910.

problematic because the linear polymer has a strong crystallineand is usually surrounded by lignin, which reduces accessibilityto hydrolytic enzymes. Many pretreatment techniques have beenused to increase the hydrolysis of lignocellulosic biomass: for in-stance, dilute acid, ammonia recycle percolation, lime, steamexplosion (Hendriks and Zeeman, 2009), alkaline and acidic wetoxidation (Varga et al., 2004). All these technologies are usuallyexecuted under severe reaction conditions with a large capitalinvestment, high processing costs and great investment risks.One novel method that was recently introduced features modestreaction conditions (50 �C and atmosphere pressure): combininga nonvolatile cellulose solvent (phosphoric acid) and a second vol-atile organic solvent (acetone), this method separates the lignocel-lulose components and easily recycles both solvents (Zhang et al.,2007). This pretreatment process is an effective method for peren-nial plants as well as hard wood from the enzymatic digestibilityresults (Kim and Mazza, 2008). Although the highest sugar yieldsafter enzymatic hydrolysis were gained because of the absence ofsugar degradation and the isolation of high-value lignocellulosecomponents, a report has been recently published on the use ofthis method to produce ethanol from cotton-based waste textiles(Jeihanipour and Taherzadeh, 2009).

We used three kinds of biomass for production of cellulosic eth-anol. First, bermudagrass (Cynodon spp.) is an enduring grass usedas forage for livestock; it has potential as an energy crop for theproduction of biofuel through a thermochemical platform (Boatenget al., 2007) and sugar platform pretreated by means of ammonia

3246 H. Li et al. / Bioresource Technology 100 (2009) 3245–3251

fiber explosion (Holtzapple et al., 1994) or dilute acid (Sun andCheng, 2005). Second, the oilseed plant rapeseed has a high con-tent of cellulose in its straw stalk and could be a candidate for bio-mass energy (Karaosmanoglu et al., 1999a). The use of slowpyrolysis technology has been studied in terms of the productionof biofuel from the stalk of rapeseed (Karaosmanoglu et al.,1999b). And, the third, Phragmites communis (reed) is a good rawmaterial for papermaking because it has high content of lignocellu-lose, but there is a paucity of literature on the bioethanol produc-tion. The conversion of these biomasses would provide relativelynew sources of energy and chemicals. Here we present our studieson the pretreatment of reed, bermudagrass and rapeseed withphosphoric acid–acetone newly developed method and on thesimultaneous saccharification and fermentation (SSF) of thesematerials for the production of bioethanol. In order to reduce theenergy consumption in the distillation step, the ethanol concentra-tion should be greater than 4% (w/w). When the high solid concen-tration is applied in the fermentation process, the inhibitorysubstances are also increased, and it reduces the ethanol yield(Jorgensen et al., 2007). So, the effect of a high solid content inthe SSF process was also investigated.

2. Methods

2.1. Raw material

Bermudagrass, reed and rapeseed stover were collected fromthe banks of the Gapcheon river near the KAIST (Korea AdvancedInstitute of Science and Technology) in the city of Daejeon, Korea.The stover was then chopped, air-dried and stored at room temper-ature. Determination of the structural carbohydrates, lignin, andmoisture contents of the starting biomass materials followed stan-dard analytical methods established by the National RenewableEnergy Laboratory (NREL) (Hames, 2005; Sluiter, 2005, 2006).The acid insoluble residue was dried at 105 �C and weighted as Kla-son lignin. The lignocellulosic materials were milled and sieved,and the fraction between a 35-mesh screen and a 20-mesh screenwas collected for further use.

2.2. Lignocellulose pretreatment

Lignocellulosic materials were pretreated following the proce-dures provided by Zhang et al. (2007). A 50 g supply of each drymaterial was placed in a 500 mL glass beaker and mixed well with400 mL of concentrated phosphoric acid (of not less than 85%). Theslurry was incubated in a rotary air bath at 120 rpm and 50 �C for1 h. After a reaction, the slurry was poured into 1.2 L of pre-coldacetone and mixed thoroughly. The mixture was centrifuged at8000 rpm for 10 min, and the supernatant was collected. The solidsediment was suspended in 1.2 L of the acetone and centrifugedthree times. After that, the solid residue was washed again in1.2 L of distilled water and centrifuged three times. In the lastwater wash step, the pH was adjusted to 5.0–6.0 with 10 M NaOH.The water-insoluble solid (WIS) was collected for further use. Theacid concentrations were changed for the purpose of evaluatinghow the phosphoric acid concentration affects the enzymaticdigestibility.

A large amount of pretreated materials was obtained by usingcentrifugation instead of pressure filtration to separate the solidand liquid substances. To increase the solid content needed infed-batch fermentation, we gently dried the water-insoluble solidand turned it frequently to avoid over-drying under room temper-ature. Excessive water removal reduces the porosity in the pre-treated materials and then decreases the accessible surface areaof cellulose to enzymes. The saccharification data show that the

drying method has little effect when the dry material content isat 36% in bermudagrass, 70% in reed and 58% in rapeseed. Thesesemi-dried materials were therefore used for high solid contentbatch and fed-batch saccharification and fermentation.

2.3. Enzymatic saccharification

Enzymatic saccharification experiments were carried out instoppered conical flasks (125 mL) in the presence of 0.01% (w/v)sodium azide. The pH was adjusted to 4.8 with a 0.05 M citrate buf-fer and 25 filter paper unit (FPU) Celluclast� 1.5 L (Novozymes A/S,Denmark) per gram of effective cellulose was added to the pre-treated raw material in a total working volume of 50 mL. The flaskswere incubated at 50 �C on a rotary shaker (Vision Co., Korea) andagitated at 150 rpm. Sample aliquots of 1 mL were taken periodi-cally and centrifuged, and the supernatants were analyzed for sol-uble sugars. High solid content fed-batch hydrolysis was used toincrease the concentration of glucose in the hydrolysate. The initialeffective cellulose concentration was 100 g/L. An equal amount ofthe initial raw material and corresponding cellulase was added at8 h and another half portion was added at 16 h. The hydrolysisreaction was ended at 24 h. Before each feeding, the sample wasanalyzed by means of high performance liquid chromatography(HPLC). The enzymatic digestibility was calculated as follows:

Enzymatic digestibility ð%Þ ¼ Soluble sugars � 0:9Carbohydrate in substrate

� 100:

2.4. Separate hydrolysis and fermentation with high solid content

High solid content separate hydrolysis and fermentation (SHF)was carried out only for pretreated reed. The condition of thehydrolysis experiment was the same as that of the high solid con-tent fed-batch hydrolysis described above, but the citrate bufferwas replaced with diluted water and the sodium azide was omit-ted. The pretreated reed was autoclaved in liquid or solid form be-fore hydrolysis and the cellulase was filtered with a 0.2 lmmembrane. After 24 h hydrolysis, the ethanol fermentation wasstarted by inoculation of yeast seed. The fermentation was carriedout at 32 �C for 96 h and the flasks were agitated at 150 rpm. Usingthe formula in NREL LAP008 (Hayward et al., 1995), we calculatedthe ethanol yield as a percentage of the theoretical yield on the ba-sis of the total effective cellulose in the pretreated materials.

2.5. Simultaneous saccharification and fermentation

The SSF experiment was conducted in accordance with NRELLAP008 (Hayward et al., 1995). The 100 g starting medium, whichcontained 3% effective cellulose and 10 g of a 10 times concen-trated YP medium (yeast extract 100 g/L and peptone 200 g/L),was used for the fermentation in a 250 mL flask. The necessaryamount of deionized water was calculated and added to makethe total weight of the biomass and enzyme mixture equal to100 g. The untreated biomass and a-cellulose were also used asthe control. The pH was adjusted to 5.0 ± 0.2 with sulfuric acid.The flasks containing an untreated biomass were autoclaved at121 �C for 60 min; the other flasks were autoclaved for 30 min.After the flasks were cooled down to room temperature, we asep-tically added a mixture of 25 FPU Celluclast� 1.5 L/g cellulose and10% (v/v) yeast inoculum. The lost weight before and after theautoclaving was replenished as a volume of sterile deionizedwater.

The fermentation step was carried out in duplicate at 38 �C for96 h, and the flasks were agitated at 150 rpm. At appropriate sam-pling times, 1 mL samples were taken aseptically with sterile large-

H. Li et al. / Bioresource Technology 100 (2009) 3245–3251 3247

mouth pipette tips. After being diluted 10 times and vortex mixed,the samples were centrifuged at 12,000 rpm for 10 min. The etha-nol concentration and the remaining monosugars were determinedby means of HPLC. The same method described in Section 2.4 wasused to calculate the ethanol yield.

2.6. Preparation of yeast inoculum

The yeast Saccharomyces cerevisiae ATCC #24858 was used. Thestrain was stored in a glycerol mixture at �80 �C. We prepared theinoculum by transferring the microorganism in a glycerol stock to amedium of 50 mL MGYP (malt extract 0.3%, glucose 1%, yeast ex-tract 0.3%, peptone 0.5%) in a 250 mL flask. The growth was carriedout at 30 �C in an orbital shaker for 18 h. The amount of addedinoculum was 10% (v/v) of the SSF medium.

2.7. Analysis

The cellulase activity was assayed in terms of FPUs in accor-dance with the NREL standard procedure (Adney and Baker,1996). The soluble sugars were analyzed by means of HPLC (Varian,Inc. USA) with a refractive index detector. All the samples were fil-tered with a 0.22 lm disposable filter before the HPLC analysis. Thesamples from the acid hydrolysate were analyzed with an AminexHPX-87P column (Bio-Rad Laboratories Inc., USA). The temperaturewas 80 �C, and Millipore water was used for the mobile phase at aflow rate of 0.5 mL/min. The samples from the pretreatment liquid,enzymatic saccharification and SSF runs were separated on anAminex HPX-87H column (Bio-Rad Laboratories Inc., USA). Thetemperature was 50 �C, and 5 mM of H2SO4 (99.999%, Sigma–Al-drich, Inc. USA) was used as an eluent at a flow rate of 0.6 mL/min. The glucose and total xylose in the liquid fraction, which wereproduced by the water washing during the pretreatment, weredetermined after the removal of acetone and hydrolysis with 4%sulfuric acid at 121 �C for 1 h.

3. Results and discussion

3.1. Characterization of the raw material

The stover of bermudagrass, reed and rapeseed was character-ized by means of two-step acid hydrolysis. In the first step, the lig-nocellulose was decrystallized by concentrated sulfuric acid, andthe soluble carbohydrate was then hydrolyzed to monosugars withdiluted sulfuric acid at high temperature. Table 1 shows the aver-age composition of the three lignocellulosic materials expressed inweight percent of the dry matter. The raw material composition ofbermudagrass and reed was within the previously reported range.However, the glucose content in the rapeseed was much lowerthan previously reported (27.6 vs. 50.83) (Karaosmanoglu et al.,1999a). The lower glucose content may be due to the differencesin the kind of reeds, the cultivation climate and the harvest season.The lignocellulosic materials are a promising option as feedstockfor ethanol production, especially in terms of their abundant avail-ability, low cost (primarily due to transport) and ethanol yield. Themost important advantage of lignocellulosic materials is that theyare not directly related to food production. Bermudagrass and reed

Table 1Composition of the bermudagrass, reed and rapeseed stover.

Raw material Glucan Xylan Klason lignin Total solid content

Bermudagrass 47.8 13.3 19.4 91.2Reed 39.5 29.8 24.0 93.8Rapeseed 27.6 20.2 18.3 89.8

always grow on wastelands without the need of fertile cultivableland for cropping. The high content of glucose in bermudagrassand reed indicate that the two materials could both be a potentialfeedstock for bioethanol production. Rapeseed is widely plantedaround the world as an oil crop. After harvest, the use of residuerapeseed stalks for bioethanol production enhances the utilizationof the crop, providing a good way to improve the rural economy.

3.2. Effect of phosphoric acid concentration on enzymatic digestibility

Bermudagrass was pretreated at 50 �C for 60 min with differentphosphoric acid concentrations and the preteated samples werehydrolyzed by cellulase for 24 h. When 50% phosphoric acid wasused, the enzymatic digestibility was slightly increased comparingwith the untreated sample (43.1% vs. 39.6%). Raising the acid con-centrations to 73% hardly changes the enzymatic digestibility(53.6%). This result indicates that the lignocellulose structure can-not be broken down at concentrations of less than 73%. The enzy-matic digestibility reached 97.3% with the concentratedphosphoric acid pretreatment, which is consistent with the resultsfrom phosphoric acid swollen microcrystalline cellulose (Avicel); italso suggests that there is a phase transition from cellulose swell-ing to cellulose dissolution at higher H3PO4 levels in lignocellulose(Zhang et al., 2006). The existence of phase transition phenomenaimply that a high liquid–solid ratio is needed in the pretreatment.However, the high viscosity of phosphoric acid gives rise to theproblem of mass diffusion and mixing, especially during the initialpretreatment phase. Therefore, a new type of reactor should bespecially designed for this application.

The enzymatic hydrolysis profiles show that cellulase readilyattacked the more accessible lignocellulosic samples (30 g/Leffective cellulose) pretreated with concentrated phosphoric acid–acetone. The pretreated bermudagrass lignocellulose was quicklyhydrolyzed by up to 90% at 2 h and by up to 96% at 24 h. The samehydrolysis profiles were also found for reed and rapeseed. The dataof hydrolysis and digestibility from these three materials are sim-ilar to those from pretreated cellulosic samples (corn stover,switchgrass and hybrid poplar) reported by Zhang et al. (2007)and four representative lignocellulosic biomasses reported byKim and Mazza (2008). This similarity suggests that the pretreat-ment can breakdown the crystalline cellulose to amorphous cellu-lose and remove the majority of the hemicellulose and lignin indifferent lignocellulosic materials, even though they have differentphysical structures and chemical compositions.

Diluted phosphoric acid (2–6%) was always used for lignocellu-lose pretreatment because the microorganism nutrient salt sodiumphosphate is formed after neutralization of the hydrolysate withNaOH. However, the diluted phosphoric acid is usually applied athigh temperature or high pressure for a long time (Gamez et al.,2006; Romero et al., 2007) and requires a high energy supply.Moreover, a large amount of furfural, a common inhibitor in ligno-cellulose pretreatment, was produced during acid hydrolysis(Vázquez et al., 2007). Compared to diluted phosphoric acid pre-treatment under high temperature and high pressure conditions,the concentrated phosphoric acid pretreatment is easier undermodest reaction conditions and may also lower the capital costsof equipment and unit operation costs.

3.3. Effect of the concentration of pretreated material on enzymaticdigestibility

The enzymatic digestibility of the three raw materials pre-treated with the phosphoric acid–acetone method was studied interms of a raw material concentration of 20 g/L and an effectivecellulose concentration of 100 g/L. A portion greater than 99% ofthe cellulose in the reed and rapeseed was converted to glucose

Table 2Enzymatic digestibility at a raw material concentration of 20 g/L (24 h).

Raw material Enzymatic digestibility (%) Concentration of glucose (g/L)

Bermudagrass 97.5 9.35Reed 99.1 7.78Rapeseed 99.4 5.57

3248 H. Li et al. / Bioresource Technology 100 (2009) 3245–3251

with a raw material concentration of 20 g/L for 24 h (Table 2). Forbermudagrass, 97.5% of the cellulose was hydrolyzed. When theeffective cellulose concentrates were 30 g/L, the enzymatic digest-ibilities of the three materials were higher than 95%. Therefore, thepretreatment of these materials was an efficient way of yielding ahigh enzymatic digestibility.

A higher sugar concentration and, consequently, a higher finalethanol concentration can be obtained by increasing the raw mate-rial concentration. However, when the hydrolysis occurs with ahigh solid concentration, the high initial viscosity makes the mix-ing difficult. The fed-batch hydrolysis avoids this problem andmakes the reaction easy. When we used this method, the maxi-mum water-insoluble solid content reached 36.1% for reed andthe maximum glucose concentration reached about 123 g/L (Table3) for both bermudagrass and reed, though the enzymatic digest-ibility was about 70%. Due to the limitation of the initial viscosityand the inhibition of harmful products in the pretreatment, the lig-nocellulose hydrolysis was always carried out with a solid contentof less than 15% (Jorgensen et al., 2007). The cellulosic materialtreated with Zhang’s technology was easily hydrolyzed and the liq-uefaction was fast; hence, it was possible to increase the solid con-tent in the reaction and produce a high glucose concentration.

3.4. Ethanol production by batch SSF with 3% effective cellulose

When enzyme hydrolysis is applied, different levels of processintegration are possible. The classic configuration, known as SHF,is used for fermenting hydrolyzates; that configuration involves asequential process where the hydrolysis and fermentation are car-ried out in a different reactor. However, most workers in the fieldprefer the SSF route, in which enzymes and fermentative organ-isms are added to the same vessel to produce ethanol from sugarsas soon as they are released. Our batch SSF experiments were con-ducted using 3% effective cellulose with an enzyme loading of25 FPU/g cellulose, an initial pH of 5.0 ± 0.2, a temperature of38 �C, and a speed of 150 rpm. After 96 h of fermentation, thequantity of ethanol obtained from each of the three pretreatedmaterials was 16.1 g/L for the bermudagrass, 16.4 g/L for the reed,and 15.8 g/L for the rapeseed, respectively (Fig. 1a). Accordingly,the ethanol yields were 94.7%, 96.4% and 92.9%, respectively(Fig. 1b). The values for the ethanol concentration and yield from

Table 3Results of high solid content fed-batch hydrolysis.

Raw material Time (h) Enzymatic digestibility (%)

Bermudagrass 8b 65.216c 75.424 72.6

Reed 8b 62.716c 73.224 67.7

Rapeseed 8b 63.416c 57.024 67.9

a WIS: water-insoluble solid.b After sampling, equal amount of initial raw material and corresponding cellulase wc After sampling, a half amount of initial raw material and corresponding cellulase w

these three treated biomasses were all higher than those of the un-treated control materials and a-cellulose. The high ethanol yieldsindicate there is a little inhibitors in hydrolysates from phosphoricacid–acetone pretreated cellulosic materials. During the SSF pro-cess, the glucose produced by enzymatic hydrolysis is quicklyassimilated by yeast for cell growth and ethanol production; hence,only a low concentration of glucose remains in all the flasks(Fig. 1c). The phosphoric acid–acetone pretreatment is thereforesuitable for ethanol production with an SSF process.

3.5. Effect of solid content on the ethanol yield

To increase the ethanol concentration and test for the pres-ence of any inhibition in the SSF, we also conducted batch andfed-batch SSF experiments at a high effective cellulose content.The cellulose content in rapeseed is much lower than that of reedand bermudagrass. Hence, the fermentation broth with rapeseedcontains a higher level of solids in the same cellulose-based con-centration, and is so more viscous and difficult to be mixed thanreed and bermudagrass. We selected 10% effective cellulose forthe reed and bermudagrass (22.1% and 13.8% WIS, respectively)and 5% for the rapeseed (14.1% WIS) for high solid batch SSF.After 96 h of SSF, all the materials produced a high concentrationof ethanol (47.5 g/L for bermudagrass, 50.5 g/L for reed and27.6 g/L for rapeseed); furthermore, the ethanol yields were high-er than 90% (93% for bermudagrass, 98.7% for reed and 97.3% forrapeseed) (Fig. 2). The high ethanol yields from the high contentpretreated materials indicate that the concentration of inhibitorsproduced by the phosphoric acid–acetone pretreatment should below and therefore have no or little effect on the ethanol produc-tion. However, we found that the ethanol yield decreased sharplywhen the total WIS concentration reached 36.1% (reed), 21.2%(bermudagrass) and 21.8% (rapeseed), and the corresponding dataof the ethanol yield were 59.9%, 65.1% and 84.7% in the fed-batchSSF because of the high residual glucose concentration that re-mained in the final broth. Nevertheless, the ethanol yield(Fig. 3) was 20% higher than that of steam pretreated wheatstraw (Jorgensen et al., 2007) at the same solid concentration.Furthermore, if the residual glucose were converted to ethanol,the yield could have been higher than 77% for all biomasses(Table 4). Note also that the temperature in the SSF was 38 �C,which inhibits the yeast growth and ethanol production and pro-duces a low ethanol titer of about 56 g/L compared with a titer of87 g/L at 30 �C in our check-up test with glucose (data notshown). In addition, because most inhibitory components are re-moved in the acetone washing step, it would seem that the lowyield in the high solid content cannot be attributed primarily tothe harmful components produced by the pretreatment methodused here.

Glucose (g/L) Effective cellulose content (g/L) WISa (%)

72.4 100 13.8116.3 138.8 19.2123.6 153.1 21.2

69.0 100 22.1118.5 145.6 32.2122.9 163.3 36.1

35.2 50 14.148.6 76.8 21.857.9 76.8 21.8

as added.as added.

0 20 40 60 80 100

Fermentation time (h)

0

5

10

15

Eth

anol

con

cen

trat

ion

(g/

L)

RapeseedBermudagrassReedAvicel PH101

Untreated BermudagrassUntreated RapeseedUntreated Reed

0 20 40 60 80 100Fermentation time (h)

0

20

40

60

80

100

Eth

anol

yie

ld (

% t

heo

rati

cal y

ield

)

RapeseedBermudagrassReedAvicel PH101

Untreated BermudagrassUntreated RapeseedUntreated Reed

0 20 40 60 80 100Fermentation time (h)

0

2

4

6

8

Glu

cose

con

cen

trat

ion

(g/

L)

RapeseedReedBermudagrassAvicel PH101

Untreated RapeseedUntreated ReedUntreated Bermudagrass

a

b

c

Fig. 1. Simultaneous saccharification and fermentation of phosphoric acid/acetonepretreated cellulosic materials at 3% effective cellulose, enzyme loading of 25 FPU/gcellulose, initial pH 5.0 ± 0.2, 38 �C. (a) ethanol concentration, (b) ethanol yield and(c) glucose concentration.

Grass Reed Rapeseed0

20

40

60

Eth

anol

and

glu

cose

con

cent

rati

on (

g/L

)

0

20

40

60

80

100

Yie

ld o

f et

hano

l (%

)

Ethanol concentrationEthanol yieldResidue glucose

Fig. 2. Results of SSF tests at high content cellulose (10% effective cellulose for reedand bermudagrass and 5% for rapeseed).

0 10 20 30 40

Solid concentration (%)

20

40

60

80

100

Eth

anol

yie

ld (

% t

heor

atic

al y

ield

)

0

20

40

60

Eth

anol

con

cent

rati

on (

g/L

)

Ethanol yieldReedBermugrassRapeseed

Ethanol concentrationReedBermudagrassRapeseed

Fig. 3. Ethanol yield at different solid contents of reed, bermudagrass and rapeseed.After 96 h of fermentation, the glucose remains in the high solid contentfermentations. The dashed lines show the theoretically calculated yields based onthe total glucose formed during the fermentation. The solid line denotes the yieldbased on the total cellulose in the fermentation (see Table 4).

H. Li et al. / Bioresource Technology 100 (2009) 3245–3251 3249

3.6. Ethanol production from pretreated materials by fed-batch SHFand SSF

In the biomass-to-ethanol process, a high ethanol concentrationis required to reduce the energy input in the distillation for ethanolrecovery so that the total cost of the bioethanol is minimized. Formost types of lignocellulosic material, a solids concentration above15% dry matter is needed in the fermentation (Jorgensen et al.,2007). There are some technical problems such as mass transferwhen operating the hydrolysis and fermentation with a high solidscontent. To solve some of the problems, we used fed-batch SSF toincrease the ethanol concentration. The pre-weighted semi-driedmaterials were autoclaved at 121 �C for 30 min and fed at 24 hand 72 h to reduce the additional water-carrying. After 96 h of fer-mentation, the bermudagrass and reed flasks produced a maxi-mum ethanol concentration of 56.1 g/L and 55.0 g/L, though alarge amount of glucose remained in the broth (Table 4). Because

Table 4Results of high solid content fed-batch SHF and SSF.

Raw material Time (h) Ethanol (g/L) Yield (%) Residual glucose (g/L) ECCa (g/L) WISb (%)

Bermudagrass 24c 41.3 73.4 15.6 100 13.848d 42.8 54.8 48.7 138.8 19.272 54.8 63.6 35.4 153.1 21.296 56.1(74.0e) 65.1(82.3e) 35.0 153.1 21.2

Reed 24c 42.9 76.2 12.2 100 22.148d 47.5 58.0 39.7 145.6 32.272 55.5 60.5 38.8 163.3 36.196 55.0(75.3e) 59.9(77.0e) 39.9 163.3 36.1

(SHF result) 96 69.3 74.7 163.3 36.1

Rapeseed 24d 20.2 71.7 9.0 50 14.196 36.6(38.3e) 84.7(88.2e) 3.3 76.8 21.8

a ECC: effective cellulose concentration.b WIS: water-insoluble solid.c After sampling, equal amount of initial raw material and corresponding cellulase was added.d After sampling, a half amount of initial raw material and corresponding cellulase was added.e Values theoretically calculated based on the conversion of residual glucose to ethanol.

Table 5Comparison of SSF results from various lignocellulosic materials pretreated by different methods.

Raw material Pretreated method Microbial Ethanol con. (g/L) Yield (%) Remarks Reference

Rice hulls Alkaline peroxide E. coli strain FBR5 8.0 86.3 pH 6.0, 35 �C Saha and Cotta(2007)

Aspen Steam-exploded Saccharomyces cerevisiae 47 85 De et al. (2002)Corn cob Steeping in 2.9 M NH4OH Saccharomyces

1400(pLNH33)45 86 48 h Cao et al. (1996)

Crystalline cellulose Klebsiella oxytoca P2 47 86 35 �C and32 �C

Doran and Ingram(1993)

Corn stover Wet oxidation S. cerevisiae 52 83 Varga et al. (2004)Wheat straw Dilute sulfuric acid S. cerevisiae 57 80 Mohagheghi et al.

(1992)Commercial yellow poplar

sawdustTwo-temperature dilute-acidprehydrolysis

S. cerevisiae D5A 40 91 Torget et al. (1996)

Microcrystalline cellulose Phosphoric acid Kluyveromyces marxianusIMB3

7.0 70 Nilsson et al. (1995)

Bermudagrass 47.5 9310% ECa

Reed H3PO4-acetone S. cerevisiae 50.5 98.7 This workRapeseed 27.6 97.3 5% ECa

a EC: effective cellulose content.

3250 H. Li et al. / Bioresource Technology 100 (2009) 3245–3251

our SSF experiment was conducted at 38 �C, the yeast strain at thistemperature produced a maximum ethanol concentration of 56 g/Lfrom glucose because of ethanol tolerance. This result is consistentwith the results of previous SSF studies (Jorgensen et al., 2007; Mo-hagheghi et al., 1992). In SHF, the ethanol concentration reached69.3 g/L because a high concentration of glucose is produced inthe hydrolysis stage and the yeast grows at its optimal tempera-ture for ethanol production. In a comparison with the SSF resultsof the other studies, the pretreatment method used in this studyproduces a higher ethanol concentration than the previously re-ported methods (Table 5).

In the phosphoric acid–acetone pretreatment, the phosphoricacid removes almost all of the hemicellulose and partial ligninand, under moderate conditions, there was no additional harmfulcompounds produced. Acetone extracts most of the organic com-ponents. Hence, the pretreatment produces ‘‘clean” cellulose,which facilitates enzymatic hydrolysis and yeast fermentation.The fed-batch mode is an effective strategy for high solid contentSSF. The added solid materials are liquefied in a timely mannerby the cellulase and then converted to ethanol. In this study, forreed, the highest content of the total water-insoluble solid reached36.1% (Tables 3 and 4), and this value does not hinder the ethanolbioconversion. To the best of our knowledge, this is the best resultfor high solid content lignocellulosic material SSF, though the poorperformance of yeast limits any further increase of the ethanol

concentration. The results showed that the pretreated materialsby phosphoric acid–acetone could be used as good feedstock forbioethanol production, and SHF process could produce a higherethanol concentration than SSF process for the lignocellulosicmaterials pretreated by used method. Future work must examineaspects such as yeast with a higher performance in high tempera-ture, in situ extraction of ethanol in the broth, or advanced pro-cesses such as the continuous high cell density culture (Changet al., 1993, 2008; Jeon et al., 2007; Lee et al., 2000; Lim et al.,2008), the multi-stage continuous high cell density culture (Kwonet al., 2001) or cogeneration of valuable co-products (Cardona andSánchez, 2007). The optimization of the SHF and SSF process with afed-batch mode is also very useful for producing a higher concen-tration of ethanol (Kim et al., 1994).

4. Conclusion

The phosphoric acid–acetone method was applied in the pre-treatment of bermudagrass, reed and rapeseed for ethanol produc-tion by SHF and SSF with batch and fed-batch mode. In SHFprocess, the ethanol concentration reached 69.3 g/L from pre-treated reed because of the respective optimal temperatures forhydrolysis and fermentation. In batch SSF process, the highest eth-anol concentration of 50.5 g/L was obtained from pretreated reed

H. Li et al. / Bioresource Technology 100 (2009) 3245–3251 3251

and the ethanol yield were 98.7%. The fed-batch SSF strategy wasadopted to increase an ethanol concentration further and the eth-anol concentration reached 56 g/L of inhibitory concentration of ayeast strain used at 38 �C.

References

Adney, B., Baker, J., 1996. Measurement of Cellulase Activities. National RenewableEnergy Laboratory, Golden, CO, USA (LAP-006).

Boateng, A.A., Anderson, W.F., Phillips, J.G., 2007. Bermudagrass for biofuels: effectof two genotypes on pyrolysis product yield. Energy Fuels 21, 1183–1187.

Cao, N.J., Krishnan, M.S., Du, J.X., Gong, C.S., Ho, N.W., Chen, Z.D., Tsao, G.T., 1996.Ethanol production from corn cob pretreated by the ammonia steeping processusing genetically engineered yeast. Biotechnol. Lett. 18, 1013–1018.

Cardona, C.A., Sánchez, Ó.J., 2007. Fuel ethanol production: process design trendsand integration opportunities. Bioresour. Technol. 98, 2415–2457.

Chang, H.N., Lee, W.G., Kim, B.S., 1993. Cell retention culture with an internal filtermodule: continuous ethanol fermentation. Biotechnol. Bioeng. 41, 677–681.

Chang, H.N., Kim, B.J., Kang, J.W., Jeong, C.M., Kim, N.J., Park, J.K., 2008. High celldensity ethanol fermentation in an upflow packed-bed cell recycle bioreactor.Biotechnol. Bioprocess. Eng. 13, 123–135.

De, B.I., Viola, E., Barisano, D., Cardinale, M., Nanna, F., Zimbardi, F., Cardinale, G.,Braccio, G., 2002. Ethanol production at flask and pilot scale from concentratedslurries of steam-exploded aspen. Ind. Eng. Chem. Res. 41, 1745–1753.

Doran, J.B., Ingram, L.O., 1993. Fermentation of crystalline cellulose to ethanol byKlebsiella oxytoca containing chromosomally integrated Zymomonas mobilisgenes. Biotechnol. Prog. 9, 533–538.

Gamez, S., Gonzalez-Cabriales, J.J., Ramirez, J.A., Garrote, G., Vazquez, M., 2006.Study of the hydrolysis of sugar cane bagasse using phosphoric acid. J. Food Eng.74, 78–88.

Hames, B., 2005. Preparation of Samples for Compositional Analysis. NationalRenewable Energy Laboratory, Golden, CO, USA (Version 2005).

Hayward, T.K., Combs, N.S., Schmidt, S.L., Philippidis, G.P., 1995. SSF ExperimentalProtocols: Lignocellulosic Biomass Hydrolysis and Fermentation. NationalRenewable Energy Laboratory, Golden, CO, USA (LAP-008).

Hendriks, A.T.W.M., Zeeman, A.G., 2009. Pretreatments to enhance the digestibilityof lignocellulosic biomass. Bioresour. Technol. 100, 10–18.

Holtzapple, M.T., Ripley, E.P., Nikolaou, M., 1994. Saccharification, fermentation, andprotein recovery from low-temperature AFEX-treated coastal bermudagrass.Biotechnol. Bioeng. 44, 1122–1131.

Jeihanipour, A., Taherzadeh, M.J., 2009. Ethanol production from cotton-basedwaste textiles. Bioresour. Technol. 100, 1007–1010.

Jeon, B.Y., Kim, S.A., Kim, D.H., Na, B.K., Park, D.H., Tran, H.T., Zhang, R., Ahn, D.H.,2007. Development of a serial bioreactor system for direct ethanol productionfrom starch using Aspergillus niger and Saccharomyces cerevisiae. Biotechnol.Bioprocess. Eng. 12, 566–573.

Jorgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction oflignocellulose at high-solids concentrations. Biotechnol. Bioeng. 96, 862–870.

Karaosmanoglu, F., Tetik, E., Gurboy, B., Sanli, I., 1999a. Characterization of the strawstalk of the rapeseed plant as a biomass energy source. Energy Sources 21, 801–810.

Karaosmanoglu, F., Tetik, E., Gollu, E., 1999b. Biofuel production using slowpyrolysis of the straw and stalk of the rapeseed plant. Fuel Process. Technol.59, 1–12.

Kim, J.W., Mazza, G., 2008. Optimization of phosphoric acid catalyzed fractionationand enzymatic digestibility of flax shives. Ind. Crops Prod. 28, 346–355.

Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K., Woo, S.I., 1994. Production ofpoly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus withglucose concentration control. Biotechnol. Bioeng. 43, 892–898.

Kwon, S.H., Yoo, I.K., Lee, W.G., Chang, H.N., Chang, Y.K., 2001. High-rate continuousproduction of lactic acid by Lactobacillus rhamnosus in a two-stage membranecell-recycle bioreactor. Biotechnol. Bioeng. 73, 25–34.

Lee, W.G., Park, B.G., Chang, Y.K., Chang, H.N., Lee, J.S., Park, S.C., 2000. Continuousethanol production from concentrated wood hydrolysates in an internalmembrane-filtration bioreactor. Biotechnol. Prog. 16, 302–304.

Lim, S.-J., Kim, B.J., Jeong, C.-M., Choi, J., Ahn, Y.H., Chang, H.N., 2008. Anaerobicorganic acid production of food waste in once-a-day feeding and drawing-offbioreactor. Bioresour. Technol. 99, 7866–7874.

Mohagheghi, A., Tucker, M., Grohmann, K., Wyman, C., 1992. High solidssimultaneous saccharification and fermentation of pretreated wheat straw toethanol. Appl. Biochem. Biotechnol. 33, 67–81.

Nilsson, U., Barron, N., Mchale, L., Mchale, A.P., 1995. The effects of phosphoric acidpretreatment on conversion of cellulose to ethanol at 45 �C using thethermotolerant yeast Kluyveromyces marxianus IMB3. Biotechnol. Lett. 17,985–988.

Romero, I., Moya, M., Sánchez, S., Ruiz, E., Castro, E., Bravo, V., 2007. Ethanolicfermentation of phosphoric acid hydrolysates from olive tree pruning. Ind.Crops Prod. 25, 160–168.

Saha, B.C., Cotta, M.A., 2007. Enzymatic saccharification and fermentation ofalkaline peroxide pretreated rice hulls to ethanol. Enzyme Microb. Technol. 41,528–532.

Sánchez, Ó.J., Cardona, C.A., 2008. Trends in biotechnological production of fuelethanol from different feedstocks. Bioresour. Technol. 99, 5270–5295.

Sluiter, A., 2005. Determination of Total Solids in Biomass. National RenewableEnergy Laboratory, Golden, CO, USA (Version 2005).

Sluiter, A., 2006. Determination of Structural Carbohydrates and Lignin in Biomass.National Renewable Energy Laboratory, Golden, CO, USA (Version 2006).

Sun, Y., Cheng, J.J., 2005. Dilute acid pretreatment of rye straw and bermudagrassfor ethanol production. Bioresour. Technol. 96, 1599–1606.

Torget, R., Hatzis, C., Hayward, T.K., Hsu, T.A., Philippidis, G.P., 1996. Optimization ofreverse-flow, two-temperature, dilute-acid pretreatment to enhance biomassconversion to ethanol. Appl. Biochem. Biotechnol. 57 (58), 85–101.

Varga, E., Klinke, H.B., Reczey, K., Thomsen, A.B., 2004. High solid simultaneoussaccharification and fermentation of wet oxidized corn stover to ethanol.Biotechnol. Bioeng. 88, 567–574.

Vázquez, M., Oliva, M., Téllez-Luis, S.J., Ramírez, J.A., 2007. Hydrolysis of sorghumstraw using phosphoric acid: evaluation of furfural production. Bioresour.Technol. 98, 3053–3060.

Zhang, Y.H.P., Cui, J., Lynd, L.R., Kuang, L.R., 2006. A transition from celluloseswelling to cellulose dissolution by o-phosphoric acid: evidence fromenzymatic hydrolysis and supramolecular structure. Biomacromolecules 7,644–648.

Zhang, Y.H.P., Ding, S.Y., Mielenz, J.R., Cui, J.B., Elander, R.T., Laser, M., Himmel, M.E.,McMillan, J.R., Lynd, L.R., 2007. Fractionating recalcitrant lignocellulose atmodest reaction conditions. Biotechnol. Bioeng. 97, 214–223.