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Processoptimizationtoconvertforageandsweetsorghumbagassetoethanolbasedonammoniafiberexpansion(AFEX)pretreatment.BioresourTechnol

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Bioresource Technology 101 (2010) 1285–1292

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Bioresource Technology

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Process optimization to convert forage and sweet sorghum bagasse to ethanol basedon ammonia fiber expansion (AFEX) pretreatment

Bing-Zhi Li a, Venkatesh Balan b,*, Ying-Jin Yuan a,*, Bruce E. Dale b

a Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology,Tianjin University, P.O. Box 6888, Tianjin 300072, PR Chinab Biomass Conversion Research Lab (BCRL), Department of Chemical Engineering and Materials Science, Michigan State University, 3900 Collins Road,MBI International Building, Lansing, MI 48910, USA

a r t i c l e i n f o

Article history:Received 18 March 2009Received in revised form 1 September 2009Accepted 10 September 2009Available online 6 October 2009

Keywords:Ammonia fiber expansion pretreatmentForage sorghumSweet sorghum bagasseCellulosic ethanolFermentation

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.09.044

* Corresponding authors. Fax: +1 517 336 4615 (V. BYuan).

E-mail addresses: balan@msu.edu (V. Balan), yjyua

a b s t r a c t

With growing demand for bio-based fuels and chemicals, there has been much attention given to the per-formance of different feedstocks. We have optimized the ammonia fiber expansion (AFEX) pretreatmentand fermentation process to convert forage and sweet sorghum bagasse to ethanol. AFEX pretreatmentwas optimized for forage sorghum and sweet sorghum bagasse. Supplementing xylanase with cellulaseduring enzymatic hydrolysis increased both glucan and xylan conversion to 90% at 1% glucan loading. Highsolid loading hydrolyzates from the optimized AFEX conditions were fermented using Saccharomyces cere-visiae 424A (LNH-ST) without any external nutrient supplementation or detoxification. The strain was bet-ter able to utilize xylose at pH 6.0 than at pH 4.8, but glycerol production was higher for the former pH thanthe latter. The maximum final ethanol concentration in the fermentation broth was 30.9 g/L (forage sor-ghum) and 42.3 g/L (sweet sorghum bagasse). A complete mass balance for the process is given.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The development of alternatives to fossil fuels is receiving in-creased attention as it becomes an urgent global priority due tothe growing concern over the effect of green house gases on theenvironment and security of the global liquid fuel supply (Rubin,2008). Cellulosic ethanol could be a key alternative fuel, but severaltypes of feedstocks that grow well in different climatic conditionsare needed to make this fuel source viable. C4 plants, such as corn(Zea mays L.), sorghum (Sorghum sp.) and sugarcane (Saccharumsp.), are promising candidates for energy plants because of theirhigh photosynthetic efficiency and higher biomass yield per hect-are (Rubin, 2008). Based on a recent economic analysis, sweet sor-ghum is considered to be one of the most drought resistant cropsand has higher biomass yield and lower production costs thanmany other plants (Corredor et al., 2009). Forage sorghum, whichis typically grown for animal feed as silage, has a very short grow-ing period (about 60 days), much shorter than the growing periodrequired for corn or other kinds of sorghum (no less than120 days), including grain sorghum and sweet sorghum (Table 1).

Research has provided some basis for improving the sugar con-tent in sweet sorghum by controlling the planting conditions(Almodares and Sharif, 2007). Solid-state fermentation of sweet

ll rights reserved.

alan), +86 22 27403888 (Y.-J.

n@tju.edu.cn (Y.-J. Yuan).

sorghum stems has been developed due to their high sugar content(Gibbons et al., 1986; Kargi et al., 1985; Kargi and Curme, 1985; Yuet al., 2008). However, scale-up of solid-state fermentation is at abottleneck because of engineering problems associated with theprocess (Holker and Lenz, 2005). Another approach to utilizingsweet sorghum is processing the harvested plant into juice and ba-gasse. Sweet sorghum juice contains high concentrations of sugarand could be used directly as a substrate for ethanol production.Liu and Shen showed promising results for the conversion of sweetsorghum juice to ethanol by immobilized yeast cells (Liu and Shen,2008). Due to the similarity between sweet sorghum and sugar-cane, large scale industrial bio-refineries producing ethanol fromsweet sorghum juice can learn much from sugarcane-based etha-nol production. A huge amount of sweet sorghum bagasse will re-sult from the large scale production of sweet sorghum juice. Thereare several techniques to utilizing sweet sorghum bagasse such asburning, converting to methane and converting to ethanol (Gnan-sounou et al., 2005; Stamatelatou et al., 2003).

Ammonia fiber expansion (AFEX) is one of the most promisingpretreatment methods for lignocellulosic biomass when comparedto other leading pretreatment processes (Eggeman and Elander,2005). AFEX pretreatment is a dry-to-dry process (i.e., there is noliquid stream produced during pretreatment) which makes it quitedifferent from and potentially less costly than other pretreatmentmethods, such as dilute-acid and steam-explosion. Recent researchindicates that AFEX pretreatment also has the potential to reducethe mean ethanol selling price by about 60% below previous

Table 1Comparison of different sorghum plants to that of corn plants.

Grain sorghuma Sweet sorghumb Forage sorghumc Corn stoverd

Growth (days) 90–120 115–125 45–60 85–145Climate (temperature) Average temperatures > 80 F and

day-time temperatures > 90 FTolerant of drought Tolerate drought Base 50 F (Max 86 F, Min 50 F)

Soil condition More tolerant of wet soils andflooding as well as drought

Adapted to a wide range of soils pH > 5.5, optimum6.0–6.5.

Silt-loam and sandy-loam soilspH > 5.5

Yield 300–2000 kg/ha of grain in India andAfrica, 4500–6500 kg/ha in theUnited States

25,000–75,000 kg/ha of green matter 10,000–30,000 kg/ha of dry matter

9661 kg/ha of grain, 4830 kg/haof dry stover (in United States)

Fertilizer 112 kg/ha of nitrogen, 15.7 kg/ha ofphosphorous, and 15.7 kg/ha ofpotassium

100 kg/ha of nitrogen, 50 kg/ha ofphosphorous and 40 kg/ha ofpotassium

67–134 kg/ha ofnitrogen

148 kg/ha of nitrogen, 47 kg/haof phosphorous, and 54 kg/ha ofpotassium

Water consumption Low Medium Low High

a Data sources: http://www.hort.purdue.edu/newcrop/afcm/sorghum.html; http://www.ag.ndsu.nodak.edu/carringt/agalerts/milo.htm.b National sweet sorghum producers and processors association (http://www.ca.uky.edu/nssppa/sorghumfaqs.html).c http://www.ext.vt.edu/pubs/forage/418-004/418-004.html; http://www.hort.purdue.edu/newcrop/AFCM/forage.html.d http://www.ag.ndsu.edu/pubs/plantsci/rowcrops/a1173/a1173w.htm; GREET 1.8b, National Agricultural Statistics Services (NASS) and Agricultural Statistics Board, US

Department of Agriculture.

1286 B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292

estimates (Sendich et al., 2008). AFEX pretreatment has led tohigher conversions of several different kinds of cellulosic biomass,including corn stover (Teymouri et al., 2005), switchgrass (Ali-zadeh et al., 2005), Miscanthus (Chinese silver grass) (Murnenet al., 2007) and rice straw (Balan et al., 2008).

The objective of this paper is to optimize the biorefining processfor both forage sorghum and sweet sorghum bagasse, includingAFEX pretreatment, hydrolysis using commercial enzymes and eth-anol fermentation using Saccharomyces cerevisiae 424A (LNH-ST)without any external nutrient supplementation or detoxification.

2. Methods

2.1. Biomass source

Whole forage sorghum was received from Florida, USA. Sweetsorghum was from Binzhou in Shandong Province, China. Sweetsorghum stems were stripped from the leaves and squeezed by athree-roller mill in Key Laboratory of Systems Bioengineering inTianjin University. The residual bagasse was dried until the mois-ture content was between 5% and 10% based on the total weight.Both forage sorghum and sweet sorghum bagasse were groundusing a Wiley mill with a 5-mm diameter sieve and then storedat 4 �C in zip-lock bags until further use.

2.2. Washing protocol

In order to remove residual soluble sugars from sweet sorghumbagasse, about 400 mL of water was used to wash 100 g of dry ba-gasse three times. The washed biomass was squeezed by handusing cheese cloth to remove residual water. Then the bagassewas placed under a fume hood until the moisture content ap-proached 120% (dry weight basis; all moisture percentages werebased on dry weight throughout the study). The wash streamwas collected and stored at �20 �C until further use.

2.3. Composition analysis

The National Renewable Energy Laboratory (NREL) LAP-004 pro-tocol was used to determine the composition of biomass and extrac-tives (http://www.nrel.gov/biomass/analytical_procedures.html).

2.4. AFEX pretreatment

For quick optimization of AFEX pretreatment conditions, exper-iments were done in parallel 22-mL stainless steel reactors accord-

ing to the method outlined previously by Teymouri et al. (2005).Large scale AFEX pretreatment for fermentation experiments wasconducted in a 2-L stainless steel reactor. AFEX pretreated biomasswas kept under the fume hood overnight to remove residualammonia, and then stored at 4 �C until further use.

2.5. Enzymatic hydrolysis

The NREL standard protocol (LAP-009) was followed for enzy-matic hydrolysis. Both commercial cellulase (Spezyme CP) andxylanase (Multifect xylanase) were generous gifts from Genencor(Palo Alto, CA). A b-glucosidase (Novo 188) was purchased fromSigma–Aldrich (St. Louis, MO). Experiments at 1% glucan loadingwere conducted with 15-mL reaction volume in 20-mL scintillationvials, with the necessary commercial cellulase (31.3 mg/g glucan)and b-glucosidase (17.1 mg/g glucan) at 50 �C and 150 rpm. About0.05 M citrate buffer (pH 4.8) with 30 mg/L cycloheximide and40 mg/L tetracycline were used in enzymatic hydrolysis. Certainsamples were also hydrolyzed using additional commercial xylan-ases (0–32.2 mg/g glucan). The samples were taken during enzy-matic hydrolysis as previously described in Balan et al. (2008).The samples were frozen at �20 �C for subsequent HPLC sugaranalysis. The protein concentrations of the enzymes were deter-mined by the BCA protein assay (Pierce, Rockford, IL). The proteinconcentrations of the respective enzymes were as follows: Spe-zyme CP (123 mg/mL; 59 FPU/mL; where FPU stands for filter pa-per units), b-glucosidase (67 mg/mL; 250 pNPGU/mL; pNPGUstands for p-nitrophenyl-glucoside units), and Multifect xylanase(42 mg/mL).

High solid loading enzymatic hydrolysis prior to fermentationand the mass balance experiments were conducted in 2000 or250 mL flasks, respectively. High solid loading enzymatic hydroly-sis was performed with 40 mg/L tetracycline addition, at 50 �C andat pH 4.8 with 250 rpm shaking. Half of the biomass and enzymeswere used to start the hydrolysis, with the second half added intothe flask after 3 h. Cellulase was supplemented with xylanase asdescribed above. The hydrolyzate was filtered against a 0.2 lm fil-ter prior to fermentation.

The glucan and xylan conversion was calculated based on thetheoretical sugar release from biomass, i.e., 1.11 g glucose/g glucanand 1.14 g xylose/g xylan.

2.6. Ethanol fermentation

S. cerevisiae 424A (LNH-ST) genetically engineered to fermentxylose (Ho et al., 2000), and provided by Dr. Nancy Ho at Purdue

Fig. 1. The effect of AFEX pretreatment on sweet sorghum bagasse before (A) andafter (B) washing. Hydrolysis experiments were done at 50 �C and 150 rpm using 1%glucan loading with 31.3 mg cellulase/g glucan and 17.1 mg b-glucosidase/g glucan.The corresponding glucose concentration (white shade) and xylose concentration(gray shade) after 72 h in the hydrolysates are shown. Here, UT-NE, untreatedsample without enzymes loading; UT-E, untreated sample with enzymes loading;AFEX-NE, AFEX treated sample without enzymes loading; AFEX-E, AFEX treatedsample with enzymes loading. AFEX condition was 2:1 ammonia loading to biomassloading, 120% moisture content and 140 �C for 5 min. Standard deviations arecalculated based on duplicate experiments.

B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292 1287

University, was used to ferment sweet sorghum bagasse and foragesorghum hydrolyzates.

A single colony of S. cerevisiae 424A (LNH-ST) was picked from aYEPD-Agar plate (10 g/L yeast extract, 20 g/L peptone, 50 g/L glu-cose, and 20 g/L agar), inoculated into a 250-mL flask with 50-mLYEPD media (10 g/L yeast extract, 20 g/L peptone and 50 g/L glu-cose), and incubated overnight at 150 rpm and 30 �C as the seedfor fermentation.

The fermentation was carried out in 250-mL flasks with 60-mLhydrolyzate at 150 rpm and 30 �C. The initial cell density of the fer-mentation was OD600 = 0.5 (about 0.275 g dry cell/L). The fermen-tation broth was sampled (0.9 mL) at regular intervals todetermine cell density, sugar and ethanol present in the broth.All the fermentation experiments were done without any externalnutrient supplementation or detoxification. Ethanol yield was esti-mated based on total consumed sugars. The theoretical maximumyield was 0.51 g EtOH/g sugar.

2.7. Monomeric sugar and ethanol analysis using HPLC

Sugar and ethanol analysis was performed using a HPLC-RIdetector system. This system consisted of a Waters (Milford, MA)515 Pump connected to a Waters 410 refractive index detector.An Aminex HPX-87P carbohydrate analysis column (Bio-rad, Her-cules, CA) equipped with a deashing guard cartridge (Bio-rad)was used for quantifying sugars in hydrolyzate. Degassed HPLCgrade water was used as the mobile phase at 0.6 mL/min at a col-umn temperature of 85 �C. The injection volume was 10 lL with arun time of 20 min. An Aminex HPX-87H column (Bio-rad) wasused to quantify both sugar and ethanol concentrations for fer-mentation samples and acid hydrolyzed samples. The temperatureof the HPX-87H column was maintained at 50 �C. The mobile phaseused for this column was 5 mM sulfuric acid (H2SO4) at a flow rateof 0.6 mL/min.

2.8. Error analysis

The hydrolysis and fermentation were performed in duplicate,and the composition analysis was done in triplicate. Error bars rep-resent the standard deviation of the replicates. For all significancetests, a student t-test was used requiring a probability p < 0.05 tobe significant.

3. Results

3.1. Composition analysis

Both forage and sweet sorghum bagasse were extracted withwater and 95% ethanol using an ASE2000 (Accelerated SolventExtractor, DIONEX, CA) to remove extractives before acid hydroly-sis. HPLC results showed that there were no free monomeric sugarsin the extractives for forage sorghum, while a considerable amountof monomeric sugar was found in sweet sorghum bagasse (Fig. 1).The composition analysis (Table 2) showed 35.6% and 38.3% of glu-can and 18.4% and 18.2% of xylan for forage and washed sweet sor-ghum bagasse, respectively.

3.2. Effect of sweet sorghum bagasse washing and AFEX pretreatment

After AFEX pretreatment the free glucose content decreased to1.5 from 5 g/L, which indicates that free monomeric sugars weredegraded to other products during pretreatment (Fig. 1A). Highconcentrations of free sugars were found in unwashed sweet sor-ghum bagasse due to residual sugar left after removing sugar juicefrom the stems. The free sugar was subsequently removed using

triple water washing. About fourfold increases in both glucanand xylan conversion could be seen in AFEX treated washed sweetsorghum bagasse samples when compared to untreated washedsamples. These results indicate that AFEX pretreatment facilitatesenzymatic hydrolysis and enhances glucan/xylan conversion inthe hydrolysis (Bradshaw et al., 2007; Teymouri et al., 2005).

3.3. Optimization of AFEX conditions

Four parameters for AFEX pretreatment were varied in the opti-mization of the conditions for both sweet sorghum bagasse andforage sorghum, including: biomass moisture content, the ratioof ammonia to dry biomass loading, reaction temperature, and res-idence time. The glucan/xylan conversion of untreated sweet sor-ghum bagasse and forage sorghum was very low; 9.9% and 21.8%glucan conversion and 6.6% and 13.1% xylan conversion, respec-tively. For both sweet sorghum bagasse and forage sorghum, AFEXpretreatment significantly increases the monomeric sugars afterenzymatic hydrolysis. In addition, glucan/xylan conversions wereconsistently higher at 2:1 ammonia to biomass loading than at1:1 loading.

Table 2Composition of forage sorghum (FS) and sweet sorghum bagasse (SSB).

Composition FS(UWUTa)

SSB(UWUTa)

SSB(WUTb)

Glucan 35.6 ± 0.8 27.3 ± 0.4 38.3 ± 0.3Xylan 18.4 ± 0.5 13.1 ± 0.1 18.2 ± 0.3Arobilan 1.8 ± 0.1 1.4 ± 0.0 1.8 ± 0.1Lignin 18.2 ± 0.2 14.3 ± 0.2 19.7 ± 1.1Extractives 18.7 ± 0.1 32.3 ± 0.7 14.5 ± 1.0

All data represent the percentage based on dry weight and are means of theduplicates ± standard deviation.

a Unwashed and untreated.b Washed and untreated.

1288 B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292

For washed sweet sorghum bagasse, by varying AFEX pretreat-ment followed by enzymatic hydrolysis (31.3 mg/g glucan of cellu-lase, 17.1 mg/g glucan of b-glucosidase, 50 �C, 150 rpm, 1% glucanloading), it was found that there was no significant increase in glu-can/xylan conversion beyond 2:1 ammonia to biomass loading andbeyond 120% moisture. On the other hand, increasing the pretreat-ment temperature from 100 to 140 �C increases both glucan andxylan conversions significantly. The residence time had very littleimpact on both glucan and xylan conversions. Based on theseexperiments, the optimal AFEX conditions for washed sweet sor-ghum bagasse are 120% moisture content, 2:1 ammonia to biomassloading, 140 �C and 30 min residence time. Under these conditions,glucan and xylan conversion reached about 80% and 90%, respec-tively, during enzyme hydrolysis.

For forage sorghum, three different temperatures (100, 120 and140 �C) and three different moisture contents (60%, 120% and 233%)were used for two different ammonia to biomass loading (1:1 and2:1) conditions. At 1:1 ammonia to dry biomass loading, glucan/xy-lan conversions after enzymatic hydrolysis were higher at 60%moisture, while, at 2:1 ammonia loading, glucan/xylan conversionswere highest at 120% moisture. This difference could be due to thechemical reaction that occurs between ammonia and water duringthe pretreatment process. At 2:1 ammonia loading, higher glucanconversions were obtained at the higher temperature (140 �C),but at low ammonia loading, the effect of temperature was not sopronounced and seemed to depend on the moisture content. Basedon both glucan and xylan conversions, the optimal AFEX conditionsfor forage sorghum were 2:1 ammonia to biomass loading, 120%moisture, 140 �C and 5 min for residence time.

3.4. Optimization of xylanase loading

Using standard enzymatic hydrolysis conditions (31.3 mg/g glu-can of cellulase and 17.1 mg/g glucan of b-glucosidase), the effectof xylanase loading on glucan/xylan conversion was investigatedat 1% glucan loading with different xylanase loadings (0, 7.8, 15.6and 32.2 mg/g glucan). Based on proteomic analysis and activityassays we found that there is very little xylanase in Spezyme CP(data not shown). Previous reports have shown that adding xylan-ase to cellulase improved both glucan and xylan conversion (Chun-dawat et al., 2007). For AFEX-treated forage sorghum samples,varying the xylanase loading improved both the glucan and xylanconversion by about 3% and 7%, respectively.

3.5. Fermentation of hydrolyzates

Fermentation results for both sweet sorghum bagasse and for-age sorghum are summarized in Table 3. The concentrations of glu-cose, xylose, ethanol and glycerol in the fermentation broth as afunction of time are given in Fig. 2. For sweet sorghum bagasse,only hydrolyzate with 6% glucan loading was used and ethanol fer-mentation was performed using S. cerevisiae at pH 6.0 with and

without the water wash stream (Fig. 2IA and IB). For forage sor-ghum, two different hydrolyzates were prepared for fermentation(1% or 6% glucan loading) and ethanol fermentations were doneat two different pH (4.8 and 6.0) conditions (Fig. 2IIA-D).

In the case of sweet sorghum bagasse, the initial glucose con-centration in the hydrolyzates was 54.3 g/L without the washstream and 66.6 g/L with the wash stream. The volume recoveryof the hydrolyzate with 6% glucan loading after centrifugationwas 79% (v/v). The supernatant was removed from unhydrolyzedsolids by centrifugation at 16,000 rpm for 30 min. The glucan andxylan conversions at 6% glucan loading were found to be 64.5%and 74.3% (without wash stream) and 66% and 82.4% (with washstream), respectively. The final concentration of ethanol withoutthe wash stream was 29.4 g/L, while it was 42.3 g/L with washstream. The xylose utilization during fermentation was found tobe 55.6% (with wash stream) and 51.3% (without wash stream).Glycerol, one of the side products, was also found in the fermenta-tion broth and tended to increase as a function of time. However,the amount of xylitol produced in the fermentation broth withthe wash stream was greater than the amount produced withoutthe wash stream. It is interesting to note that cell density in thehydrolysis with the wash stream was higher than the one withoutthe wash stream (Fig. 3A). One can see a logarithmic increase incell density in the first 24 h, after which it levels off. As we cansee in Fig. 2, glucose was consumed within the first 24 h of fermen-tation while it took a longer time for the xylose to be consumed.The ethanol yield was found to be 96.9% (for hydrolyzate withwash stream) and 82.2% (for hydrolyzate without wash stream).

For forage sorghum hydrolyzates, the final ethanol concentra-tions were found to be 3.9 g/L (1% glucan loading, pH 4.8), 30.3 g/L (6% glucan loading, pH 4.8), 4.1 g/L (1% glucan loading, pH 6.0)and 30.9 g/L (6% glucan loading, pH 6.0) (Fig. 2IIA and B). Hereagain, glucose was consumed within the first 12 h (1% glucan load-ing) and 24 h (6% glucan loading) during the fermentation ofhydrolyzates, while it took longer to consume xylose. Glycerolwas also produced during fermentation and the final concentrationof glycerol was higher at pH 6.0 than that at pH 4.8. About 40%more glycerol was produced during fermentation at pH 6.0 thanthat at pH 4.8.

Cell density was found to be higher at pH 6.0 when compared topH 4.8 for 6% glucan loading hydrolyzates, while no significant dif-ference was noticed for the 1% glucan loading hydrolyzates(Fig. 3B). For 6% glucan loading hydrolyzates, unutilized xylose lev-els were about 17.7 g/L (pH 4.8) and 13.0 g/L (pH 6.0) (i.e., onlyabout 40% of the available xylose was consumed in the fermenta-tion at pH 4.8, while more than 50% of the available xylose wasconsumed at pH 6.0).

3.6. Mass balance

A mass balance was developed for the complete conversion ofboth sweet sorghum bagasse and forage sorghum to ethanol basedon enzymatic hydrolysis at 6% glucan loading (Fig. 4). There is noevident mass loss during AFEX pretreatment, which is a dry-to-dry process. The conversions during enzymatic hydrolysis for for-age sorghum were 74% for glucan and 73% for xylan; 29.2 g of glu-cose and 15.2 g of xylose were obtained with 6% glucan loadingusing 100 g of AFEX-treated forage sorghum. The initial cell densityof S. cerevisiae in the fermentation was OD600 = 0.5. The ethanolconcentration produced from the fermentation was 30.8 g/L at96 h. After 96 h of inoculation, all of the glucose and 53% of the xy-lose was consumed. From 100 g of dry forage sorghum, 17.1 g eth-anol was produced, giving a 90.0% ethanol yield from thefermentation (Fig. 4A).

The conversion for sweet sorghum bagasse enzymatic hydroly-sis was 67% for glucan and 76% for xylan without wash stream while

Table 3Summary of the fermentation performance for both forage sorghum (FS) and sweet sorghum bagasse (SSB).

Biomass Glucan loading (g/mL) Fermentation pH cglya (g/L) ceth

b (g/L) cxylc (g/L) consxyl

d (%) Yethe (%)

FS 1% 4.8 0.3 ± 0.0 3.9 ± 0.3 0.7 ± 0.0 85.4 ± 0.6 61.6 ± 5.3FS 6% 4.8 2.6 ± 0.4 30.3 ± 0.1 17.7 ± 2.5 38.0 ± 2.5 96.0 ± 0.4FS 1% 6.0 0.3 ± 0.0 4.1 ± 0.1 0.6 ± 0.0 88.9 ± 0.7 66.9 ± 1.4FS 6% 6.0 3.6 ± 0.0 30.9 ± 0.1 13.0 ± 0.2 57.0 ± 1.1 86.2 ± 0.2SSB1f 6% 6.0 6.1 ± 0.2 42.3 ± 0.3 15.0 ± 0.6 55.6 ± 2.7 96.9 ± 0.7SSB2g 6% 6.0 3.5 ± 0.0 29.3 ± 1.0 14.6 ± 0.7 51.3 ± 2.8 82.4 ± 2.7

All data are means of the duplicates ± standard deviation.a Concentration of glycerol produced in the fermentation.b Concentration of ethanol produced in the fermentation.c Concentration of xylose left at the end of fermentation.d Xylose consumption based on the initial concentration of xylose.e Ethanol yield based on the theoretical ethanol production of the consumed sugars.f With wash stream.g Without wash stream.

Fig. 2. Ethanol fermentation of both sweet sorghum bagasse and forage sorghum biomass hydrolyzates using genetically modified Saccharomyces cerevisiae 424A (LNH-ST).Here IA, sweet sorghum bagasse hydrolyzate without wash stream fraction and IB, sweet sorghum bagasse hydrolyzates (1% glucan loading) with water washed fraction; IIAand IIC, Forage sorghum hydrolyzates (1% and 6% glucan loading, respectively) at pH 4.8; IIB and IID, forage sorghum hydrolyzates (1% and 6% glucan loading, respectively) atpH 6.0. All the fermentations were done in duplicates and the average data from those experiments are presented here. The standard deviations were less than 2%.

B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292 1289

0

3

6

9

12

without washing stream with washing stream

OD

600

Fermentation Progress (hour)

A. Sweet sorghum bagasse

0 25 50 75 100

0 25 50 75 100

1% (pH4.8) 6% (pH4.8)

Fermentation Progress (hour)

B. Forage sorghum

OD

600

1% (pH6.0)

0

3

6

9

12

6% (pH6.0)

Fig. 3. Measurement of yeast cell density using spectrophotometer (OD600) duringethanol fermentation experiments. Here, (A) Cell density of yeast in sweet sorghumbagasse hydrolyzate fermentation broth. (B) Cell density of yeast in forage sorghumhydrolyzate fermentation broth.

1290 B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292

68% for glucan and 88% for xylan with wash stream. About 20.4 g(25.0 g) of glucose and 11.3 g (12.7 g) of xylose were obtained with6% glucan loading using 100 g of AFEX treated sweet sorghum ba-gasse without (with) wash stream in the hydrolyzates. From100 g of dry pretreated sweet sorghum bagasse, 11.0 g (15.9 g) eth-anol was produced with 82.2% (96.9%) ethanol yield in the fermen-tation using the hydrolyzates without (with) the wash stream.

4. Discussion

4.1. AFEX pretreatment and enzymatic hydrolysis

AFEX is an efficient pretreatment method that improves the su-gar release from cellulose and hemicellulose (Chundawat et al.,2007). For forage sorghum, the glucan conversion after enzymatichydrolysis was 80% for AFEX-treated biomass and 20% for untreatedmaterial. Xylan conversion increased to 83% for treated biomassfrom 13% for the untreated material. Similar improvements for glu-can/xylan conversion were also found for other types of biomass,such as corn stover, switchgrass, Miscanthus, rice straw, reed canary-grass and distillers’ grains (Alizadeh et al., 2005; Balan et al., 2008;Bradshaw et al., 2007; Murnen et al., 2007; Teymouri et al., 2005).The optimal AFEX conditions for forage sorghum and sweet sorghumbagasse were not the same as those for other biomass types, whichcan be explained by variation in composition as well as differencesin the lignin/cellulose/hemicellulose matrix structure. Further stud-

ies are necessary to determine the precise reasons for these differ-ences in optimal conditions. Sweet sorghum bagasse containedfree sugars which were found to be destroyed during AFEX pretreat-ment. Washing the biomass prior to AFEX pretreatment was an effi-cient way to remove the free sugars in sweet sorghum bagasse.

Different types of biomass have different cell wall structureswith different architectures of cellulose, hemicellulose and lignin(Mosier et al., 2005). Even for the same type of biomass, many fac-tors can affect the composition of the biomass, such as growth per-iod, environmental conditions (pH, temperature, availability ofsunlight and rain), and storage conditions (Bradshaw et al., 2007;Weimer and Springer, 2007). Because of these differences, the ratiosof the enzymes needed during hydrolysis can vary with the type ofbiomass. The most widely-used enzymes for hydrolysis of celluloseare commercial enzymes such as the enzymes used here: SpezymeCP, Novozym 188, and Multifect xylanase. These enzymes are notpure enzymes, but a mixture of several enzymes including varyingamounts of cellulases and hemicellulases. The amount of xylanasein Spezyme CP is relatively low and supplementation of xylanaseis necessary in order to improve xylan conversion (Chundawatet al., 2007). When xylanase was added to the hydrolysis systemalong with the cellulase enzymes, the glucan conversion was in-creased by about 7%. The increase in glucan conversion may alsobe due to synergistic effects between cellulase, b-glucosidase, andxylanase (Murashima et al., 2003; Kumar and Wyman, 2009).

4.2. Ethanol fermentation

S. cerevisiae 424A (LNH-ST) has been shown to be an excellentstrain for cellulosic ethanol production (Govindaswamy and Vane,2007; Lau and Dale, 2009; Zhong et al., 2009) because of its abilityto consume both glucose and xylose during anaerobic fermenta-tion. We used this strain to ferment the hydrolyzates from AFEX-treated forage and sweet sorghum bagasse without any externalnutrient supplementation or detoxification. It is very importantto keep the pH within the optimal range for yeast during the fer-mentation. We found that the production of glycerol, the mainby-product in glucose consumption, was higher at pH 6.0 than atpH 4.8. It was shown in earlier work that higher pH could stimulatethe biosynthesis of glycerol (Ingledew, 1999). The optimal pH forxylose utilization was not the same as that for glucose fermenta-tion. Higher xylose conversion was observed at pH 6.0 than at pH4.8, as reported in previous work (Sedlak and Ho, 2004). Althoughxylose conversion was much higher at pH 6.0, ethanol concentra-tion only increased slightly (Table 3). This was due to increasedglycerol production in the glucose consumption stage. More re-search is needed to find an optimal pH point for maximum ethanolproduction from both glucose and xylose consumption.

In the fermentation of hydrolyzates at 1% glucan loading, xylosewas almost completely consumed at both pH 4.8 and pH 6.0(Fig. 2IIA and B). This indicates that this strain has a very good abil-ity to utilize glucose and xylose. For the hydrolyzates from 6% glu-can loading, however, a high xylose concentration remained at theend of fermentation. The consumption curves for xylose show thatthe rate of xylose consumption following the glucose disappear-ance decreased as the fermentation progressed. These results agreewith previously reported results (Lau and Dale 2009; Sedlak andHo, 2004).

One possible reason for the incomplete xylose utilization maybe inhibitors in the fermentation broth. Degradation products pro-duced during AFEX pretreatment, such as various phenolic acids,are present in lower concentrations compared to other pretreat-ment methods, but they may still have negative effects on the fer-mentation (unpublished results). Additionally, ethanol is alsoknown to inhibit the microorganism (Dasari et al., 1990).

Fig. 4. Complete mass balances for the entire process (pretreatment, hydrolysis and fermentation). (A) Forage sorghum; (B) sweet sorghum bagasse without wash steam and(C) sweet sorghum bagasse with wash stream.

B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292 1291

The ethanol yield was very high (about 96.9%) when fermenta-tion was done using AFEX treated sweet sorghum bagasse hydroly-zate with wash stream. Many researchers believe that hydrolyzatesfrom biomass are not a rich media for microorganisms (Kadam andNewman, 1997) and have added different nutrients to supplementthe fermentation (Kim et al., 2006; Klinke et al., 2003; Okuda et al.,2007). Our experiments have shown that this is not true; most bio-mass contains adequate nutrients but harsh pretreatments (suchas dilute-acid) and subsequent washing destroy or remove thesenutrients. AFEX does not destroy these nutrients, so fermentationexperiments using AFEX-treated biomass can be performed with-out any external nutrient supplementation.

The genetic backgrounds of sweet sorghum and forage sorghumare quite different, and these genetic differences might affect thechemical components of the biomass and theoretic ethanol yield(Shaug and Lo, 1997; Tew et al., 2008). Therefore, the differencesin the optimal AFEX conditions and fermentation performancefor the two kind feed stocks are reasonable.

4.3. Mass balance

From the mass balance, it is clear that glucan/xylan conversionduring enzymatic hydrolysis and the utilization of xylose duringethanol fermentation were the limiting steps during conversionof both sweet sorghum bagasse and forage sorghum to ethanol.Glucan/xylan conversion during enzymatic hydrolysis depends on

the pretreatment conditions and enzymes used in the hydrolysis(Eijsink et al., 2008). Although sorghum was treated using effectivepretreatment conditions, a better cocktail of enzymes is needed inorder to get higher conversion of glucan and xylan (Berlin et al.,2007; Zhang et al., 2006). Although glucose and xylose were uti-lized by S. cerevisiae 424A (LNH-ST) during the fermentation, therate of xylose consumption is much slower than that of glucose.

In conclusion, a complete process for producing ethanol fromforage and sweet sorghum bagasse has been reported in this work.AFEX conditions for forage sorghum and sweet sorghum bagasse,enzyme loading during hydrolysis and pH in fermentation wereoptimized in this work. The yeast strain S. cerevisiae 424A (LNH-ST) was used in the fermentation of hydrolyzates from AFEX-trea-ted forage and sweet sorghum bagasse without nutrient supple-mentation or detoxification. Wash stream from sweet sorghumbagasse was proved to be an efficient supplementation for the fer-mentation of the hydrolyzates from cellulosic biomass. Xylose con-sumption was slow and not complete during fermentation andfurther microbial improvements are needed to consume both glu-cose and xylose rapidly and completely.

Acknowledgements

The authors thank Rebecca Garlock and Tiffany Draut for theirassistance during the editing and revision process and Leonardoda costa Sousa and Ming W. Lau for their help while carrying out

1292 B.-Z. Li et al. / Bioresource Technology 101 (2010) 1285–1292

the experiments. The authors are grateful for the financial supportfrom Michigan State University Research Foundation, NationalNatural Science Foundation of China (Key Program Grant No.20736006), the National Basic Research Program of China (‘‘973”Program: 2007CB714301), the International Collaboration Projectof MOST (2006DFA62400) and key projects in the National Scienceand Technology Pillar Program (No. 2007BAD42B02). This workwas also funded in part by the DOE Great Lakes Bioenergy ResearchCenter (DOE Office of Science BER DE-FC02-07ER64494). We alsothank Nancy Ho, Purdue University for generously providing us S.cerevisiae 424A (LNH-ST) strain.

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