Bioresource Technology 102 (2011) 6479–6486

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Bioresource Technology

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Enhancing alfalfa conversion efficiencies for sugar recovery and ethanolproduction by altering lignin composition q

Bruce S. Dien a,⇑, David J. Miller b, Ronald E. Hector a, Richard A. Dixon c, Fang Chen c, Mark McCaslin d,Peter Reisen d, Gautam Sarath e, Michael A. Cotta a

a Bioenergy Research Unit, United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, United Statesb Pioneer Hi-Bred International, Inc., W8131 State Road 60, Arlington, WI 53911-9709, United Statesc Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, United Statesd Forage Genetics, International, 812 1st Street South, Nampa, ID 83651-3808, United Statese Forage and Bioenergy Research Unit, United States Department of Agriculture, Agricultural Research Service, Biochemistry Hall, University of Nebraska, East Campus, Lincoln,NE 68583-0737, United States

a r t i c l e i n f o

Article history:Received 16 December 2010Received in revised form 4 March 2011Accepted 8 March 2011Available online 15 March 2011

Keywords:BiomassAlfalfaBioethanolPretreatmentLignin-modified transgenics

0960-8524/$ - see front matter Published by Elsevierdoi:10.1016/j.biortech.2011.03.022

q Names are necessary to report factually on availaneither guarantees nor warrants the standard of thename by USDA implies no approval of the product tomay also be suitable.⇑ Corresponding author.

E-mail address: Bruce.Dien@ars.usda.gov (B.S. Dien

a b s t r a c t

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cul-tivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-typecontrol. Both were harvested at two maturities. All the samples had similar carbohydrate contents includ-ing a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, whichcorresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated withdilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pre-treated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinantxylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, furtherincreased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232–278 l/ton and weresignificantly enhanced for the reduced S lignin cultivars.

Published by Elsevier Ltd.

1. Introduction

There is considerable interest in replacing petroleum withrenewable biomass feedstocks for production of chemicals andtransportation fuels as a strategy for reducing greenhouse gas emis-sions and to increase energy security (Dien and Bothast, 2009). Tomeet the ambitious goals targeted by the United States Federal gov-ernment, considerable amounts of biomass will need to be producedas dedicated bioenergy crops. The United States Department ofAgriculture has targeted that by 2022, 13.4 billion gallons/year ofethanol should be produced using dedicated bioenergy crops(www.usda.gov/documents/USDA_Biofuels_Report_ 6232010.pdf,accessed December, 2010).

Production of biomass at this capacity will present opportuni-ties for development of multiple biomass crops selected basedupon local conditions, including water availability, terrain, soil,

Ltd.

ble data; however, the USDAproduct, and the use of thethe exclusion of others that

).

and climate. One potential biofuels feedstock is the stem fractionfrom alfalfa (Medicago sativa). Alfalfa is already a commercial cropcultivated domestically throughout the United States and has sev-eral characteristics that would recommend it as a bioenergy crop(Lamb et al., 2007; Samac et al., 2006). It is a perennial with a deeproot system that enhances soil quality and carbon content. It is alegume and requires no nitrogen fertilizer input. When used inrotation with a row crop, such as corn, alfalfa eliminates the needfor nitrogen input during the following production year and isassociated with a 5–15% boost in corn yield. Finally, alfalfa leavesare high in protein content (26–30%). As such, alfalfa is uniqueamong bioenergy crops in having a readily available co-product,whereby the leaves can be marketed as animal feed and the stemsconverted to biofuels.

One disadvantage of alfalfa is that its biomass production yieldis lower than those cited for other bioenergy crops, most of whichare warm season grasses. However, in the past alfalfa has beenmanaged for forage quality to the detriment of biomass yield. Morerecent studies have started to focus on improving biomass yield. Inone study that emphasized biomass production (Lamb et al., 2007),the yield per hectare was doubled, while maintaining productionof the protein rich leaf biomass.

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From an agronomic viewpoint, alfalfa has also been the subjectof intense research because of its historic use as high quality for-age. Especially relevant to its use for biofuels production are effortsto genetically engineer its plant cell wall for greater forage digest-ibility. This includes successful efforts to alter lignin compositionand content (Boerjan et al., 2003). Lignin is a three dimensionalphenolic polymer linked by carbon–carbon and ether bonds thatis present in mature plant cell walls. As might be expected fromits aromatic (i.e. hydrophobic) nature and very stable linkages, lig-nin is a formidable barrier to enzymatic saccharification (Simmonset al., 2010; Weng et al., 2008).

In a landmark study (Chen and Dixon, 2007), reducing lignincontent was examined as a tool for enhancing sugar recovery.Beginning with a common parent, a set of six alfalfa lines was con-structed with varied lignin composition and/or content and evalu-ated for sugar yields following dilute-acid pretreatment andenzymatic saccharification. Pretreatment is required prior to enzy-matic saccharification to open up the cell wall matrix and allow en-zymes (e.g. cellulase) access to the individual cellulose fibers(Mosier et al., 2005). In this alfalfa study, the authors were success-ful in doubling the sugar yield compared to the parent cultivar byreducing lignin content (Chen and Dixon, 2007). However, lower-ing lignin content as a strategy for improving conversion qualitycan have the undesirable consequence of reducing biomass pro-duction (Simmons et al., 2010). An alternate strategy would be toalter lignin composition as opposed to content so as to make thelignin more amendable to pretreatment and enzymatic saccharifi-cation (Grabber et al., 2010; Simmons et al., 2010; Weng et al.,2008).

Lignin principally consists of p-hydroxyphenyl [H], guaiacyl [G],and syringyl [S] phenylpropanoid units that are polymerized byradical chemistry coupling reactions. For herbaceous dicots (e.g. al-falfa) G and S units predominate. There is evidence that enrichingfor G lignin composition while keeping total lignin content con-stant increases alfalfa digestibility (Guo et al., 2001a). G lignincan be enriched by reducing the activities of enzymes specific forbiosynthesis of S lignin, such as caffeic acid O-methyltransferase(Comt). Specifically, Comt methylates the 5-hydroxyl positionson the aromatic ring of monolignal precursor 5-hydroxy coniferyl-aldehyde, which is subsequently converted to sinapyl aldehyde,and finally to sinapyl alcohol by the enzyme cinnamyl alcoholdehydrogenase (Chen and Dixon, 2007). Antisense down-regula-tion of COMT has been observed to dramatically reduce S lignincontent relative to WT without reducing total lignin content orcompromising biomass yield (Guo et al., 2001b). Furthermore,down-regulation of COMT improves forage quality by making alfal-fa hay more digestible for cattle and dairy cows (Guo et al., 2001a).As such, comt alfalfa (e.g. down-regulated for COMT expression) isunder consideration for commercial development.

This study evaluates a pre-commercial released comt cultivarfor improved recovery of sugars and conversion to ethanol com-pared to the wild-type (WT) control. As alfalfa fields can be repeat-edly harvested during a single season, the sample set included twoharvest times (i.e. Early and Late) for each cultivar. Production ofsugars and ethanol from biomass is a multistep process that in-volves a pretreatment step, followed by enzymatic hydrolysis forproduction of sugars, and fermentation of the sugars into ethanol(Dien et al., 2005). Often when processing pretreated biomass intoethanol, the hydrolysis enzymes are co-added with the yeast to al-low for simultaneous saccharification and fermentation (SSF) (Dienet al., 2005), as was done in this study. SSF reduces the required en-zyme loading by avoiding end-product inhibition of the hydrolysisenzymes (e.g. cellulases) (Dien and Bothast, 2009).

Separate experiments for sugar and ethanol production wereconducted using both dilute-sulfuric acid and dilute-ammonia pre-treatments so as to observe the affect of the comt genotype on both

pretreatments. SSF experiments were conducted using a nativeSaccharomyces cerevisiae yeast (strain D5A), which can only fer-ment hexose sugars such as glucose, so as to directly measurethe conversion of cellulose into ethanol without interference fromother (pentose) sugars. However, to judge the full yield potential,dilute ammonia pretreated alfalfa samples were also convertedto ethanol using a novel S. cerevisiae strain (YRH400), which hasbeen engineered to also ferment xylose- the next most commoncarbohydrate present in alfalfa hydrolysates after glucose. There-fore, from a broader perspective, this paper also evaluates alfalfastems as a promising feedstock for recovery of sugars and ethanolproduction using both dilute-acid and ammonia pretreatments.

2. Methods

2.1. Materials

Transgenic alfalfa lines down-regulated for COMT were devel-oped as previously described (Guo et al., 2001b). Isogenic lines ofalfalfa with and without the transgene to down regulate COMTwere field grown in summer of 2007 in Nampa, Idaho. Samplesfrom two harvest times were evaluated for each alfalfa line, August16 and September 7. The two harvest times are differentiated inthe text as Early and Late. Harvested alfalfa was dried at 50 �C,the leaves manually separated from the stems, and the stemsground with a knife mill (SM100, Retsch Cutting Mill, Germany)equipped with a 4 mm screen. Cellulase GC220 and Multifect Pec-tinase were kindly donated by Genencor, Inc. (Rochester, NY) andNovo188 b-glucosidase (Novozymes A/S, Denmark) was purchasedfrom Sigma–Aldrich Chemical Co. (St. Louis, MO). Other reagentsand chemicals were purchased from Sigma–Aldrich Chemical Co.or Fisher Scientific (Pittsburgh, PA) and were research quality.The yeast strains used were S. cerevisiae D5A and YRH400. StrainYRH400 was constructed by integrating the genes for xylose utili-zation (xylose reductase, xylitol dehydrogenase, and xylulose ki-nase) into the D5A genome as previously described (Hector et al.,in press). Strain D5A has been recommended for fermentationevaluations of biomass (Dowe and McMillan, 2001).

2.2. Compositional analysis

Starch was measured following treatment with amylases (Hall,2001). Structural carbohydrates (cellulose, xylan, and arabinan),Klason lignin, acetate, and ash were determined according to theanalytical procedure of the National Renewable Energy Laboratory(NREL) (Sluiter et al., 2008). Ether extractables (e.g. lipids andwaxes) were measured using a standard protocol (AOAC 2003.05.Crude Fat in Feeds, Cereal Grains, and Forages). Nitrogen contentwas determined by combustion, and crude protein concentrationwas estimated as Nx6.25 (Padmore, 1990). Soluble sugars were ex-tracted by combining 200 mg alfalfa with 3.0 ml distilled water in aglass test tube and sonicating in an ultrasound bath for 30 min. Ex-tracted sugars were measured using high-performance liquid chro-matography (HPLC). Uronic acids were determined as previouslydescribed (Dien et al., 2006).

2.3. Dilute-acid pretreatment, enzymatic hydrolysis, and ethanolfermentation

Alfalfa stem samples were pretreated in dilute sulfuric acidsolution (3.0% w/v) by combining 1.5 g of alfalfa stems (dry basis,db) with 8.5 ml of the acid solution (15% w/v solid loading). Corn-ing Pyrex™ solution bottles (25 ml) were used as reaction vessels,and lids were tightened prior to heating. Samples were heated at

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121 �C in an autoclave for 1 h. The whole hydrolysates were ad-justed to pH 4.5–5.0 by adding Ca(OH)2.

Cellulose digestibility was determined using a modified versionof a NREL procedure (Selig et al., 2008). The whole hydrolysate wasdiluted to 3% solids by adding distilled water, 1 M sodium citratebuffer (final concentration 50 mM, pH 4.8) supplemented withthe antibacterial agent thymol (500 mg/l). The enzymatic digestionwas initiated by adding GC220 cellulase (either 10 or 30 FPU/g cel-lulose), Novo188 b-glucosidase (40 U/g cellulose), and Multifectpectinase (84 mg/g biomass). The reaction was carried out at50 �C for 72 h while stirring at 125 rpm using an incubator/shaker(Innova�, New Brunswick Scientific, NJ). Digested samples wereanalyzed for soluble carbohydrates and monosaccharides.

Ethanol conversion efficiencies were determined using a modi-fied version of the NREL procedure (Dowe and McMillan, 2001).The whole hydrolysates was neutralized by adding Ca(OH)2 andwas diluted by adding 0.55 ml 1 M sodium citrate buffer, and1.1 ml 10� YP stock (100 g/l yeast extract and 200 g/l Bacto pep-tone). The buffer and YP stock were sterilized separately (15 or20 min at 121 �C). Next, the following filter-sterilized enzymeswere added: GC220 cellulase (15 FPU/g cellulose), Novo188 b-glu-cosidase (40 U/g cellulose), and Multifect pectinase (84 mg/g bio-mass). The enzyme loadings are reduced from those used for thedigestions because simultaneous saccharification and fermentation(SSF) was conducted to minimize end product inhibition. The cul-ture was inoculated with S. cerevisiae strain D5A (American TypeCulture Collect strain 200062) to an OD600 of 1.0.

The inoculum was prepared by transferring a colony grown onsolid YP2D (YP supplemented with 20 g/l dextrose and 2.0% Bactoagar) to a liquid YP2D culture followed by an YP5D culture (YP sup-plemented with 50 g/l dextrose). The liquid cultures were grownfor 18 h at 35 �C and 200 rpm, and the cells were harvested fromthe YP5D culture by centrifugation and concentrated to a cell solu-tion of 50 OD600 in PPB (per L: 8.5 g NaCl, 0.3 g KH2PO4, 0.6 g Na2H-PO4, 0.4 g peptone).

Cultures were incubated at 32 �C for 96 h while mixing at150 rpm using a 3=4’’ Teflon™ coated cross shaped stirring bar anda 6 position magnetic stirrer (Vero Variomag Poly 6 MultipointStirrer, Cole-Parmer, Vernon Hills, Illinois). The fermentation vesselconsisted of a 100 ml threaded Pyrex™ bottle and capped with apressure sensor top (see below). Following the fermentation, thecultures were analyzed for ethanol, xylitol, and residual sugar con-centrations using HPLC.

Fermentation progress was continually monitored by measur-ing carbon dioxide gas production; one carbon dioxide is producedper ethanol. Carbon dioxide production was monitored using theAnkom wireless gas production system (Ankom Technology,Macedon, NY) set to vent at 1.0 psi over-pressure. The system in-cludes pressure monitoring caps, a wireless receiver for collectingpressure data equipped with a USB connection and a softwareprogram for recording the pressure and setting the device param-eters. The cap fits on top of the threaded fermentation bottle with agas-tight fit and contains a pressure transducer that continuallymonitors gas pressure in the headspace, a wireless transmitter, arechargeable battery, and a valve for venting gas to preventover-pressuring. Cumulative gas production was calculated fromthe recorded gas pressure data using the ideal gas law.

2.4. Alkaline pretreatment and fermentation

Alfalfa samples were treated at 15% w/w solids in 8.0% w/vammonium hydroxide solution with 100 ml volumes at 170 �Cfor 20 min. The reactions were carried out using an infra red heat-ing oven equipped with stainless steel rotating vessels set at60 rpm (Mathis Labomat, Werner Mathis AG, Zurich, Switzerland).After cooling, samples were air dried at 25 �C for 48 h to remove

ammonia. Cellulose digestibility was measured using the sameprocedure as for dilute-acid except extra distilled water was addedto achieve the targeted solids loading. The same enzymes and load-ing were used, expect GC220 cellulase was only tested at 30 FPU/gcellulose. Fermentations were evaluated by combining 4.00 g dbpretreated alfalfa stems with 30.0 ml sterile dH2O in a screw capbottle. The sodium citrate buffer, YP stock, and enzymes wereadded to the same concentrations or loadings as used for evaluat-ing dilute-acid pretreated samples. Fermentations were conductedas described above using either S. cerevisiae strain D5A or YRH400.

2.5. Analytical methods

Samples were dried at 105 �C for 18 h for moisture determina-tions; all solids loadings are referenced to these moisture contents.Samples used for moisture determinations were discarded and notused for further analysis.

Thioacidolysis followed by GCMS was used to determine therelative composition of H-, G-, and S-lignin components as previ-ously described (Palmer et al., 2008). Cellulase, b-glucosidase,and xylanase activities were measured as previously reported(Dien et al., 2006, 2008). Total soluble carbohydrates were ana-lyzed by HPLC after being hydrolyzed by treating with 2 M trifluo-roacetic acid for 1 h at 100 �C. Samples were analyzed for sugars,acetic and lactic acids, and ethanol using a SpectraSYSTEM™ liquidchromatography system (Thermo Electron Corporation, CA)equipped with an automatic sampler, column heater, isocraticpump, refractive index detector, and computer based integratorrunning Chromquest ver. 2.5 (Thermo Electron Corporation, CA).Samples (20 ll) were injected onto a sugar column (AminexHPX-87H Column, 300 � 7.8 mm, Bio Rad Laboratories, Inc., Hercu-les, CA) and eluted with 5 mM sulfuric acid at 0.6 ml/min and 65 �Cas previously described (Dien et al., 2004).

2.6. Calculations

Glucose conversion efficiencies were then calculated from theratio of glucose released following cellulase treatment and thebeginning cellulose content, adjusted for the weight gainedthrough hydrolysis. Ethanol efficiencies from the fermentationsof pretreated samples were calculated from the ratio of ethanolproduced and maximum theoretic ethanol. The latter was calcu-lated for the native S. cerevisiae D5A by multiplying the beginningglucan content by the theoretic conversion coefficient for ethanolproduction (0.567 g ethanol/g glucan) and for xylose-fermentingS. cerevisiae YRH400 by multiplying the beginning glucan and xylancontents by 0.567 and 0.580 g ethanol per grams of glucan and xy-lan, respectively. Experiments were performed in at least duplicateand statistical differences (p < 0.05) analyzed using a two factor(maturity and genotype) ANOVA (Design-Expert version 7.0.3,State-Ease, Inc., Minneapolis, MN). Results for which the interac-tion term was significant were further analyzed for differencesusing least square means (SAS software, SAS Institute Inc., Cary,NC).

3. Results and discussion

Obtaining the highest conversion yields to biofuels is essentialfor making cellulosic biofuels commercially feasible (Aden et al.,2002). Maximizing production requires overcoming natural recalci-trance of plant cell walls to biochemical conversion. Lignin is a ma-jor barrier to enzymatic hydrolysis of cellulose (Mosier et al., 2005)and lowering lignin content directly improves enzymatic sugaryields in monocots (Dien et al., 2009) and dicots (Chen and Dixon,2007). Chen and Dixon (2007) evaluated enzymatic digestibility

Fig. 1. Analysis of alfalfa stems for lignin composition. Alfalfa stems were analyzedfor lignin composition by thioacidolysis followed by GC–MS and measured asnormalized ion signal to give the relative ratios of each lignin type.

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for a set of alfalfa plants genetically altered for lignin content andobserved a linear relationship between reduced lignin and glucoseyield with a correlation of 0.852. Likewise, Dien et al. (2009) exam-ined the effect of reduced lignin on forage sorghum and also ob-served a linear correlation between reduced lignin content andglucose yield following enzymatic hydrolysis (0.971). The affect ofmodifying lignin composition (i.e. the ratio of G lignin to S lignincontents) on biomass recalcitrance is less certain. The alfalfa linescompared in the present study can help to address this uncertaintybecause they vary in lignin composition, but all have similar Klasonlignin contents.

In this study, an alfalfa line enriched for G lignin was comparedto the WT line as feedstocks for biofuels production. This trans-genic alfalfa line is referenced as either comt or down-regulatedCOMT; Comt refers to the enzyme. Samples were field grown andcollected at early and late dates in the growing season. Leaves wereseparated from the stems because their relatively high-proteincontent makes them a valuable feed co-product. The samples wereextensively analyzed for composition and compared for theoreticethanol yields based upon carbohydrate compositions. Subse-quently, the experimental sugar and ethanol yields were deter-mined by enzymatic hydrolysis to sugars and by SSF to ethanol,respectively. Selective conversion yields of cellulose to ethanolwere measured by using a native S. cerevisiae – unable to fermentxylose- or the SSF protocol. Xylose at the end of the fermentationwas measured as a separate product. The samples were first eval-uated using a low severity dilute-acid pretreatment for sugar andethanol yields. Next, the samples were evaluated using dilute-ammonium pretreatment. Finally, the ammonium pretreated sam-ples were also evaluated for the maximum experimental ethanolyield by using a novel S. cerevisiae strain engineered to ferment xy-lose along with glucose.

3.1. Effect of comt genotype on biomass composition

The four alfalfa samples (early harvested WT, early harvestedcomt, late harvested WT, and late harvested comt) were extensivelyanalyzed for chemical composition to see if down regulation ofCOMT would affect gross cell wall composition. The chemical com-positions of the stem fractions are listed in Table 1. All the sampleswere remarkably similar in composition. Neutral carbohydrates ac-counted for most of the biomass, 48.9–51.3% w/w, followed bycrude protein (12.2–13.2% w/w), Klason lignin (11.9–14.6% w/w),and uronic acid (from pectin, 7.5–7.8% w/w). The Klason ligninmeasurement is of particular interest because the COMT down-reg-ulation directly affects the lignin biosynthesis pathway (seeSection 1). The early harvested comt material had 12% less Klasonlignin than the early harvested WT control. This reduction in ligninmight be associated with delayed development in the comt plantsbecause the later harvested comt and WT materials only differed by2%. Other than this small difference, down-regulation of COMT didnot appear to affect gross plant composition.

The alfalfa samples were next analyzed for the ratios of lignintypes by thioacidolysis analysis. The comt phenotype is expectedto enrich for G lignin because reducing Comt activity slows

Table 1Chemical composition of alfalfa stems (g per kg of alfalfa stems, dry basis).a

Genotype Harvest maturity Ether extractables Carbohydrates Acetate

comt Early 8 ± 1 489 ± 4 45.0 ± 6WT Early 8 ± 1 489 ± 2 41.0 ± 1comt Late 11 ± 1 498 ± 3 54.0 ± 2WT Late 10 ± 0 513 ± 1 54.0 ± 0

a Mean ± standard deviation based upon duplicate determinations.

production of sinapylaldehyde, which is an intermediate in the for-mation of S type lignin (Chen and Dixon, 2007). And. when lignincomposition was measured using GC–MS (Fig. 1), the comt lineswas dramatically reduced in S lignin relative to the WT controls.In addition, levels of 5-hydroxyconiferyl lignin (5-OH G Lignin)were increased relative to the WT controls. Incorporation of 5-OH G has also been previously observed in comt down-regulatedalfalfa plants and poplar trees (Lu et al., 2010; Marita et al.,2003). It should be noted that unlike Klason lignin, thioacidolysisanalysis does not measure total lignin; it only measures b-O-4linked lignin monomers. This is why it is possible to observe a dra-matic reduction in S lignin relative to G- and H- and no change inthe total Klason lignin content. That Klason lignin contents weresimilar for comt and WT samples suggests that slowing S ligninbiosynthesis increased the flux of intermediates to the G ligninbranch of the pathway.

The breakdown of the carbohydrate fraction into individualneutral sugars is listed in Table 2. The neutral sugar compositionswere remarkably similar for the entire group of samples and thestandard deviations for the sample set were 1.0–3.3% w/w. Theone exception was that the starch content of the early comt samplewas double that of the early WT sample; the lignin content for thiscomt sample was also lower than the others (Table 1). One possibil-ity is that the earlier harvested comt plants were physiologically atan earlier developmental stage as compared to the WT plants.However, starch only comprised 0.4–1.8% w/w of the biomass.The maximum (or theoretic) ethanol yields for the biomass sam-ples were calculated directly from the neutral sugar data (Table2) for two scenarios: conversion of only hexoses (e.g. glucose)and conversion of all neutral carbohydrates – including pentoses(e.g. xylose). Fermentation of hexoses were considered separatelybecause they are more readily fermented to ethanol than pentoses(Dien et al., 2003). The theoretic ethanol yields were 245–256 l/tonfor fermentation of the hexoses and 375–391 l/ton for fermenta-

Total uronic acid Crude protein Klason lignin Ash Sum

.4 76.0 ± 2.7 132 ± 1 118.8 ± 1.9 75 ± 0 952.8

.0 77.0 ± 2.6 127 ± 0 133.4 ± 0.1 64 ± 6 939.3

.4 78.0 ± 7.1 129 ± 6 142.8 ± 0.2 66 ± 2 967.1

.8 75.0 ± 0.8 122 ± 1 145.9 ± 1.9 71 ± 0 959.4

Table 2Carbohydrate composition of alfalfa stems (g/kg of alfalfa, db).a

Genotype Harvest Soluble sugars Storage Structural carbohydrates Theoretic ethanol yield (l/ton, db)

Sucrose Glucose Fructose Starch Cellulose Xylan Arabinan From hexoses From all sugars

comt Early 28.0 ± 5.4 3.2 ± 0.1 5.0 ± 0.1 18.5 ± 2.1 306 ± 2 155 ± 0 28 ± 1 256 391WT Early 23.0 ± 2.2 3.2 ± 0.2 5.8 ± 0.4 9.0 ± 8.5 304 ± 0 150 ± 6 27 ± 1 245 375comt Late 25.7 ± 3.9 6.6 ± 0.3 7.4 ± 0.3 5.0 ± 0.0 307 ± 0 147 ± 2 29 ± 1 250 379WT Late 23.6 ± 3.5 5.0 ± 0.1 6.0 ± 0.1 4.0 ± 1.4 311 ± 2 150 ± 0 32 ± 0 249 382

a Mean ± standard deviation based upon duplicate determinations.

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tion of hexoses and pentoses. Clearly conversion of both glucoseand xylose is important to maximize ethanol yields. It is notablethat the maximum ethanol yields were similar for all the samplesregardless of genotype and harvest date. While pectin represents7% w/w of the biomass samples (Table 1) and can be convertedto ethanol by some microorganisms (Doran et al., 2000), it wasnot included in the ethanol calculations because most ethanol pro-ducing microbes – including the yeasts used in this study – are un-able to ferment it.

3.2. Dilute-acid pretreatment for recovery of sugars and ethanolproduction

The alfalfa samples were pretreated by reacting them at 121 �Cfor 1 h in the presence of dilute sulfuric acid. As discussed in theintroduction, pretreatment is necessary for efficient enzymatic sac-charification. Following dilute-acid pretreatment, the neutralizedsamples were evaluated by treating them with commercial carbo-hydrolases (e.g. cellulase, b-glucosidase, and pectinase) for 72 hand measuring the released glucose, xylose, and soluble xylan olig-omers. Pretreated samples were saccharified at high (30 FPU/g glu-can) and low (10 FPU/g glucan) cellulase loadings to betterevaluate the affect of COMT down-regulation on recoveries of glu-cose and xylose. The glucose and xylose conversion efficiencies (i.e.% of available glucose or xylose enzymatically extracted) are listedin Table 3. Glucose yields for both enzyme loadings followed thesame trend among the sample set, albeit doubling the amount ofcellulase added led to 1.5� higher glucose yields than for the lowercellulase loading. Glucose yields were higher for the later har-vested samples and were unaffected by the comt genotype. The xy-lose conversion efficiencies varied much less, approximately 3%,and followed no general pattern for the lower and higher enzymeloading experiments. For example, at the lower enzyme loading,

Table 3Comparison of conversion efficiencies to sugars and ethanol for dilute-acid pretreatedalfalfa stems.A

Harvest maturityGenotype

Conversion efficiencies (% of maximum)

Early Late

WT comt WT comt

Low enzyme loading (10 FPU/g glucan)Glucose 35.7 ± 3.9bB 37.5 ± 2.4b 43.3 ± 2.3a 46.1 ± 2.3aXylose 75.8 ± 0.2b 78.7 ± 0a 75.5 ± 1.1b 79.3 ± 0.3a

High enzyme loading (30 FPU/g glucan)Glucose 59.5 ± 2.7b 58.9 ± 3.6b 63.7 ± 1.8a 67.5 ± 1.6aXylose 79.4 ± 1.9abC 76.1 ± 3.1b 78.2 ± 1.4b 83.1 ± 0.8a

SSF using native D5A S. cerevisiaeEthanol 83.8 ± 5.5bC 86.9 ± 3.9b 80.4 ± 3.4b 93.7 ± 3.8aXylose 62.8 ± 5.7a 59.7 ± 5.4a 60.4 ± 4.8a 62.2 ± 4.6a

A Mean ± standard deviation based upon at least triplicate determinations.B Means within each row followed by the same letter are not significantly dif-

ferent based on the ANOVA F test of significance at p < 0.05.C Means within each row followed by the same letter are not significantly dif-

ferent based on differences of least square means at p < 0.05.

comt samples had higher xylose extraction efficiencies than theWT controls; but this trend was not apparent at the higher enzymeloading. Therefore, based upon these results, it can be concludedthat comt had a minimal, if any, effect on recovered sugar yieldsfrom dilute acid pretreated samples.

The pretreated samples were also directly evaluated for conver-sion to ethanol by simultaneous saccharification and fermentation(SSF) using an industrial S. cerevisiae yeast strain, D5A. Ethanolconversion efficiencies (Table 3) were calculated for just glucosecontent because native S. cerevisiase strain D5A cannot metabolizexylose. The results were mixed for the comt lines. Ethanol conver-sion efficiencies were significantly improved for the comt vs. WTfor the late but not early harvested samples. Overall, the ethanolconversion efficiencies afforded by the dilute-acid pretreatmentwere favorable (80.4-93.7%). Notably they were higher than ex-pected from the enzyme digestion results (in the absence of fer-mentation) based upon the percent glucose recoveries (Table 3).One possible explanation is that the SSF avoided end-product inhi-bition of the added cellulases because glucose was fermented toethanol as soon as it formed; glucose and cellobiose are potentinhibitors of cellulases.

Therefore, the effect of lowering S lignin content (e.g. comt phe-notype) on convertibility was mixed for dilute-acid pretreatedsamples: there was no detectable influence on enzymatic releaseof glucose or SSF of early harvested samples. Conclusions fromprior studies are also conflicted on the effects of lowering S lignin.Chen and Dixon (2007) observed that COMT down-regulation wasassociated with significantly greater enzymatic sugar release.However, this result could not be linked to altered lignin composi-tion because the greenhouse grown comt transgenic plants alsohad 21.7% less total lignin than the control. As previously dis-cussed, increased sugar release is highly correlated with reducedlignin content. Additionally, lowering the ratio of S lignin to G lig-nin, introduced via down-regulation of alternate lignin biosynthe-sis enzymes to Comt, has even been observed to reduce sugaryields (Jackson et al., 2008).

Enzymatic glucose yields reported in the present study (58.9–67.5% of maximum) are comparable to previously reported values:57–67% (Rock et al., 2009) and 63–72% (Dien et al., 2006) of max-imum. In contrast, saccharification efficiencies up to 79% were re-ported following dilute-acid pretreatment of reduced ligninmutants (Chen and Dixon, 2007); however, these yields are not di-rectly comparable to this study because their results are basedupon the washed solids and not the starting material. Samplewashing is known to improve cellulose conversion by removal ofsoluble inhibitors (Merino and Cherry, 2007).

3.3. Alkaline pretreatment for recovery of sugars and ethanolproduction

Alkaline pretreatment is an effective alternative to dilute-acidfor pretreatment of herbaceous biomass (Dien et al., 2005). Weare unaware of any prior reports on the affect of comt on the con-version of alkaline pretreated alfalfa biomass. We favor using

Fig. 3. Effect of maturity and comt phenotype on ethanol conversion efficiency ofalfalfa stems. Stems were pretreated with ammonia and fermented to ethanol bySSF using xylose-fermenting Saccharomyces strain YRH400. Error bars showstandard deviations.

Table 5Fermentation yields from ammonia pretreated alfalfa stemsb.

Genotype Harvestmaturity

Xylosea

(g/l)Xylitol(g/l)

Ethanol(g/l)

Yield-ethanol(l/ton)

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ammonium hydroxide for alkaline pretreatment because theammonia catalyst can be recovered by evaporation for reuse inthe process and the resulting hydrolysate is readily fermented bySaccharomyces (Dien et al., 2009). Therefore, the samples were pre-treated with ammonia and evaluated for enzymatic saccharifica-tion and ethanol fermentation.

Ammonia pretreated samples were saccharified with the stan-dard (high) cellulase loading (30 FPU/g glucan) for 72 h and evalu-ated for released glucose and xylose. The observed glucose yields(Table 4) were 72.3–77.8% of maximum. These are higher than ob-served for dilute-acid pretreatment and are comparable to previ-ously published results. Ammonia fiber expansion (AFEX)pretreated alfalfa gave conversion efficiencies of 60–80% basedupon total sugar yields; glucose yields are unreported (Belkacemiet al., 1998).

The comt lines improved both glucose and xylose hydrolysisefficiencies compared to the WT lines (Table 4). The comt versusWT samples were improved by 5.8% and 6.8% for glucose and xy-lose recoveries, respectively (Fig. 2). Unlike the trend observedfor dilute-acid pretreatment, harvest date had no affect on enzy-matic saccharification.

The same pretreated samples were also evaluated for directconversion to ethanol using native S. cerevisiae D5A (Table 4). Onceagain ethanol efficiencies were calculated based solely on glucancontents. The fermentation results followed the same pattern

Fig. 2. Effect of maturity and comt phenotype on enzymatic digestibility of alfalfa stems.glucose and xylose. Key: glucose = dark gray and xylose = light gray. Error bars show sta

comt Early 4.2 ± .2 3.2 ± 0.1 22.9 ± 1.0 277.5 ± 20.1WT Early 3.2 ± .2 2.3 ± 0.1 18.6 ± 0.0 232.0 ± 1.8comt Late 3.2 ± .1 2.9 ± 0.3 21.7 ± 1.3 262.4 ± 12.7WT Late 3.6 ± .2 2.3 ± 0.1 19.4 ± 0.5 239.9 ± 8.0

bMean ± standard deviation based upon duplicate determinations.a Residual xylose.

Table 4Comparison of conversion efficiencies to sugars and ethanol for ammonia pretreatedalfalfa stems.A

Harvest maturity Genotype Conversion efficiencies (% of maximum)

Early Late

WT comt WT comt

High enzyme loading digestion (30 FPU/g glucan)Glucose 72.3 ± 1.1aB 78.9 ± 0.1b 72.7 ± 2.0a 77.8 ± 0.7bXylose 71.6 ± 0.5a 79.8 ± 0.0b 72.7 ± 2.0a 78.0 ± 0.9b

EthanolC 86.4 ± 2.8 94.4 ± 0.8b 89.21 ± 0.4a 94.7 ± 2.3bXylose 28.7 ± 1.2a 33.1 ± 3.2b 29.0 ± 1.52a 32.5 ± 0.7bEthanolC 66.0 ± 0.5a 79.6 ± 5.8 b 71.8 ± 2.4a 79.6 ± 3.8b

A Mean ± standard deviation based upon duplicate determinations.B Means within each row followed by the same letter are not significantly dif-

ferent based on the ANOVA F test of significance at p < 0.05.C Ethanol efficiency is based upon total glucose content for S. cerevisiae D5A and

upon total glucan and xylan contents for YRH400; YRH400 produced more ethanolthan D5A.

observed for the enzyme digestions. For both ethanol and xylose,the conversion efficiencies were significantly improved for thecomt lines. On average, the ethanol yields for the comt lines were6.8% higher than the WT lines and 4.0% higher for xylose recover-ies. Xylose recoveries were much lower for the fermentations thanfor the enzymatic digestions. Reduced xylose recoveries for the fer-mentations resulted most likely from end-product inhibition of thexylanases because the SSFs used a higher concentration of pre-treated biomass than the saccharifications (10% vs. 3% w/w). Thelower enzyme loading used for the SSF vs. enzyme digestion wouldhave accentuated this inhibition effect. For the case of dilute-acid

Stems were pretreated with ammonia and enzymatically digested for production ofndard deviations.

Fig. 4. Fermentation rate of ammonia pretreated alfalfa stems using Saccharomyces YRH400 using a SSF. Fermentation progress was tracked by measuring gas production.Plots are average of duplicates fermentations.

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pretreatment, while the SSFs also had lower xylose yields than theenzyme digestions (Table 3), the decrease was much less. Whenpretreating with dilute-acid, the primary mechanism for xyloseformation is acid hydrolysis, which is not subjected to end-productinhibition. Therefore, down-regulation of COMT is advantageous incombination with dilute ammonia pretreated alfalfa stems forrecovery of sugars and ethanol production.

Down regulation of COMT also increases rumen digestibility ofalfalfa in fistulated steers in the absence of reduced lignin contents(Guo et al., 2001a). The specific mechanism that leads to improveddigestion is unknown. Conversely, reducing the ratio of S lignin toG lignin is expected to result in a more condensed lignin with in-creased carbon–carbon linkages (Guo et al., 2001b), which wouldbe expected to impede enzymatic conversion. However, COMTdown regulation also leads to the incorporation of novel units intolignin that arise from intermediates in the lignin synthesis path-way (Marita et al., 2003), such as 5-hydroxyconiferyl alcohol(Fig. 1). It might be that inclusion of these novel intermediatesmight ‘‘loosen’’ the overall lignin structure and be responsible forimproving digestibility.

For commercial applications, both glucose and xylose will needto be converted into ethanol. Therefore, the ammonia pretreatedalfalfa samples were subsequently converted to ethanol using thexylose-fermenting Saccharomyces strain YRH400. This yeast strainwas constructed by inserting the genes needed for xylose fermen-tation into the genome of D5A (Hector et al., in press). For thisexperiment, the ethanol conversion efficiencies were calculatedbased upon the glucan and xylan contents. Once again, the COMTdown-regulated lines outperformed the WT controls; this timethe conversion efficiencies were improved by 10.7% (Table 4,Fig. 3). The increase in efficiencies led directly to a 3.3 g/l or17.4% increase in the final ethanol concentrations for the comt bio-mass containing fermentations (Table 5). Furthermore, no lagphase was observed for any of the fermentations as monitoredby gas product (one CO2 is produced per ethanol) (Fig. 4).

Saccharomyces YRH400 cultures gave a higher final ethanol titerthan the D5A cultures because of the ability of YRH400 to also fer-ment xylose. However, the gain in ethanol yield was lower thanmight be expected given the significant amount of xylose presentin alfalfa stems. This was because some of the xylose was leftunfermented and some was converted to the side-product xylitol.

In this regard, the results are typical of those reported for other xy-lose-fermenting Saccharomyces strains (Hahn-Hagerdal et al.,2007). Further work is in progress to lower xylitol formation andincrease the rate of xylose consumption. Still ethanol yields were232.0–277.5 l/ton with the highest yield observed for the earlierharvested comt material (Table 5).

The ethanol yields reported here are superior to previously pub-lished results evaluating alfalfa biomass. The best fermentation re-ported here produced 22.9 g/l ethanol with a conversion efficiencyof 79.6% (Table 5). AFEX pretreated alfalfa stems fermented withPachysolen tannophilus produced only 5.7 g/l ethanol or a 56% con-version efficiency based upon initial sugars (Belkacemi et al.,1998). Previously reported results from studies where alfalfa stemswere pretreated with dilute-acid and liquid hot-water suggeststhese pretreatments are inefficient because of the formation ofinhibitors that stall fermentation. Alfalfa stems pretreated with di-lute-acid, required an extensive 36–48 h biologic abatement pro-cess to remove inhibitory compounds before being fermentedusing S. cerevisiae and the final ethanol concentration was limitedto 6.1 g/l (Nichols et al., 2010). Likewise, when treated with liquid-hot water, the inhibitor enriched liquid phase has to be removedprior to fermentation (Sreenath et al., 2001). While fermentationof the solids produced 17.9 g/l ethanol, it can be expected thatmuch of the xylose was lost when the sirup was discarded. Hydrol-ysates prepared using dilute-acid in the present study were fer-mentable without any special conditioning to remove inhibitors.The observed difference is most likely because of the lower tem-perature applied here (121 �C) for pretreatment. However, thesamples were treated for an hour, which may not be commerciallyrealistic. The ammonia pretreatment was operated at a higher tem-perature for 20 min and the resulting whole hydrolysates werereadily fermented by S. cerevisiae. In summary, pretreating with di-lute ammonia produced a more fermentable whole hydrolysatewith higher conversion efficiencies to ethanol than previously re-ported and yields were highest for the comt alfalfa plants.

4. Conclusions

Stems from reduced S-lignin (comt) and wild-type alfalfa lineswere evaluated for production of sugars and ethanol. The comt

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lines were greatly reduced in S-lignin content but otherwisesimilar in composition to the WT. Sugar and ethanol yields fromdilute-acid pretreated samples were affected by harvest date butnot lignin content. In contrast, for dilute-ammonia pretreatedsamples, comt improved product yields and harvest date had noaffect. Ethanol yields from fermenting with xylose-fermentingS. cerevisiae YRH400 were 232.0–227.5 l/ton and the comt linesyielded on average 34.0 l/ton more ethanol than WT.

Acknowledgements

The authors would like to thank Ms. Patricia J. O’Bryan,Mr. Loren Iten, and Nathan A. Palmer for their fine technical assis-tance. The authors also thank Ms. Debra Plamquist for statisticalhelp. The work was supported by grants from Forage GeneticsInternational (to Richard A. Dixon).

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